U.S. patent application number 11/635186 was filed with the patent office on 2007-05-03 for crystal structures of mk2 and uses thereof.
This patent application is currently assigned to Wyeth, a Delaware corporation. Invention is credited to Lidia Mosyak, Kevin D. Parris, Tania Shane, Mark L. Stahl, Kristine Svenson, Meggin L. Taylor, Kathryn W. Underwood.
Application Number | 20070100560 11/635186 |
Document ID | / |
Family ID | 32229768 |
Filed Date | 2007-05-03 |
United States Patent
Application |
20070100560 |
Kind Code |
A1 |
Parris; Kevin D. ; et
al. |
May 3, 2007 |
Crystal structures of MK2 and uses thereof
Abstract
This invention is directed to the crystal structures of MK2, and
to the use of these structures in rational drug design methods to
identify agents that may interact with active sites of MK2
proteins. Such agents may be useful as therapeutic agents.
Inventors: |
Parris; Kevin D.;
(Auburndale, MA) ; Underwood; Kathryn W.; (Quincy,
MA) ; Stahl; Mark L.; (Lexington, MA) ;
Mosyak; Lidia; (Newton, MA) ; Svenson; Kristine;
(Andover, MA) ; Shane; Tania; (Newton, MA)
; Taylor; Meggin L.; (Wakefield, MA) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
P.O BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Wyeth, a Delaware
corporation
|
Family ID: |
32229768 |
Appl. No.: |
11/635186 |
Filed: |
December 6, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10294027 |
Nov 13, 2002 |
7167802 |
|
|
11635186 |
Dec 6, 2006 |
|
|
|
Current U.S.
Class: |
702/19 ; 435/194;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C07K 2299/00 20130101;
C07H 21/04 20130101; Y02A 90/10 20180101; C12N 9/1205 20130101 |
Class at
Publication: |
702/019 ;
435/194; 435/069.1; 435/320.1; 435/325; 536/023.2 |
International
Class: |
G06F 19/00 20060101
G06F019/00; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 9/12 20060101 C12N009/12 |
Claims
1-10. (canceled)
11. A method for obtaining crystallized native MK2, crystallized
MK2 polypeptide or crystallized MK2 analogue comprising contacting
native MK2, MK2 polypeptide or MK2 analogue with a buffer solution
comprising at least one of cacodylate, Tris, Tris-HCL, acetate,
malonate, sodium phosphate, potassium phosphate, citrate, HEPES and
MES, at a salt concentration of 0.1 M to 2.4 M, and at a pH of 4.5
to 8.5, under conditions permitting the formation of crystallized
MK2, crystallized MK2 polypeptide or crystallized MK2 analogue.
12. The method of claim 11, wherein the salt has an anion selected
from the group consisting of sulfate, citrate, chloride, acetate,
phosphate, malonate and tartrate.
13. The method of claim 11, wherein the salt concentration is 0.8 M
or higher.
14. The method of claim 11, wherein native MK2, MK2 polypeptide or
MK2 analogue is contacted with the buffer solution in the presence
of PEG or a PEG substitute having a molecular weight up to
3350.
15. The method of claim 14, wherein PEG or PEG substitute is
selected from the group consisting of PEG-200, PEG-400,
PEG-500-MME, PEG-1000, PEG-1500, PEG-2000-MME, PEG-3350, Jeffamine
M-600, ethylene glycol, glycerol and 1-6 hexanediol,
2-methyl-2,4-pentanediol (MPD).
16. The method of claim 14, wherein PEG is PEG-400.
17. A crystallized complex comprising an MK2 polypeptide and
staurosporine.
18. The crystallized complex of claim 17, characterized as having
space group P6.sub.3, unit cell parameters of a=b=160.20 .ANG.,
c=133.48 .ANG..
19. A crystallized complex comprising an MK2 polypeptide and
ADP.
20. The crystallized complex of claim 19, characterized as having
space group F4.sub.132, unit cell parameters of a=b=c=253.05
.ANG..
21-57. (canceled)
58. The crystallized complex of claim 17, wherein the complex
comprises four molecules of MK2 polypeptide per asymmetric
unit.
59. The crystallized complex of claim 17, wherein the complex
further comprises selenomethionine.
60. The crystallized complex of claim 17, wherein the complex
diffracts to at least 3.1 .ANG. resolution.
61. The crystallized complex of claim 17, wherein the complex
diffracts to at least 1.1 .ANG. resolution.
62. The crystallized complex of claim 19, wherein the complex
comprises one molecule of MK2 polypeptide per asymmetric unit.
63. The crystallized complex of claim 19, wherein the complex
diffracts to at least 1.0 .ANG. resolution.
64. The crystallized complex of claim 17, wherein the MK2
polypeptide comprises amino acid residues Leu70, Gly71, Leu72,
Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141,
Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 as set forth in
SEQ ID NO:1.
65. The crystallized complex of claim 64, wherein the MK2
polypeptide further comprises amino acid residues Val69, Ile74,
Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95,
Glu104, His108, Arg 119, Ile136, Val137, Asp142, Gly143, Gly144,
His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204,
Leu205, Phe208, and Gly209 as set forth in SEQ ID NO: 1.
66. The crystallized complex of claim 17, wherein the crystallized
complex has the structural coordinates set forth in FIGS. 2 through
2A-204 .+-.a root mean square deviation from the backbone atoms of
said amino acids of not more than 1.5 .ANG..
67. The crystallized complex of claim 17, wherein the MK2
polypeptide comprises an active site comprising the relative
structural coordinates of amino acid residues Leu70, Gly71, Leu72,
Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141,
Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 of molecules A,
B, C or D according to the sequence set forth in SEQ ID NO:1, and
the structural coordinates as set forth in FIGS. 2 through 2A-204
.+-.a root mean square deviation from the backbone atoms of said
amino acids of not more than 1.5 .ANG..
68. The crystallized complex of claim 67, wherein the active site
of the MK2 polypeptide further comprises the relative structural
coordinates of amino acid residues Val69, Ile74, Gly76, Ala77,
Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108,
Arg119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186,
Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208, and
Gly209 of molecules A, B, C or D according to the sequence set
forth in SEQ ID NO:1, and the structural coordinates as set forth
in FIGS. 2 through 2A-204 .+-.a root mean square deviation from the
backbone atoms of said amino acids of not more than 1.5 .ANG..
69. The crystallized complex of claim 19, wherein the MK2
polypeptide comprises amino acid residues Leu70, Gly71, Leu72,
Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141,
Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 as set forth in
SEQ ID NO:1.
70. The crystallized complex of claim 69, wherein the MK2
polypeptide further comprises amino acid residues Val69, Ile74,
Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95,
Glu104, His108, Arg119, Ile136, Val237, Asp142, Gly143, Gly144,
His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204,
Leu205, Phe208, and Gly209 as set forth in SEQ ID NO:1.
71. The crystallized complex of claim 19, wherein the crystallized
complex has the structural coordinates set forth in FIGS. 3 through
3A-40 .+-.a root mean square deviation from the backbone atoms of
said amino acids of not more than 1.5 .ANG..
72. The crystallized complex of claim 19, wherein the MK2
polypeptide comprises an active site comprising the relative
structural coordinates of amino acid residues Leu70, Gly71, Leu72,
Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141,
Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 of molecule A
according to the sequence set forth in SEQ ID NO:1, and the
structural coordinates as set forth in FIGS. 3 through 3A-40 .+-.a
root mean square deviation from the backbone atoms of said amino
acids of not more than 1.5 .ANG..
73. The crystallized complex of claim 72, wherein the active site
of the MK2 polypeptide further comprises the relative structural
coordinates of amino acid residues Val69, Ile74, Gly76, Ala77,
Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108,
Arg119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186,
Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208, and
Gly209 of molecule A according to the s set forth in SEQ ID NO:1,
and the structural coordinates as set forth in FIGS. 3 through
3A-40 .+-.a root mean square deviation from the backbone atoms of
said amino acids of not more than 1.5 .ANG..
Description
RELATED APPLICATION
[0001] This application is a continuation application of U.S.
patent application Ser. No. 10/294,027, filed Nov. 13, 2002. The
contents of the prior application is hereby incorporated by
reference in its entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to the identification of the
crystal structures of MK2, and the use of the structures for
designing new drugs.
BACKGROUND OF THE INVENTION
[0003] Mitogen activated protein (MAP) kinases are a large and
diverse group of Ser/Thr kinases separated into three major
subgroups, which include the extracellular signal regulated kinases
(ERKs), the c-Jun N-terminal kinases (JNKs)/stress-activated
protein kinases (JNKs) and p38/reactivating kinases (RK). The ERKs
are activated by mitogens and growth factors, whereas the
JNKs/SAPKs and p38/RK are activated by bacterial lipopolysaccharide
(LPS, interleukin-1 (IL-1), tumor necrosis factor-.alpha.
(TNF-.alpha.) and cellular stresses such as heat shock, osmotic
shock, or UV damage. Exposure of cells to these factors results in
the increased production of proinflammatory cytokines. Analysis of
a specific inhibitor of p38 MAP kinase, SB203580, reveals that it
inhibits LPS-induced cytokine synthesis in human monocytes, thus
indicating that p38 is the MAP kinase responsible for
stress-induced cytokine production (1). SB203580 also prevents the
activation of MAP kinase activated protein kinase 2 (MK2),
suggesting that this kinase is activated by P38 (2).
[0004] Mice engineered to be homozygously-deficient in MK2 show a
reduction in TNF-.alpha., interferon-.gamma., IL-1.beta., and IL-6
production and an increased rate of survival upon challenge with
LPS, suggesting that this enzyme is a key component in the
inflammatory process and a potential target for anti-inflammatory
therapy (3). Activation of MK2 results in the production of
cytokines by regulating the translation and or stability of the
encoding mRNAs through the AU-rich elements of the 3'-untranslated
regions of the gene (4). MK2 also phosphorylates the transcription
factor CREB, as well as leukocyte specific protein-1 and heat shock
protein 25/27, which are involved in the regulation of actin
polymerization (5-8) and cell migration (9, 10).
[0005] MK2 is a multi-domain protein consisting of an N-terminal
proline-rich domain, a catalytic domain, an autoinhibitory domain
and at the C-terminus a nuclear export signal (NES) and nuclear
localization signal (NLS) (11-15). Two isoforms of human MK2 have
been characterized. One isoform consists of 400 amino acids and the
other isoform 370 residues which is thought to be a splice variant
missing the C-terminal NLS (11, 16, 12). MK2 is located in the
nucleus of the cell and upon binding and phosphorylation by p38,
the MK2 NES becomes functional and both kinases are co-transported
out of the nucleus to the cytoplasm (8, 12, 13, 17). Interestingly,
transport of the MK2/p38 complex does not require catalytically
active MK2, as the active site mutant, Asp207Ala, is still
transported to the cytoplasm (13). Phosphorylation of human MK2 by
p38 on residues T222, S272 and T334 is thought to activate the
enzyme by inducing a conformational change of the autoinhibitory
domain thus exposing the active site for substrate binding (8, 18).
Mutations of two autoinhibitory domain residues W332A and K326E in
murine MK2 demonstrate an increase in basal activity and a
C-terminal deletion of the autoinhibitory domain renders the enzyme
constitutively active, providing additional evidence to the role of
this domain in inhibition of MK2 activity (18).
[0006] Recently, Meng, et al., published the structure of the
autoinhibited, inactive form of MK2 47-400. However, since MK2
47-400 used by Meng, et al. included the autoinhibitory domain and
was otherwise inactive, that structure is less useful for drug
design.
SUMMARY OF THE INVENTION
[0007] The present invention provides an isolated MK2 polypeptide,
having an amino acid sequence corresponding to a portion of MK2, in
which the N-terminus begins at amino acid 41 to 55 and the
C-terminus ends at 338 to 365, or an MK2 analogue thereof. This MK2
polypeptide, unlike the MK2 polypeptide used by Meng, et al., does
not include the complete autoinhibitory domain and is catalytically
active.
[0008] The present invention also provides nucleic acids encoding
the forgoing MK2 polypeptides or MK2 analogues thereof, vectors
comprising the nucleic acids, as well as host cells transformed,
transfected or infected with the vectors. Additionally, the present
invention provides a method for preparing an MK2 polypeptide or an
MK2 analogue that comprises transforming, transfecting or infecting
a host cell with the vector, and culturing the host cell under
conditions permitting the production of MK2 polypeptide or MK2
analogue by the host cell.
[0009] The present invention also provides a method for obtaining
crystallized native MK2, an MK2 polypeptide or an MK2 analogue
comprising contacting native MK2, an MK2 polypeptide or an MK2
analogue with a buffer solution comprising at least one of
cacodylate, Tris, Tris-HCL, acetate, malonate, sodium phosphate,
potassium phosphate, citrate, HEPES and MES, at a salt
concentration of 0.1 M to 2.4 M, and at a pH of 4.5 to 8.5, under
conditions permitting the formation of crystallized MK2,
crystallized MK2 polypeptide or crystallized MK2 analogue.
[0010] The present invention also provides a crystallized complex
of MK2 polypeptide and staurosporine, having four molecules of MK2
in the asymmetric unit.
[0011] Additionally, the present invention provides a crystallized
complex of MK2 polypeptide and ADP, having one molecule of MK2 in
the asymmetric unit.
[0012] The present invention further provides a three dimensional
model of MK2, defined by the relative structural coordinates for:
(i) molecules A, B, C or D of MK2 according to FIGS. 2 through
2A-204, (ii) a portion of molecules A, B, C, or D of MK2 according
to FIGS. 2 through 2A-204, (iii) molecule A of MK2 according to
FIGS. 3 through 3A-40, or (iv) a portion of molecule A of MK2
according to FIGS. 3 through 3A-40, .+-.a root mean square
deviation from the backbone atoms of said amino acids of not more
than 1.5 .ANG..
[0013] Also provided by the present invention is an active site of
MK2, and particularly the site on MK2 in which staurosporine binds.
The active site comprises the relative structural coordinates of
amino acid resides Leu70, Gly71, Leu72, Gly73, Val78, Ala91,
Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191,
Leu192, Thr206 and Asp207 of molecules A, B, C or D according to
FIGS. 2 through 2A-204, .+-.a root mean square deviation from the
backbone atoms of said amino acids of not more than 1.5 .ANG.. In
another embodiment, the active site further comprises, in addition
to the above relative coordinates, the relative structural
coordinates for amino acid residues Val69, Ile74, Gly76, Ala77,
Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108,
Arg119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186,
Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208 and
Gly209 of molecules A, B, C or D according to FIGS. 2 through
2A-204, .+-.a root mean square deviation from the backbone atoms of
said amino acids of not more than 1.5 .ANG..
[0014] The present invention also provides a method for designing a
putative agent that interacts with an active site of MK2, and
particularly the staurosporine binding site on MK2. This method
comprises the steps of: (a) generating a three dimensional model of
said active site using the relative structural coordinates of amino
acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118,
Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192,
Thr206, and Asp207 of molecules A, B, C or D of MK2 according to
FIGS. 2 through 2A-204, .+-.a root mean square deviation from the
backbone atoms of said amino acids of not more than 1.5 521; and
(b) designing a putative agent that interacts with said active site
by performing computer fitting analysis of said putative agent with
the three dimensional model generated in step (a). In another
embodiment, the active site further comprises, in addition to the
above relative coordinates, the relative structural coordinates for
amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80,
Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136,
Val137, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189,
Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208 and Gly209 of
molecules A, B, C or D according to FIGS. 2 through 2A-204, .+-.a
root mean square deviation from the backbone atoms of said amino
acids of not more than 1.5 .ANG..
[0015] Still further, the present invention provides a method for
identifying an agent that interacts with MK2, comprising the steps
of: (a) generating a three dimensional model of MK2 using the
relative structural coordinates of (i) molecules A, B, C or D of
MK2 according to FIGS. 2 through 2A-204, (ii) a portion of
molecules A, B, C or D of MK2 according to FIGS. 2 through 2A-204,
(iii) molecule A of MK2 according to FIGS. 3 through 3A-40, or (iv)
a portion of molecule A of MK2 according to FIGS. 3 through 3A-40,
.+-.a root mean square deviation from the backbone atoms of said
amino acids of not more than 1.5 .ANG.; and (b) employing said
three-dimensional model to identify an agent that interacts with
MK2.
[0016] The present invention also provides a method for designing a
putative agent that interacts with an active site of MK2, and
particularly the staurosporine binding site on MK2. This method
comprises the steps of: (a) generating a three dimensional model of
said active site using the relative structural coordinates of amino
acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118,
Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192,
Thr206, and Asp207 of molecules A, B, C or D of MK2 according to
FIGS. 2 through 2A-204, .+-.a root mean square deviation from the
backbone atoms of said amino acids of not more than 1.5 .ANG.; and
(b) designing a putative agent that interacts with said active site
by performing computer fitting analysis of said putative agent with
the three dimensional model generated in step (a). In another
embodiment, the active site further comprises, in addition to the
above relative coordinates, the relative structural coordinates for
amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80,
Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136,
Val137, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189,
Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208 and Gly209 of
molecules A, B, C or D according to FIGS. 2 through 2A-204, .+-.a
root mean square deviation from the backbone atoms of said amino
acids of not more than 1.5 .ANG..
[0017] Additionally, the present invention provides a method for
identifying a putative agent that interacts with an active site of
MK2, and particularly the site on MK2 in which ADP binds. This
method comprises the steps of: (a) generating a three dimensional
model of said active site using the relative structural coordinates
of amino acid residues Leu70, Gly71, Leu72, Gly73, Ile74, Asn75,
Val78, Ala91, Lys93, Met138, Glu139, Cys140, Leu141, Asn191,
Thr206, Asp207 of molecule A of MK2 according to FIGS. 3 through
3A-40, .+-.a root mean square deviation from the backbone atoms of
said amino acids of not more than 1.5 .ANG.; and (b) designing a
putative agent that interacts with said active site by performing
computer fitting analysis of said putative agent with the three
dimensional model generated in step (a). In another embodiment, the
active site further comprises, in addition to the above relative
coordinates, the relative structural coordinates for amino acid
residues Val69, Gly76, Ala77, Leu79, Gln80, Phe90, Leu92, Met94,
Leu95, Glu104, His108, Val118, Ala119, Ile136, Val137, Asp142,
Gly143, Gly144, Glu145, His184, Asp186, Lys188, Pro189, Glu190,
Leu192, Leu193, Tyr194, Thr195, Leu205, Phe208, Gly209, Phe210 of
molecule A of MK2 according to FIGS. 3 through 3A-40, .+-.a root
mean square deviation from the backbone atoms of said amino acids
of not more than 1.5 .ANG..
[0018] Finally, the present invention provides agents identified
using the foregoing methods. Small molecules or other agents which
activate, inhibit or otherwise interfere with substrate binding to
MK2 may be useful as therapeutic agents in inflammatory based
diseases.
[0019] Additional objects of the present invention will be apparent
from the description which follows.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 is a sequence alignment of MK2 (SEQ ID NO:1), MK3
(SEQ ID NO:2) and MK5 (SEQ ID NO:3). Residues highlighted in bold
are identical. The GXGXXG motif (SEQ ID NO:6), the catalytic RD
residues and the bipartite nuclear localization sequence (KKI . . .
RKK) are boxed. The residues that are phosphorylated are donated by
stars.
[0021] FIGS. 2 through 2A-204 provide the atomic structural
coordinates for MK2 and staurosporine as derived by X-ray
diffraction of the MK2/staurosporine crystal complex. "Atom type"
refers to the atom whose coordinates are being measured. "Residue"
refers to the type of residue of which each measured atom is a
part--i.e., amino acid, cofactor, ligand or solvent. The "x, y and
z" coordinates indicate the Cartesian coordinates of each measured
atom's location in the unit cell (.ANG.). "Occ" indicates the
occupancy factor. "B" indicates the "B-value", which is a measure
of how mobile the atom is in the atomic structure (.ANG..sup.2).
Under "Molecules", A, B, C, and D refer to each molecule of MK2, E,
F G and H refer to each molecule of staurosporine, molecule I
corresponds to SO.sub.4, and W corresponds to water.
[0022] FIGS. 3 through 3A-40 provide the atomic structural
coordinates for MK2 and ADP as derived by X-ray diffraction of the
MK2/ADP crystal complex. "Atom type" refers to the atom whose
coordinates are being measured. "Residue" refers to the type of
residue of which each measured atom is a part--i.e., amino acid,
cofactor, ligand or solvent. The "x, y and z" coordinates indicate
the Cartesian coordinates of each measured atom's location in the
unit cell (.ANG.). "Occ" indicates the occupancy factor. "B"
indicates the "B-value", which is a measure of how mobile the atom
is in the atomic structure (.ANG..sup.2) Under "Molecule", A refers
to MK2 and B corresponds to ADP.
[0023] FIGS. 4 through 4A-1 provide the nucleic acid sequence for
MK2 (SEQ ID NO:5) with the corresponding MK2 amino acid sequence
(SEQ ID NO:4).
DETAILED DESCRIPTION OF THE INVENTION
[0024] As used herein, the following terms and phrases shall have
the meanings set forth below:
[0025] Unless otherwise noted, "MK2" includes: (i) native MK2
having the amino acid sequence (residues 1-400) set forth in FIG.
1, including conservative substitutions thereof; (ii) modeled MK2,
including conservative substitutions thereof; and (iii) an MK2
polypeptide, including conservative substitutions thereof.
[0026] "Modeled MK2" corresponds to molecules A, B, C and D of MK2
according to FIGS. 2 through 2A-204, and molecule A of MK2
according to FIGS. 3 through 3A-40.
[0027] An "MK2 polypeptide" is an amino acid sequence that defines
a portion or fragment of residues 1-400 of MK2 set forth in FIG. 1.
An MK2 polypeptide includes but is not limited to an amino acid
sequence of MK2 in which the N-terminus begins at amino acid 41 to
55 and the C-terminus ends at 338 to 365, in accordance with the
residue numbering shown in FIG. 1.
[0028] An "MK2 analogue, is a polypeptide having at least 80%
homology with "MK2" defined above, more preferably at least 90%
homology with "MK2" defined above, and most preferably at least 95%
homology with "MK2" defined above. In the preferred embodiment, an
"MK2 analogue" also has MAP kinase activated protein kinase
activity.
[0029] A "portion" of molecules A, B, C, or D of MK2 set forth in
FIGS. 2 through 2A-204, or a "portion" of molecule A of MK2 set
forth in FIGS. 3 through 3A-40, refers to the relative structural
coordinates of amino acid residues that include less than all the
amino acid residues shown. Preferably, the portion of molecules A,
B, C or D of MK2 set forth in FIGS. 2 through 2A-204, or a portion
of molecule A of MK2 set forth in FIGS. 3 through 3A-40 includes,
at a minimum, a sufficient portion of the MK2 molecule to define an
active site of MK2.
[0030] An "MK2 complex" is MK2 complexed to another molecule,
including but not limited to staurosporine or ADP.
[0031] Unless otherwise indicated, "protein" or "molecule" shall
include a protein, protein domain, polypeptide or peptide.
[0032] "Structural coordinates" are the Cartesian coordinates
corresponding to an atom's spatial relationship to other atoms in a
molecule or molecular complex. Structural coordinates may be
obtained using x-ray crystallography techniques or NMR techniques,
or may be derived using molecular replacement analysis or homology
modeling. Various software programs allow for the graphical
representation of a set of structural coordinates to obtain a three
dimensional representation of a molecule or molecular complex. The
structural coordinates of the present invention may be modified
from the original sets provided in FIGS. 2 through 2A-204 and FIGS.
3 through 3A-40 by mathematical manipulation, such as by inversion
or integer additions or subtractions. As such, it is recognized
that the structural coordinates of the present invention are
relative, and are in no way specifically limited by the actual x,
y, z coordinates of FIGS. 2 through 2A-204 and FIGS. 3 through
3A-40.
[0033] An "agent" shall include a protein, polypeptide, peptide,
nucleic acid (including DNA or RNA), molecule, compound or
drug.
[0034] "Root mean square deviation" is the square root of the
arithmetic mean of the squares of the deviations from the mean, and
is a way of expressing deviation or variation from the structural
coordinates described herein. The present invention includes all
embodiments comprising conservative substitutions of the noted
amino acid residues resulting in same structural coordinates within
the stated root mean square deviation. It will be obvious to the
skilled practitioner that the numbering of the amino acid residues
of MK2 may be different than that set forth herein, and may contain
certain conservative amino acid substitutions that yield the same
three dimensional structures as those defined by FIGS. 2 through
2A-204 and FIGS. 3 through 3A-40 herein. Corresponding amino acids
and conservative substitutions in other isoforms or analogues are
easily identified by visual inspection of the relevant amino acid
sequences or by using commercially available homology software
programs (e.g., MODELLAR, MSI, San Diego, Calif.).
[0035] "Conservative substitutions" are those amino acid
substitutions which are functionally equivalent to the substituted
amino acid residue, either by way of having similar polarity,
steric arrangement, or by belonging to the same class as the
substituted residue (e.g., hydrophobic, acidic or basic), and
includes substitutions having an inconsequential effect on the
three dimensional structure of MK2 with respect to the use of said
structures for the identification and design of agents which
interact with MK2, as well as for molecular replacement analyses
and/or for homology modeling.
[0036] The present invention first provides an MK2 polypeptide
having an amino acid sequence corresponding to a portion of MK2, in
which the N-terminus begins at amino acid 41 to 55 and the
C-terminus ends at 338 to 365. The present inventors have found
that sequences which lack amino acid residues 366-400 of MK2, are
active. Preferably, a MK2 polypeptide has the amino acid sequence
corresponding to amino acid residues 41-364 of MK2 according to
FIG. 1.
[0037] The present invention also provides an MK2 analogue of the
foregoing MK2 polypeptide that is at least 80% homologous, more
preferably at least 90% homologous and most preferably at least 95%
homologous, with the foregoing MK2 polypeptide having an amino acid
sequence corresponding to a portion of MK2, in which the N-terminus
begins at amino acid 41-to 55 and the C-terminus ends at 338 to
365. In the more preferred embodiment, the MK2 analogue is at least
80% homologous, more preferably at least 90% homologous and most
preferably at least 95% homologous, with the MK2 polypeptide having
the amino acid sequence corresponding to amino acid residues 41-364
of MK2 according to FIG. 1.
[0038] The present invention also provides a nucleic acid encoding
the MK2 polypeptide having an amino acid sequence corresponding to
a portion of MK2, in which the N-terminus begins at amino acid 41
to 55 and the C-terminus ends at 338 to 365, as well as a nucleic
acid encoding amino acid residues 41-364 of MK2 according to FIG.
1. The nucleic acid sequence for MK2 is known, and is shown in
FIGS. 4 through 4A-1. Such nucleic acids can be introduced into the
appropriate vectors, and then transformed, transfected or infected
into the appropriate host cells, which can be cultured to produce
recombinant MK2 polypeptide. The recombinant MK2 polypeptide can
then be isolated and purified according to known techniques.
Suitable vector and expression systems are well known and
commercially available.
[0039] The present invention also provides a nucleic acid encoding
an MK2 analogue that is at least 80% homologous, more preferably at
least 90% homologous and most preferably at least 95% homologous,
with the MK2 polypeptide having an amino acid sequence
corresponding to a portion of MK2, in which the N-terminus begins
at amino acid 41 to 55 and the C-terminus ends at 338 to 365. In
the more preferred embodiment, the nucleic acid encodes an MK2
analogue that is at least 80% homologous, more preferably at least
90% homologous and most preferably at least 95% homologous, with
the MK2 polypeptide having the amino acid sequence corresponding to
amino acid residues 41-364 of MK2 according to FIG. 1. Such nucleic
acids can be introduced into the appropriate vectors, and then
transformed, transfected or infected into the appropriate host
cells, which can be cultured to produce recombinant MK2
polypeptide. The recombinant MK2 analogue can then be isolated and
purified according to known techniques. Suitable vector and
expression systems are well known and commercially available.
[0040] The present invention also provides a method for
crystallizing native MK2, an MK2 polypeptide or an MK2 analogue.
The method comprises contacting native MK2, an MK2 polypeptide or
an MK2 analogue with a buffer solution comprising at least one of
cacodylate, Tris, Tris-HCL, acetate, malonate, sodium phosphate,
potassium phosphate, citrate, HEPES and MES, at a salt
concentration of 0.1 M to 2.4 M, and at a pH of 4.5 to 8.5, under
conditions permitting the formation of crystallized native MK2,
crystallized MK2 polypeptide or crystallized MK2 analogue. In a
preferred embodiment, the salt has an anion selected from the group
consisting of sulfate, citrate, chloride, acetate, phosphate,
malonate and tartrate. In another preferred embodiment, the salt
concentration is 0.8 M or higher. It is also within the confines of
the present invention that the native MK2, the MK2 polypeptide or
the MK2 analogue is contacted with the buffer solution in the
presence of PEG or a PEG substitute having a molecular weight up to
3350. The PEG includes but is not limited to PEG-200, PEG-400,
PEG-500-MME, PEG-1000, PEG-1500, PEG-2000-MME and MEG-3350, and is
preferably PEG-400. The PEG substitute includes but is not limited
to Jeffamine M-600, ethylene glycol, glycerol and 1-6 hexanediol,
2-methyl-2,4-pentanediol (MPD). The more specific crystallization
conditions are exemplified in example which follows, as well as in
Table 4.
[0041] The present invention also provides a crystallized complex
of MK2 polypeptide and staurosporine, having four molecules of MK2
polypeptide in the asymmetric unit. This crystal effectively
diffracts X-rays for the determination of the structural
coordinates of MK2, and is characterized as having space group
P6.sub.3, unit cell parameters of a=b=160.20 .ANG., c=133.48
.ANG..
[0042] Additionally, the present invention provides a crystallized
complex of MK2 polypeptide and ADP, having one molecule of MK2
polypeptide in the asymmetric unit. This crystal effectively
diffracts X-rays for the determination of the structural
coordinates of MK2, and is characterized as having space group
F4.sub.132, unit cell parameters of a=b=c=253.05 .ANG..
[0043] Using the crystals of the present invention, X-ray
diffraction data can be collected by a variety of means in order to
obtain the atomic coordinates of the molecules in the crystals.
With the aid of specifically designed computer software, such
crystallographic data can be used to generate a three dimensional
structure. Various methods used to generate and refine a three
dimensional structure of a molecular structure are well known to
those skilled in the art, and include, without limitation,
multiwavelength anomalous dispersion (MAD), multiple isomorphous
replacement, reciprocal space solvent flattening, molecular
replacement, and single isomorphous replacement with anomalous
scattering (SIRAS).
[0044] Accordingly, the present invention also provides a three
dimensional model of MK2 as derived by x-ray diffraction data of
the MK2/staurosporine crystal. The three dimensional model of MK2
derived from the MK2/staurosporine crystal is preferably defined by
the relative structural coordinates for molecules A, B, C or D of
MK2 according to FIGS. 2 through 2A-204, .+-.a root mean square
deviation from the backbone atoms of said amino acids of not more
than 1.5 .ANG., preferably not more than 1.0 .ANG., and most
preferably not more than 0.5 .ANG.. The three dimensional model
also includes the relative structural coordinates of a portion of
molecules A, B, C or D of MK2 according to FIGS. 2 through 2A-204,
.+-.a root mean square deviation from the backbone atoms of said
amino acids of not more than 1.5 .ANG., preferably not more than
1.0 .ANG., and most preferably not more than 0.5 .ANG.. The three
dimensional model of MK2 is useful for a number of applications,
including, but not limited to, the visualization, identification
and characterization of various active sites of MK2. The active
site structures may then be used to design agents with interact
with MK2.
[0045] The present invention also provides a three dimensional
model of MK2 as derived by x-ray diffraction data of the MK2/ADP
crystal. The three dimensional model of MK2 derived from the
MK2/ADP crystal is preferably defined by the structural coordinates
for molecule A of MK2 shown in FIGS. 3 through 3A-40, .+-.a root
mean square deviation from the backbone atoms of the amino acids of
not more than 1.5 .ANG., preferably not more than 1.0 .ANG., and
most preferably not more than 0.5 .ANG.. The three dimensional
model also includes the relative structural coordinates of a
portion of molecule A of MK2 according to FIGS. 3 through 3A-40,
.+-.a root mean square deviation from the backbone atoms of said
amino acids of not more than 1.5 .ANG., preferably not more than
1.0 .ANG., and most preferably not more than 0.5 .ANG.. The three
dimensional model of MK2 derived from the MK2/ADP crystal is useful
for a number of applications, including, but not limited to, the
visualization, identification and characterization of various
active sites of MK2. The active site structures may then be used to
design agents with interact with MK2.
[0046] The present invention also provides a machine, such as a
computer, programmed in memory with the coordinates of FIGS. 2
through 2A-204, or FIGS. 3 through 3A-40, or portions thereof,
together with a program capable of converting the coordinates into
a three dimensional graphical representation of the structural
coordinates on a display connected to the machine. A machine having
a memory containing such data aids in the rational design or
selection of inhibitors or activators of MK2 activity, including
the evaluation of ability of a particular chemical entity to
favorably associate with MK2 or an MK2 complex as disclosed herein,
as well as in the modeling of compounds, proteins, complexes, etc.
related by structural or sequence homology to MK2.
[0047] For storage, transfer and use with such programs, a machine,
such as a computer, is provided for that produces a three
dimensional representation of the MK2 molecule, a portion thereof
(such as an active site or a binding site), an MK2 molecular
complex, or an MK2 analogue. The machine of the present invention
comprises a machine-readable data storage medium comprising a data
storage material encoded with machine-readable data.
Machine-readable storage media comprising data storage material
include conventional computer hard drives, floppy disks, DAT tape,
CD-ROM, and other magnetic, magneto-optical, optical, and other
media which may be adapted for use with a computer. The machine of
the present invention also comprises a working memory for storing
instructions for processing the machine-readable data, as well as a
central processing unit (CPU) coupled to the working memory and to
the machine-readable data storage medium for the purpose of
processing the machine-readable data into the desired three
dimensional representation. Finally, the machine of the present
invention further comprises a display connected to the CPU so that
the three dimensional representation may be visualized by the user.
Accordingly, when used with a machine programmed with instructions
for using said data, e.g., a computer loaded with one or more
programs of the sort identified below, the machine provided for
herein is capable of displaying a graphical three-dimensional
representation of any of the molecules or molecular complexes, or
portions of molecules of molecular complexes, described herein.
[0048] Molecular modeling methods known in the art may be used to
identify an active site or binding pocket of MK2, MK2 complex or an
MK2 analogue. Specifically, the structural coordinates provided by
the present invention may be used to characterize a three
dimensional model of the MK2, MK2 complex or MK2 analogue. From
such a model, putative active sites may be computationally
visualized, identified and characterized based on the surface
structure of the molecule, surface charge, steric arrangement, the
presence of reactive amino acids, regions of hydrophobicity or
hydrophilicity, etc. Such putative active sites may be further
refined using chemical shift perturbations of spectra generated
from various and distinct MK2 complexes, competitive and
non-competitive inhibition experiments, and/or by the generation
and characterization of MK2 mutants to identify critical residues
or characteristics of the active site. The identification of
putative active sites of a molecule or molecular complex is of
great importance, as most often the biological activity of a
molecule or molecular complex results from the interaction between
an agent and one or more active sites of the molecule or molecular
complex. Accordingly, the active sites of a molecule or molecular
complex are the best targets to use in the design or selection of
activators or inhibitors that affect the activity of the molecule
or molecular complex.
[0049] As such, the present invention also provides an active site
of MK2, and particularly the site of binding of staurosporine to
MK2. In one embodiment, the active site comprises the relative
structural coordinates of amino acid residues Leu70, Gly71, Leu72,
Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141,
Glu145, Glu190, Asn191, Leu192, Thr206 and Asp207 of molecules A,
B, C or D according to FIGS. 2 through 2A-204, .+-.a root mean
square deviation from the backbone atoms of said amino acids of not
more than 1.5 .ANG., preferably not more than 1.0 .ANG., and most
preferably not more than 0.5 .ANG.In another embodiment, the active
site further comprises, in addition to the above relative
coordinates, the relative structural coordinates for amino acid
residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90,
Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136, Val137,
Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193,
Tyr194, Thr195, Lys204, Leu205, Phe208 and Gly209 of molecules A,
B, C or D according to FIGS. 2 through 2A-204, .+-.a root mean
square deviation from the backbone atoms of said amino acids of not
more than 1.5 .ANG., preferably not more than 1.0 .ANG., and most
preferably not more than 0.5 .ANG..
[0050] Still further, the present invention provides an active site
of MK2, and particularly the site of binding of staurosporine to
ADP. In one embodiment, the active site comprises the relative
structural coordinates of amino acid residues Leu70, Gly71, Leu72,
Gly73, Ile74, Asn75, Val78, Ala91, Lys93, Met138, Glu139, Cys140,
Leu141, Asn191, Thr206, Asp207 of molecule A of MK2 according to
FIGS. 3 through 3A-40, .+-.a root mean square deviation from the
backbone atoms of said amino acids of not more than 1.5 .ANG.,
preferably not more than 1.0 .ANG., and most preferably not more
than 0.5 .ANG.. In another embodiment, the active site further
comprises, in addition to the above relative coordinates, the
relative structural coordinates for amino acid residues Val69,
Gly76, Ala77, Leu79, Gln80, Phe90, Leu92, Met94, Leu95, Glu104,
His108, Val118, Ala119, Ile136, Val137, Asp142, Gly143, Gly144,
Glu145, His184, Asp186, Lys188, Pro189, Glu190, Leu192, Leu193,
Tyr194, Thr195, Leu205, Phe208, Gly209, Phe210 of molecule A of MK2
according to FIGS. 3 through 3A-40, .+-.a root mean square
deviation from the backbone atoms of said amino acids of not more
than 1.5 .ANG., preferably not more than 1.0 .ANG., and most
preferably not more than 0.5 .ANG..
[0051] Another aspect of the present invention is directed to a
method for identifying an agent that interacts with MK2. In this
method, a three dimensional model of MK2 is first generated using
the relative structural coordinates of (i) molecules A, B, C or D
of MK2 according to FIGS. 2 through 2A-204, (ii) a portion of
molecules A, B, C or D of MK2 according to FIGS. 2 through 2A-204,
(iii) molecule A of MK2 according to FIGS. 3 through 3A-40, or (iv)
a portion of molecule A according to FIGS. 3 through 3A-40, .+-.a
root mean square deviation from the backbone atoms of said amino
acids of not more than 1.5 .ANG., preferably not more than 1.0
.ANG., and most preferably not more than 0.5 .ANG.. The
three-dimensional model is then used to identify an agent that
interacts with MK2.
[0052] The present invention also provides a method for designing a
putative agent that interacts with an active site of MK2, and
particularly the site on MK2 to which staurosporine binds. In this
method, a three dimensional model of the active site is first
generated using the relative structural coordinates of amino acid
residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138,
Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, and
Asp207 of molecules A, B, C or D of MK2 according to FIGS. 2
through 2A-204, .+-.a root mean square deviation from the backbone
atoms of said amino acids of not more than 1.5 .ANG., preferably
not more than 1.0 .ANG., and most preferably not more than 0.5
.ANG.. A putative agent that interacts with the active site is then
designed, generated or identified by performing computer fitting
analysis of the putative agent with the three dimensional model
generated above. In another embodiment, the active site further
comprises, in addition to the above relative coordinates, the
relative structural coordinates for amino acid residues Val69,
Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93,
Leu95, Glu104, His108, Arg119, Ile136, Val137, Asp142, Gly143,
Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195,
Lys204, Leu205, Phe208 and Gly209 of molecules A, B, C or D
according to FIGS. 2 through 2A-204, .+-.a root mean square
deviation from the backbone atoms of said amino acids of not more
than 1.5 .ANG., preferably not more than 1.0 .ANG., and most
preferably not more than 0.5 .ANG..
[0053] The present invention also provides a method for designing a
putative agent that interacts with an active site of MK2, and
particularly the site on MK2 to which staurosporine binds. In this
method, a three dimensional model of the active site is first
generated using the relative structural coordinates of amino acid
residues Leu70, Gly71, Leu72, Gly73, Val78, Ala9, Val118, Mse138,
Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, and
Asp207 of molecules A, B, C or D of MK2 according to FIGS. 2
through 2A-204, .+-.a root mean square deviation from the backbone
atoms of said amino acids of not more than 1.5 .ANG., preferably
not more than 1.0 .ANG., and most preferably not more than 0.5
.ANG.. A putative agent that interacts with the active site is then
designed, generated or identified by performing computer fitting
analysis of the putative agent with the three dimensional model
generated above. In another embodiment, the active site further
comprises, in addition to the above relative coordinates, the
relative structural coordinates for amino acid residues Val69,
Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93,
Leu95, Glu104, His108, Arg119, Ile136, Val137, Asp142, Gly143,
Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195,
Lys204, Leu205, Phe208 and Gly209 of molecules A, B, C or D
according to FIGS. 2 through 2A-204, .+-.a root mean square
deviation from the backbone atoms of said amino acids of not more
than 1.5 .ANG., preferably not more than 1.0 .ANG., and most
preferably not more than 0.5 .ANG..
[0054] Using the active site, the agent may be designed or
evaluated using computer fitting analyses utilizing various
computer software programs that evaluate the "fit" between the
putative active site and the identified agent, by (a) generating a
three dimensional model of the putative active site of a molecule
or molecular complex using homology modeling or the atomic
structural coordinates of the active site, and (b) determining the
degree of association between the putative active site and the
identified agent. The degree of association may be determined
computationally by any number of commercially available software
programs, or may be determined experimentally using standard
binding assays.
[0055] Three dimensional models of the putative active site may be
generated using any one of a number of methods known in the art,
and include, but are not limited to, homology modeling as well as
computer analysis of raw data generated using crystallographic or
spectroscopy data. Computer programs used to generate such three
dimensional models and/or perform the necessary fitting analyses
include, but are not limited to: GRID (Oxford University, Oxford,
UK), MCSS (Molecular Simulations, San Diego, Calif.), AUTODOCK
(Scripps Research Institute, La Jolla, Calif.), DOCK (University of
California, San Francisco, Calif.), Flo99 (Thistlesoft, Morris
Township, N.J.), Ludi (Molecular Simulations, San Diego, Calif.),
QUANTA (Molecular Simulations, San Diego, Calif.), Insight
(Molecular Simulations, San Diego, Calif.), SYBYL (TRIPOS, Inc.,
St. Louis, Mo.) and LEAPFROG (TRIPOS, Inc., St. Louis, Mo.). The
structural coordinates also may be used to visualize the
three-dimensional structure of MK2 using MOLSCRIPT (Kraulis, P J,
J. Appl. Crystallogr. 24: 946-950 (1991)) and RASTER3D (Bacon, D.
J. and Anderson, W. F., J. Mol. Graph. 6: 219-220 (1998)), for
example.
[0056] The agent, whether an inhibitor or activator, may be
selected by screening an appropriate database, may designed de novo
by analyzing the steric configurations and charge potentials of an
empty MK2 active site in conjunction with the appropriate software
programs, or may be designed using characteristics of known
inhibitors or activators to MK2 or other mitogen activated protein
kinases in order to create "hybrid" activators or inhibitors. The
method of the present invention is preferably used to design or
select inhibitors of MK2. In this case, the potential inhibitor or
activator is designed to incorporate chemical or steric features
favorable for association with the active site. The inhibitor or
activator may be selected by screening an appropriate database, may
designed de novo by analyzing the steric configurations and charge
potentials of empty active sites in conjunction with the
appropriate software programs, or may be designed using
characteristics of known inhibitors or activators to MK2 or other
mitogen activated protein kinases in order to create "hybrid"
activators or inhibitors.
[0057] Once the agent has been designed or identified, it may be
obtained or synthesized and further evaluated for its affect on MK2
activity. For example, the agent may be evaluated by contacting the
identified agent with MK2 and measuring the effect of the agent on
MK2 activity. Depending upon the action of the agent on MK2, the
agent may act either as an inhibitor or activator of MK2 activity.
With respect to the specific active sites identified above, the
agent also may be contacted with MK2 in the presence of
staurosporine or ADP in order to determine whether or not the agent
inhibits binding between MK2 and staurosporine or ADP,
respectively.
[0058] Various molecular analysis and rational drug design
techniques are further disclosed in U.S. Pat. Nos. 5,834,228,
5,939,528 and 5,865,116, as well as in PCT Application No.
PCT/US98/16879, published WO 99/09148, the contents of which are
hereby incorporated by reference.
[0059] The present invention is also directed to the agents,
activators or inhibitors identified using the foregoing methods.
Such agents may be a protein, polypeptide, peptide, nucleic acid,
including DNA or RNA, molecule, compound, or drug. Small molecules
or other agents which interact with MK2 may be useful in the
treatment of diseases or conditions associated with MK2.
[0060] The present invention may be better understood by reference
to the following non-limiting Example. The following Example is
presented in order to more fully illustrate the preferred
embodiments of the invention, and should in no way be construed as
limiting the scope of the present invention.
EXAMPLE 1
1. Materials and Methods.
[0061] Sequence Selection for MK2 Crystallization. Various
constructs were made, expressed and the resulting MK2 were purified
and crystalized. After analyzing a large number of constructs for
expression of MK2 in E. coli, it was found that deleting the first
40 residues of the proline rich domain greatly enhanced the
expression levels and solubility of the enzyme. Analyses of the
resulting MK2 also indicated that constructs lacking amino acids
366 to 400 were not active in vitro. It was also found that
crystals could be obtained for constructs of MK2 in which the
N-terminus begins at amino acid 41 to 55 and the C-terminus ends at
338 to 365. The construct 41-364 resulted in the best crystals for
determining the crystal structure of MK2, and details concerning
its expression, purification and crystallization are provided
below.
[0062] Cloning and expression of MK2. The MAPKAP kinase 2 gene was
PCR cloned into the NcoI and XhoI sites of pET16b (Novagen) using
Hot tub polymerase (Amersham Pharmacia Biotech). The expressed
protein contains residues 41-364, excluding the N-terminal
proline-rich sequence. To produce selenomethionine labeled MK2, the
protein was expressed in BL21(DE3) (Novagen) E. coli at 25.degree.
C. Precultures were grown in shake flasks in LeMaster media
supplemented with L-methionine and expression cultures were grown
in LeMaster media supplemented with L-selenomethionine that was
replenished upon culture induction. Cultures were induced with 0.5
mM IPTG for four hours. Unlabelled MK2 was also expressed in E.
coli BL21 (DE3). The culture was induced with 0.5 mM IPTG and cells
were harvested 4 hours post-induction.
[0063] Purification of MK2 41-364. The purification was performed
at 4.degree. C. 5 g of bacterial cells were homogenized in 200 ml
of Buffer A (50 mM Tris pH 7.5, 10 mM DTT, 0.24 mg/ml AEBSF)+90
.mu.g/ml TPCK, 2.5 mM Aminobenzamidine, 500 .mu.L protease
inhibitor cocktail (without EDTA) for use in purification of
poly-(Histidine) tagged proteins (Sigma), RNase, DNase. Cells were
lysed by four passages through a Microfluidics microfluidizer
submerged in ice. The lysate was collected and centrifuged at
20,000.times.g for 30 min. The supernatant was applied to a Poros
HQ column (Applied Biosystems) that was equilibrated in Buffer A.
The flow through was loaded onto a Poros HS column (Applied
Biosystems) and the bound protein was eluted with a gradient of
Buffer A+1M NaCl. Ammonium sulfate to 0.8 M was added to the peak
fraction and the protein was loaded onto a polypropyl aspartamide
column (Nest Group) equilibrated with Buffer B (50 mM Hepes pH 7.5,
10 mM DTT, 0.8 M ammonium sulfate). The protein was eluted with a
gradient and the peak fraction was concentrated in a Millipore
Ultrafree concentrator. The protein was applied to a Superdex 200
column (Amersham Pharmacia Biotech) equilibrated with 20 mM Hepes
pH 7.5, 200 mM NaCl, 10 mM DTT. Protein purity was >95%.
Kinetic Analysis.
[0064] Materials. ATP, ADP, phosphoenolpyruvate (PEP), NADH, and
pyruvate kinase/lactate dehydrogenase enzymes were purchased from
Sigma Chemical Co. (St. Louis, Mo.). Activated p38 MAP kinase was
purchased from Upstate Biotech (Lake Placid, N.Y.). LSP-1 peptide
(RTPKLARQASIELPSM) (SEQ ID NO:10) was purchased from AnaSpec Inc.
(San Jose, Calif.).
[0065] Activation of MK2 Constructs. The MK2 constructs 41-400 and
41-364 were activated by the phosphorylation of the constructs by
p38 MAP kinase. The activation was done in 20 mM HEPES (pH 7.5), 10
mM MgCl.sub.2, 2 mM DTT, 0.50 mM ATP, 0.2 mg/ml MK2 and 0.125 .mu.g
activated p38. The reaction was incubated at 25.degree. C. for 1-2
hour then placed on ice to be used for kinetic analysis.
[0066] Kinase Kinetics. The rate of MK2 kinase was characterized in
20 mM HEPES (pH 7.5), 10 mM MgCl.sub.2, 2 mM DTT and 100 mM NaCl.
The kinetics was followed by linking the turnover of ATP is to the
turnover of NADH to NAD. This was followed spectrophotometrically
at 340 nm. The continuos assay contained 20 units pyruvate kinase,
30 units lactate dehydrogenase, 0.25 mM NADH, 2 mM PEP and 1.6 to
8.0 .mu.g/mL MK2. For determining the apparent K.sub.m for ATP, the
ATP concentration was varied from 0.005 to 0.25 mM while the
peptide was held constant at 0.2 mM for activated MK-2 or 1.0 mM
for the constitutively active form of MK-2 (41-364). For
determining the apparent K.sub.m for LSP-1, the LSP-1 concentration
was varied from 0.01 to 0.5 mM while ATP was held constant at 2 mM.
The kinetic analysis was carried out in a 96-well plate at
25.degree. C., on a Molecular Devices spectrophotometer.
[0067] Substrate Kinetics. Peptide LSP, based on the protein
substrate of MK-2 lymphocyte/leukocyte specific protein, was used
for the investigation of the kinetic mechanism for the enzyme MK-2.
Data was fit to equation 1 for normal Michaelis-Menten kinetics.
v=V.sub.max[S]/K.sub.m+[S] (1) where [S] is the substrate,
V.sub.max is the maximum enzyme velocity, K.sub.m is the Michaelis
constant.
[0068] Crystallization. The selenomethionine labeled protein was
concentrated to .about.5 mg/mL according to the Bradford method
(31) in a solution containing 20 mM HEPES pH 7.5, 200 mM NaCl, 10
mM DTT, and 5 mM MgCl.sub.2. Prior to crystallization,
staurosporine (0.375 mM) was added from a DMSO stock. Diffraction
quality conical crystals were obtained at 18.degree. C. from 2M
ammonium sulfate, 100 mM HEPES pH 7.5, 2% PEG 400. These crystals,
which appeared in 7-10 days, belonged to space group P6.sub.3 with
cell dimensions of a=b=160.20 .ANG., c=133.48 .ANG. and contained
four molecules of MK2 in the asymmetric unit. Native MK2 was
crystallized in the presence of ADP and the protein solution was
prepared as above except that 5 mM ADP was added instead of the
staurosporine. Diffraction quality native MK2/ADP co-crystals were
obtained at 18.degree. C. from 2.0M ammonium sulfate. These
bipyramidal crystals belonged to space group F4.sub.132 with cell
dimensions a=b=c=253.05 .ANG. and contained one molecule of MK2 and
one molecule of ADP in the asymmetric unit.
[0069] MK2 41-364 also was successfully crystallized using the
various conditions described in Table 4. Based on these
experiments, it was discovered that MK2 could be crystallized over
a broad pH range (4.5 to 8.5). It also was discovered that over
that pH range, MK2 could crystallize using a variety of buffers
(Cacodylate, Tris, Tris-HCL, Acetate, Malonate, Sodium/Potassium
Phosphate, Citrate, HEPES, MES). Additionally, salts of the anions
(Sulfate, Citrate, Chloride, Acetate, Phosphate, Malonate and
Tartrate) were preferred for crystallization. Still further, the
amount of salt needed to crystallize MK2 ranged from 0.1 M to 2.4
M, with less salt required in the presence of polyethylene glycol.
However, MK2 preferred to crystallize in the presence of high salt
concentrations, defined as 0.8 M or higher, as only three
conditions (#1, #14 and #16) have an organic (PEG 400) as the
precipitant. Finally, it was discovered that PEG-400, in the range
of 2 to 30%, could aid in the crystallization of MK2. It is
envisioned that any PEG (39) or its equivalent (e.g., PEG MME, MPD)
up to a molecular weight of 3350 could be substituted for PEG
400.
[0070] Data Collection.
[0071] MK2-Staurosporine. MAD data were collected on beamline 5.O.2
at the ALS, Berkley using an ADSC Quantum-4 CCD detector from a
single crystal of the hexagonal SeMet-MK2. The crystal was cooled
to -180.degree. C. for data collection and in order to minimize the
exposure of the crystal to x-rays, the strategy option within
MOSFLM (32) was used to determine the settings that gave the most
complete MAD data using the shortest total exposure time. The
wavelengths used can be found in Table 2. These data were then used
as input to the programs Shake and Bake (33) and ShelX (34) for
determination of the Selenium atom positions. Heavy atom parameters
for each were refined with SHARP (35). In addition to the MAD data,
a higher resolution data set was collected at 1.1 .ANG..
[0072] MK2-ADP. Single-wavelength (1.0 .ANG.) data for the MK2/ADP
co-crystals were collected on beamline 5.O.1 at the ALS, Berkley
using an ADSC Quantum-4 CCD detector. A single crystal, cooled to
-180.degree. C., was used to collect the data set. The data were
processed using DENZO/Scalepack (HKL Research, Inc.,
Charlottesville, Va.) and the statistics from refinement are
given-in Table II.
[0073] Model Building and Refinement.
[0074] MK2-Staurosporine. The structure of the MK2 was built into
the original 3.1 .ANG. resolution solvent flattened
symmetry-averaged MAD map using the X-AUTOFIT features within
QUANTA (Molecular Simulations Inc., San Diego, Calif.). The phases
were then extended from 3.1 .ANG. to 2.7 .ANG. with symmetry
averaging in DM. This model was then used as the initial model for
refinement using the program CNX (35) against the 2.7 .ANG. data.
Prior to refinement, 5% of the data were randomly selected and
designated as a R.sub.free test set to monitor the progress of the
refinement. Following seven cycles of refining and rebuilding the
refinement converged with a model which contained four molecules of
MK2, four staurosporine molecules, 43 water molecules, and two
sulfate ions at an R.sub.cryst of 23.9% (R.sub.free 27.4%). The
refinement statistics are given in Table 2.
[0075] MK2-ADP. The structure of the MK2/ADP complex was solved
using molecular replacement. A composite consisting of the
overlapped MK2 monomers from the staurosporine structure was
utilized as a molecular replacement probe with AMORE (37). The
molecular replacement solution was then rebuilt into a 3.2 .ANG.
resolution solvent averaged map. After the initial placement of the
protein chain into density, the model was rebuilt utilizing omit
maps calculated with BUSTER (38) in order to eliminate the bias
from the molecular replacement solution. The structure was refined
in CNX using methods as described above. Refinement converged after
six rebuilding cycles with a R.sub.cryst of 25.9% and a R.sub.free
of 29.2%. The final model consisted of residues 46-152, 159-217,
227-265, 274-345, and the ADP moiety. The refinement statistics are
given in Table 2.
2. Results and Discussion
[0076] Alignment of homologues. Map Kap kinase 2 (MK2) is an enzyme
that belongs to a family of Map kinase activated protein kinases.
Human members of this family include MK2, MK3 and MK5 (23, 24).
There is also a MK4 from sea urchin (25). These proteins are highly
homologous and, in addition, all have shown to be activated by the
Map kinase p38, although to date the only well studied enzyme is
MK2 (26, 24). MK2 is phosphorylated on T222, S272, and T334 by p38
and has a putative autophosphorylation site at T338. All four of
the phosphorylation sites are conserved in MK3 but only T222 is
present in MK5. All of the isozymes have the ATP binding site motif
GXGXXG (SEQ ID NO:6), (residues 71-76 in MK2), but only MK2 has the
bipartite nuclear localization signal KKIEDDASNPLLLKRRKK (SEQ ID
NO:7) (residues 373-389). The putative activation segment is highly
conserved in all three isozymes (residues 207-233 in MK2),
including the conserved motifs found to flank the activation
segments of many kinases, DFG and APE, with the latter being APQ in
MK5 (27). Interestingly, the conserved p38 phosphorylation site,
T222, is in the activation loop. MK2 and MK3 contain an N-terminal
proline rich domain that is absent in MK5 and the C-terminal
extension thought to contain the autoinhibitory domain in MK2 is
elongated in MK5. Overall, MK2 shares 75% identity with MK3 and 42%
identity with MK5.
[0077] Analysis of protein constructs. MK2 is a 400 amino acid
protein consisting of five domains, an N-terminal proline rich
domain, a kinase catalytic domain, a C-terminal kinase
autoinhibitory domain, a nuclear export signal, and a nuclear
localization sequence, which also the postulated site for p38
binding (28). After analyzing a large number of constructs for
expression of MK2 in E. coli, it was found that deleting the first
40 residues of the proline rich domain greatly enhanced the
expression levels and solubility of the enzyme. MK2 41-364, a
constitutively active form of the enzyme in which a portion of the
C-terminal autoinhibitory domain was removed, was purified and
produced crystals that diffracted to 2.7 .ANG.. As shown in Table
1, the K.sub.m for ATP is very similar for MK2 41-364 as compared
to p38 activated MK2 41-400, 7 .mu.M versus 15 .mu.M, respectively.
However, binding of the peptide substrate, leukocyte specific
protein 1, to MK2 41-364 is more than 40-fold weaker than to the
p38 activated MK2 41-400, 584 .mu.M vs. 13 .mu.M K.sub.m. In
addition, the V.sub.max for MK2 41-364 is also much lower when
compared to the p38 activated MK2 41-400 (0.076 vs. 8.9
.mu.mol/min/mg, respectively). These data indicate that MK2 41-364,
which lacks a portion of the autoinhibitory domain, binds ATP
normally but peptide substrate binding has been affected
dramatically. However, when MK2 41-364 is phosphorylated by p38,
the V.sub.max of the enzyme is greatly increased to 11
.mu.mol/min/mg, which is comparable to the V.sub.max of p38
activated MK2 41-400, 8.9 .mu.mol/min/mg. Phosphorylation of MK2
41-364 also alters the K.sub.m for peptide substrate such that the
K.sub.m decreases from 584 .mu.M to 20 .mu.M, similar to the
K.sub.m observed in activated MK2 41-400. These data suggest that
for optimal substrate binding and activity MK2 must be
phosphorylated. Clearly, phosphorylation of key residues within the
autoinhibitory C-terminal .alpha. helix and T222 in the activation
segment induce conformational changes in the enzyme that allow
highly efficient binding of peptide substrate (22). Analysis of the
MK2 structures in conjunction with other known Ser/THR kinase
structures suggests how this may occur.
[0078] Analysis of MK2 structures. The MK2 kinase core domain
contains an overall fold that is very similar to the structures of
other protein kinases. It is bilobal, consisting of a smaller
N-terminal domain that is largely .beta. sheet and a larger
C-terminal domain dominated by .alpha. helices. MK2 shares two
structural features within the N-terminal lobe that have been shown
to be important in the regulation of many protein kinases (29).
These structural elements are the highly conserved .alpha.C helix
(residues 99-113), and the .beta.1-.beta.2 loop (residues 13-32),
that has been termed the phosphate binding loop or "P loop". The P
loop contains the highly conserved ATP binding motif, GXGXXG (SEQ
ID NO:6).
[0079] The C-terminal lobe contains another important regulatory
feature, the MK2 activation segment (residues 207-233), which
extends outward from the surface of the catalytic domain into
solvent. Electron density was not observed for part of the segment,
residues 216-226 including T222, which is one of the p38
phosphorylation sites. The activation segment is likely to be
dynamic due to the role the loop is expected to play in the
regulation of the enzyme. As discussed previously, MK2 41-364 is a
constitutively active truncated version of the enzyme and lacks
part of the C-terminal autoinhibitory sequence. Of the four MK2
structures in the asymmetric unit, C-terminal residues 345-364 are
disordered in three and residues 358-364 are disordered in the
fourth structure. The additional residues ordered in the latter
structure are in contact with a symmetry related molecule.
[0080] Comparison of the MK2 binary ADP and staurosporine complex
structures reveals a conformation change in the P loop where the
loop shifts inward to bind to staurosporine and shifts outward to
accommodate ADP.
[0081] Comparison with other kinases. Although the overall
structure of the catalytic domain is highly conserved in many
protein kinases, the mechanisms of regulation are quite diverse and
in some cases, require dramatic conformational changes (29). The
N-terminal and C-terminal lobes of many protein kinases are
connected by a flexible hinge (residues 142-145, DGGE (SEQ ID NO:9)
in MK2) thus the relative positions of the domains determine
whether the kinase is in the "open" or inactive state versus the
"closed" or active conformation (29). MK2 can be classified as an
"RD" kinase as the catalytic aspartate, D186, is immediately
preceded by an arginine, R185. Activation of a number of RD kinases
requires phosphorylation on one or more threonine, serine, or
tyrosine residues within the activation segment. Activation of MK2
requires phosphorylation, by p38, of the activation segment residue
T222. Three protein kinases closely related to MK2, both by
structure and shared regulatory mechanisms, are cAMP dependent
protein kinase (PKA) (25), Ca.sup.++/calmodulin dependent protein
kinase (cAMK) (19) and Titin (21). All are Ser/Thr kinases and all
require phosphorylation of a residue within the activation segment
for activation of the kinase. Like MK2, all share an additional
level of negative regulation by either a C-terminal autoinhibitory
domain or a regulatory subunit. Although PKA lacks an
autoinhibitory domain, a bound regulatory subunit maintains the
kinase in an inactive conformation. Activation of PKA requires
binding of an allosteric regulator, cAMP, to the regulatory subunit
resulting in a conformation change and release of the catalytically
active kinase subunit. cAMK is maintained in an inactive
conformation by a C-terminal autoinhibitory domain that blocks both
substrate and ATP binding. Activation of cAMK requires
Ca.sup.++/calmodulin binding which is thought to induce a
conformation change resulting in the displacement of the
autoinhibitory domain from the peptide and ATP binding sites. Titin
also contains a C-terminal autoinhibitory domain that sterically
blocks both substrate and ATP binding. Titin activation also
requires Ca.sup.++/calmodulin binding which is also thought to
induce a conformation change resulting in the displacement of the
autoinhibitory domain from the peptide and ATP binding sites.
[0082] The recently published structure of the autoinhibited,
inactive form of MK2 47-400 reveals a unique mechanism of kinase
regulation (22). The C-terminal autoinhibitory .alpha. helix
extends along the entire surface of one face of the C-terminal lobe
towards the active site and binds, hypothetically, as a
pseudosubstrate. Asp366 acts as a phosphothreonine mimetic by
coordinating with the basic residues R185 and K212 within the
active site of the enzyme. This "pseudosubstrate" region is thought
to be positioned in a manner that would effectively block binding
of both protein and peptide substrates. The N-terminal lobe of the
kinase domain has significant structural differences when compared
to the active MK2 structures presented here. The .beta.-2 strand in
the MK2 structures of the present invention is replaced by an
.alpha. helix which effectively disrupts the 5 strand .beta. sheet
observed in the structures of the present invention and many other
kinases. Additionally, the .alpha.C and .alpha.D helices are
shorter by 1.5 and 1.0 turns, respectively, in the autoinhibited
structure.
[0083] Analysis of the MK2 active site. The ATP binding site of
protein kinases is located in a deep cleft between the N-terminal
and C-terminal lobes of the catalytic domain. The .beta.1 and
.beta.2 strands in the N-terminal lobe constitute the P loop, which
contains a glycine rich motif, GXGXXG (SEQ ID NO:6), is highly
conserved in all protein kinases. The conserved glycines confer two
important structural properties to the P loop, lack of side chains,
which allow loop backbone amides to interact with ATP phosphates
without steric hindrance, and backbone flexibility, which allows
the P loop to adopt multiple conformations. Conformational
flexibility of the P loop is an important factor in the regulation
of many protein kinases. The structure of the MK2 P loop, within
the context of a 5 strand .beta.-sheet, and the spatial
relationship of the .alpha.C2 helix are highly consistent with the
structures of many protein kinases and also constitute an important
part of the kinase active site.
[0084] A number of catalytic residues conserved in all protein
kinases are spatially oriented to allow efficient transfer of
phosphate from ATP to a protein substrate. The binary complex
structure of MK2 41-364 and staurosporine is highly consistent with
the ternary complex structure of PKA, PKI inhibitor peptide and
staurosporine (30) thus forms the basis for a comparative analysis
of the MK2 and PKA active sites. The analysis revealed that all
active site residues discussed below are spatially conserved.
Arg-185 and Asp-186 (R165, D166 in PKA) are catalytic loop residues
that are invariant in all "RD" kinases. The catalytic aspartate,
D186, is thought to act as a base to remove a proton from the
protein substrate hydroxyl group. The phosphates of ATP are
positioned for hydrolysis by interactions with backbone amide
protons in the P loop and by ionic interactions with K93 (K72 in
PKA), which is positioned and stabilized properly by E104 (E91 from
PKA), a residue from the .alpha.C helix.
[0085] The MK2 activation segment, residues 207-233, extends
outward from the kinase catalytic domain in a conformation
partially stabilized by interactions with another symmetry related
MK2 C-terminal domain. The activation segment is also disordered in
the structure of the autoinhibited form of MK2 (22). The structural
data suggest that the activation segment in unphosphorylated MK2 is
highly dynamic and solvent accessible.
[0086] The MK2 structures of the present invention are of
unphosphorylated enzyme. Although unphosphorylated MK2 41-364 is
catalytically active and shows normal binding of ATP, the K.sub.m
for LSP-1 peptide substrate 45-fold higher and the V.sub.max is
100-fold lower when compared to p38 phosphorylated MK2 41-400 (See
Table 1). MK2 41-364 phosphorylated by p38, however, exhibits
virtually identical peptide substrate binding and catalytic rates
as compared to phosphorylated MK2 41-400. These data suggest that
p38 phosphorylation of the activation segment residue T222 shifts
the equilibrium of the activation segment from an unbound highly
dynamic state, as observed in the MK2 structures of the present
invention and in the inactive MK2 (22), to a more stable bound
state required for efficient binding of peptide substrate and a
high catalytic rate. Constitutively active MK2 41-364, however,
lacks key residues within the pseudosubstrate region and as
observed in the structures of the present invention, the majority
of the autoinhibitory C-terminal helix is disordered and probably
highly mobile thus allowing a lower level of peptide substrate
binding and catalysis. Phosphorylation of MK2 41-364 by p38
restores full enzymatic activity presumably by shifting the
equilibrium of the phosphorylated activation segment to the bound
state allowing efficient binding of peptide substrate. These data
also clearly show that residues 365-400 are not required for full
catalytic activity in vitro.
[0087] As observed in many other RD kinases, including PKA, that
require phosphorylation of a threonine residue within the
activation segment for activation and efficient catalysis, two
basic residues, R165 and K189 in PKA, are required for coordination
and charge neutralization of the phosphoryl threonine. These
residues are also spatially conserved in the MK2 structures (R185
and K212).
[0088] Differences in ADP and staurosporine structures. Comparison
of the MK2 structures co-crystallized with ADP and staurosporine
show that the only significant differences lie in the .beta.-sheet
containing the GKGING (SEQ ID NO:8) (71-76) loop. This loop,
between .beta. strand 1 and 2, and the loop on the other side of
.beta. strand 2 that connects to .beta. strand 3 (residues 83-88)
move extensively. Ile74 moves 4.7 A closer to Gly209 in the
activation loop when bound with staurosporine as compared to ADP
where the loop is shifter outward to accommodate ADP. Conversely in
the structure with ADP Thr86, found in the loop between .beta.
strand 2 and 3, moves closer to the protein core by 2.5 .ANG. as
compared to the loop in the structure with staurosporine. This
coupled movement of the loops connecting strands 1, 2 and 3 allows
the structural integrity of the b-sheet to remain while moving to
accommodate different entities in the active site. Excluding the
residues in the glycine rich loop, Asp207 and Lys93 make the only
significant movement of residues in the active site. In the
structure with ADP, these residues adopt orientations in close
proximity to the ADP moiety thereby revealing their importance in
the catalytic mechanism (29).
[0089] Biological implications. Cells exposed to heat shock,
cytokines (TNF, IL-1.beta.) or ultraviolet light display an
increase in p38 MAP kinase activity due to phosphorylation by the
upstream kinases MKK3 and MKK6. p38 in turn phosphorylates a
variety of substrates including the transcription factors ATF-2 and
CHOP-1, and other kinases such as MK2 and MK3. The p38/MK2 signal
transduction cascade plays a pivotal role in the production of
proinflammatory cytokines. Mice homozygously deficient in MK2 show
a reduction in TNF, IL1.beta., IL-6 and IFN-.gamma. synthesis and
an increased rate of survival upon exposure to LPS, as compared to
wild-type mice. MK2 controls the synthesis of cytokines by
regulating the translation and/or stability of the encoding mRNAs
through the AU-rich elements of the 3'-untranslated regions of the
gene. These data indicate that MK2 is a vitally important enzyme in
inflammatory based diseases and is a target for anti-inflammatory
drug design.
[0090] The present invention is based on the determination of a
catalytically active MK2 41-364 in complex with ADP and
staurosporine. From the structures of the present invention, as
well as the previous structure of the inactive enzyme 47-400 (22),
it is observed that MK2 is regulated quite differently from other
kinases that have a similar fold. Specifically, the autoinhibitory
domain of MK2 does not block the nucleotide binding site and that
phosphorylation of the residues in the activation domain are
necessary for optimal binding of the substrate and activity of the
enzyme. The structures of the present invention will permit the
design of inhibitors to MK2. TABLE-US-00001 TABLE 1 Apparent
K.sub.m Apparent K.sub.m V.sub.max Construct p38 (ATP) (.mu.M)
(LSP-1) (.mu.M) .mu.mole/min/mg 41-400 - not determined not
determined no activity 41-400 + 7.0 + 0.7 13 + 1 8.9 + 0.1 41-364 -
15 + 1.5 584 + 50 0.076 + 0.004 41-364 + not determined 20 + 2 11 +
14 Conditions: p38 (1.25 .mu.g/ml), MK-2 (200 .mu.g/mL), ATP (500
.mu.M), incubate 1-2 hours at 25.degree. C.
[0091] TABLE-US-00002 TABLE 2 Statistics for data collection and
phase determination MK2-Staurosporine MK2-Staurosporine MK2-ADP
Remote Peak Inflection SeMet Native Wavelength (.ANG.) .lamda.3 =
0.95666 .lamda.1 = 0.97926 .lamda.2 = 0.97955 1.1 1.0 Resolution
Range (.ANG.) 50-3.1 50-3.1 50-3.1 50-2.7 25-3.1 R.sub.merge* (%)
6.7(49.2) 6.6(37.7) 6.0(37.9) 4.7(48.1) 5.5(63.1) Completeness (%)
99.6(99.4) 100(100) 100(100) 100(100) 99.3(100.0) Total Reflections
255,756 427,587 449,393 215,726 90,852 Unique Reflections 82,015
42,754 42,927 52,352 12,997 I/.sigma.(I) 10.8(1.7) 16.2(3.4)
18.0(3.6) 20.2(1.6) 34.9(3.3) F' (e-).dagger. -2.95 -4.60 -7.21 F''
(e-) 3.70 6.44 1.81 MAD phasing Statistics for MK2-Staurosporine
Resolution limits (.ANG.) 6.92 5.24 4.62 4.00 3.70 3.46 3.27 3.10
Overall Phasing power.dagger-dbl. .lamda. 3 anomalous 3.36 2.08
1.72 1.07 0.82 0.68 0.55 0.52 1.08 .lamda. 1 isomorphous 0 0 0 0 0
0 0 0 0 .lamda. 1 anomalous 5.48 4.36 3.58 2.57 1.99 1.64 1.34 1.12
2.39 .lamda. 2 isomorphous 2.72 2.35 1.96 1.59 1.35 1.14 0.93 0.80
1.57 .lamda. 2 anomalous 2.99 1.67 1.33 0.83 0.62 0.53 0.40 0.36
0.80 Mean FOM.sup..sctn. 0.85 0.75 0.70 0.60 0.55 0.48 0.42 0.39
0.59 *R.sub.merge = .SIGMA.|I.sub.h -
<I.sub.h>|/.SIGMA.I.sub.h where <I.sub.h> is the
average intensity over symmetry equivalents. Numbers in parentheses
reflect statistics for the last shell. .dagger.F' and F'' reported
values were refined by SHARP. .dagger-dbl.Phasing power =
.SIGMA.|F.sub.h|/.SIGMA.|||F.sub.PHobs| - |F.sub.PHcalc||, where
F.sub.h is the calculated heavy atom structure-factor amplitude.
.sup..sctn.Figure of Merit =
<.SIGMA.P(a)e.sup.ia/.SIGMA.|P(.alpha.)|>, where .alpha. is
the phase and P(.alpha.) is the phase probability distribution.
[0092] TABLE-US-00003 TABLE 3 Residues of MK2 within 4 .ANG. of
staurosporin in MK2/staurosporine complex Leu70, Gly71, Leu72,
Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141,
Glu145, Glu190, Asn191, Leu192, Thr206, Asp207 Residues of MK2
within 8 .ANG. of staurosporin in MK2/staurosporine complex Val69,
Leu70, Gly71, Leu72, Gly73, Ile74, Gly76, Ala77, Val78, Leu79,
Gln80, Lys89, Phe90, Ala91, Leu92, Lys93, Leu95, Glu104, His108,
Val118, Arg119, Ile136, Val137, Mse138, Glu139, Cys140, Leu141,
Asp142, Gly143, Gly144, Glu145, His184, Asp186, Lys188, Pro189,
Glu190, Asn191, Leu192, Leu193, Tyr194, Thr195, Lys204, Leu205,
Thr206, Asp207, Phe208, Gly209 Residues of MK2 within 4 .ANG. of
ADP in MK2/ADP complex Leu70, Gly71, Leu72, Gly73, Ile74, Asn75,
Val78, Ala91, Lys93, Met138, Glu139, Cys140, Leu141, Asn191,
Thr206, Asp207 Residues of MK2 within 8 .ANG. of ADP in MK2/ADP
complex Val69, Leu70, Gly71, Leu72, Gly73, Ile74, Asn75, Gly76,
Ala77, Val78, Leu79, Gln80, Phe90, Ala91, Leu92, Lys93, Met94,
Leu95, Glu104, His108, Val118, Ala119, Ile136, Val137, Met138,
Glu139, Cys140, Leu141, Asp142, Gly143, Gly144, Glu145, His184,
Asp186, Lys188, Pro189, Glu191, Asn191, Leu192, Leu193, Tyr194,
Thr195, Leu205, Thr206, Asp207, Phe208, Gly209, Phe210
[0093] TABLE-US-00004 TABLE 4 Precipitant Buffer Additional Salt #1
30% (v/v) 0.1M Cacodylate pH 6.5 0.2M Lithium Sulfate
polyethyleneglycol-400 #2 1.0 M sodium citrate 0.1M Tris pH 7.0
0.2M Sodium chloride #3 0.8 M sodium dihydrogen 0.2 M Acetate pH
4.5 phosphate 1.2M K.sub.2HPO.sub.4 dipotassium hydrogen phosphate
#4 1.0M sodium citrate 0.1M Cacodylate pH 6.5 #5 1.9 M sodium
malonate pH 5.0 #6 1.5 M sodium malonate pH 6.0 #7 1.5 M sodium
malonate pH 7.0 #8 2.0 M ammonium Sulfate #9 1/17M
NaH.sub.2PO.sub.4 0.63M K.sub.2HPO.sub.4 (Final: 1.8M Sodium/
Potassium Phosphate pH 6.3) #10 0.27M NaH.sub.2PO.sub.4 1.53M
K.sub.2HPO.sub.4 (Final: 1.8M Sodium/ Pottassium Phosphate pH 7.5)
#11 0.072M NaH.sub.2PO.sub.4 1.728M K.sub.2HPO.sub.4 (Final: 1.8M
Sodium/ Pottassium Phosphate pH 8.2) #12 1.6M ammonium sulfate 0.1M
citric acid pH 5.0 #13 2.4M ammonium sulfate 0.1M HEPES pH 7.0 #14
30% v/v polyethyleneglycol- 0.1M Tris hydrochloride pH 8.5 0.2M
tri-Sodium citrate 400 dihydrate #15 1M potassium sodium 0.1M
HEPES-Na pH 7.5 tartrate tetrahydrate #16 18% v/v
polyethyleneglycol- 0.1M HEPES-Na pH 7.5 0.1M ammonium sulfate 400
#17 1M tri-sodium citrate 0.1M HEPES-Na pH 7.5 dihydrate #18 1.6M
Sodium/Potassium 0.1M MES pH 6.5 Tartrate #19 2.0M Ammonium Sulfate
0.1M HEPES pH 7.5 2% PEG 400
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[0133] All publications mentioned herein above, whether to issued
patents, pending applications, published articles, or otherwise,
are hereby incorporated by reference in their entirety. While the
foregoing invention has been described in some detail for purposes
of clarity and understanding, it will be appreciated by one skilled
in the art from a reading of the disclosure that various changes in
form and detail can be made without departing from the true scope
of the invention in the appended claims.
Sequence CWU 1
1
10 1 400 PRT Homo sapiens MISC_FEATURE MK2 1 Met Leu Ser Asn Ser
Gln Gly Gln Ser Pro Pro Val Pro Phe Pro Ala 1 5 10 15 Pro Ala Pro
Pro Pro Gln Pro Pro Thr Pro Ala Leu Pro His Pro Pro 20 25 30 Ala
Gln Pro Pro Pro Pro Pro Pro Gln Gln Phe Pro Gln Phe His Val 35 40
45 Lys Ser Gly Leu Gln Ile Lys Lys Asn Ala Ile Ile Asp Asp Tyr Lys
50 55 60 Val Thr Ser Gln Val Leu Gly Leu Gly Ile Asn Gly Lys Val
Leu Gln 65 70 75 80 Ile Phe Asn Lys Arg Thr Gln Glu Lys Phe Ala Leu
Lys Met Leu Gln 85 90 95 Asp Cys Pro Lys Ala Arg Arg Glu Val Glu
Leu His Trp Arg Ala Ser 100 105 110 Gln Cys Pro His Ile Val Arg Ile
Val Asp Val Tyr Glu Asn Leu Tyr 115 120 125 Ala Gly Arg Lys Cys Leu
Leu Ile Val Met Glu Cys Leu Asp Gly Gly 130 135 140 Glu Leu Phe Ser
Arg Ile Gln Asp Arg Gly Asp Gln Ala Phe Thr Glu 145 150 155 160 Arg
Glu Ala Ser Glu Ile Met Lys Ser Ile Gly Glu Ala Ile Gln Tyr 165 170
175 Leu His Ser Ile Asn Ile Ala His Arg Asp Val Lys Pro Glu Asn Leu
180 185 190 Leu Tyr Thr Ser Lys Arg Pro Asn Ala Ile Leu Lys Leu Thr
Asp Phe 195 200 205 Gly Phe Ala Lys Glu Thr Thr Ser His Asn Ser Leu
Thr Thr Pro Cys 210 215 220 Tyr Thr Pro Tyr Tyr Val Ala Pro Glu Val
Leu Gly Pro Glu Lys Tyr 225 230 235 240 Asp Lys Ser Cys Asp Met Trp
Ser Leu Gly Val Ile Met Tyr Ile Leu 245 250 255 Leu Cys Gly Tyr Pro
Pro Phe Tyr Ser Asn His Gly Leu Ala Ile Ser 260 265 270 Pro Gly Met
Lys Thr Arg Ile Arg Met Gly Gln Tyr Glu Phe Pro Asn 275 280 285 Pro
Glu Trp Ser Glu Val Ser Glu Glu Val Lys Met Leu Ile Arg Asn 290 295
300 Leu Leu Lys Thr Glu Pro Thr Gln Arg Met Thr Ile Thr Glu Phe Met
305 310 315 320 Asn His Pro Trp Ile Met Gln Ser Thr Lys Val Pro Gln
Thr Pro Leu 325 330 335 His Thr Ser Arg Val Leu Lys Glu Asp Lys Glu
Arg Trp Glu Asp Val 340 345 350 Lys Glu Glu Met Thr Ser Ala Leu Ala
Thr Met Arg Val Asp Tyr Glu 355 360 365 Gln Ile Lys Ile Lys Lys Ile
Glu Asp Ala Ser Asn Pro Leu Leu Leu 370 375 380 Lys Arg Arg Lys Lys
Ala Arg Ala Leu Glu Ala Ala Ala Leu Ala His 385 390 395 400 2 382
PRT Homo sapiens MISC_FEATURE MK3 2 Met Asp Gly Glu Thr Ala Glu Glu
Gln Gly Gly Pro Val Pro Pro Pro 1 5 10 15 Val Ala Pro Gly Gly Pro
Gly Leu Gly Gly Ala Pro Gly Gly Arg Arg 20 25 30 Glu Pro Lys Lys
Tyr Ala Val Thr Asp Asp Tyr Gln Leu Ser Lys Gln 35 40 45 Val Leu
Gly Leu Gly Val Asn Gly Lys Val Leu Glu Cys Phe His Arg 50 55 60
Arg Thr Gly Gln Lys Cys Ala Leu Lys Leu Leu Tyr Asp Ser Pro Lys 65
70 75 80 Ala Arg Gln Glu Val Asp His His Trp Gln Ala Ser Gly Gly
Pro His 85 90 95 Ile Val Cys Ile Leu Asp Val Tyr Glu Asn Met His
His Gly Lys Arg 100 105 110 Cys Leu Leu Ile Ile Met Glu Cys Met Glu
Gly Gly Glu Leu Phe Ser 115 120 125 Arg Ile Gln Glu Arg Gly Asp Gln
Ala Phe Thr Glu Arg Glu Ala Ala 130 135 140 Glu Ile Met Arg Asp Ile
Gly Thr Ala Ile Gln Phe Leu His Ser His 145 150 155 160 Asn Ile Ala
His Arg Asp Val Lys Pro Glu Asn Leu Leu Tyr Thr Ser 165 170 175 Lys
Glu Lys Asp Ala Val Leu Lys Leu Thr Asp Phe Gly Phe Ala Lys 180 185
190 Glu Thr Thr Gln Asn Ala Leu Gln Thr Pro Cys Tyr Thr Pro Tyr Tyr
195 200 205 Val Ala Pro Glu Val Leu Gly Pro Glu Lys Tyr Asp Lys Ser
Cys Asp 210 215 220 Met Trp Ser Leu Gly Val Ile Met Tyr Ile Leu Leu
Cys Gly Phe Pro 225 230 235 240 Pro Phe Tyr Ser Asn Thr Gly Gln Ala
Ile Ser Pro Gly Met Lys Arg 245 250 255 Arg Ile Arg Leu Gly Gln Tyr
Gly Phe Pro Asn Pro Glu Trp Ser Glu 260 265 270 Val Ser Glu Asp Ala
Lys Gln Leu Ile Arg Leu Leu Leu Lys Thr Asp 275 280 285 Pro Thr Glu
Arg Leu Thr Ile Thr Gln Phe Met Asn His Pro Trp Ile 290 295 300 Asn
Gln Ser Met Val Val Pro Gln Thr Pro Leu His Thr Ala Arg Val 305 310
315 320 Leu Gln Glu Asp Lys Asp His Trp Asp Glu Val Lys Glu Glu Met
Thr 325 330 335 Ser Ala Leu Ala Thr Met Arg Val Asp Tyr Asp Gln Val
Lys Ile Lys 340 345 350 Asp Leu Lys Thr Ser Asn Asn Arg Leu Leu Asn
Lys Arg Arg Lys Lys 355 360 365 Gln Ala Gly Ser Ser Ser Ala Ser Gln
Gly Cys Asn Asn Gln 370 375 380 3 473 PRT Homo sapiens MISC_FEATURE
MK5 3 Met Ser Glu Glu Ser Asp Met Asp Lys Ala Ile Lys Glu Thr Ser
Ile 1 5 10 15 Leu Glu Glu Tyr Ser Ile Asn Trp Thr Gln Lys Leu Gly
Ala Gly Ile 20 25 30 Ser Gly Pro Val Arg Val Cys Val Lys Lys Ser
Thr Gln Glu Arg Phe 35 40 45 Ala Leu Lys Ile Leu Leu Asp Arg Pro
Lys Ala Arg Asn Glu Val Arg 50 55 60 Leu His Met Met Cys Ala Thr
His Pro Asn Ile Val Gln Ile Ile Glu 65 70 75 80 Val Phe Ala Asn Ser
Val Gln Phe Pro His Glu Ser Ser Pro Arg Ala 85 90 95 Arg Leu Leu
Ile Val Met Glu Met Met Glu Gly Gly Glu Leu Phe His 100 105 110 Arg
Ile Ser Gln His Arg His Phe Thr Glu Lys Gln Ala Ser Gln Val 115 120
125 Thr Lys Gln Ile Ala Leu Ala Leu Arg His Cys His Leu Leu Asn Ile
130 135 140 Ala His Arg Asp Leu Lys Pro Glu Asn Leu Leu Phe Lys Asp
Asn Ser 145 150 155 160 Leu Asp Ala Pro Val Lys Leu Cys Asp Phe Gly
Phe Ala Lys Ile Asp 165 170 175 Gln Gly Asp Leu Met Thr Pro Gln Phe
Thr Pro Tyr Tyr Val Ala Pro 180 185 190 Gln Val Leu Glu Ala Gln Arg
Arg His Gln Lys Glu Lys Ser Gly Ile 195 200 205 Ile Pro Thr Ser Pro
Thr Pro Tyr Thr Tyr Asn Lys Ser Cys Asp Leu 210 215 220 Trp Ser Leu
Gly Val Ile Ile Tyr Val Met Leu Cys Gly Tyr Pro Pro 225 230 235 240
Phe Tyr Ser Lys His His Ser Arg Thr Ile Pro Lys Asp Met Arg Arg 245
250 255 Lys Ile Met Thr Gly Ser Phe Glu Phe Pro Glu Glu Glu Trp Ser
Gln 260 265 270 Ile Ser Glu Met Ala Lys Asp Val Val Arg Lys Leu Leu
Lys Val Lys 275 280 285 Pro Glu Glu Arg Leu Thr Ile Glu Gly Val Leu
Asp His Pro Trp Leu 290 295 300 Asn Ser Thr Glu Ala Leu Asp Asn Val
Leu Pro Ser Ala Gln Leu Met 305 310 315 320 Met Asp Lys Ala Val Val
Ala Gly Ile Gln Gln Ala His Ala Glu Gln 325 330 335 Leu Ala Asn Met
Arg Ile Gln Asp Leu Lys Val Ser Leu Lys Pro Leu 340 345 350 His Ser
Val Asn Asn Pro Ile Leu Arg Lys Arg Lys Leu Leu Gly Thr 355 360 365
Lys Pro Lys Asp Ser Val Tyr Ile His Asp His Glu Asn Gly Ala Glu 370
375 380 Asp Ser Asn Val Ala Leu Glu Lys Leu Arg Asp Val Ile Ala Gln
Cys 385 390 395 400 Ile Leu Pro Gln Ala Gly Lys Gly Glu Asn Glu Asp
Glu Lys Leu Asn 405 410 415 Glu Val Met Gln Glu Ala Trp Lys Tyr Asn
Arg Glu Cys Lys Leu Leu 420 425 430 Arg Asp Thr Leu Gln Ser Phe Ser
Trp Asn Gly Arg Gly Phe Thr Asp 435 440 445 Lys Val Asp Arg Leu Lys
Leu Ala Glu Ile Val Lys Gln Val Ile Glu 450 455 460 Glu Gln Thr Thr
Ser His Glu Ser Gln 465 470 4 326 PRT Homo sapiens 4 Met Gly Gln
Gln Phe Pro Gln Phe His Val Lys Ser Gly Leu Gln Ile 1 5 10 15 Lys
Lys Asn Ala Ile Ile Asp Asp Tyr Lys Val Thr Ser Gln Val Leu 20 25
30 Gly Leu Gly Ile Asn Gly Lys Val Leu Gln Ile Phe Asn Lys Arg Thr
35 40 45 Gln Glu Lys Phe Ala Leu Lys Met Leu Gln Asp Cys Pro Lys
Ala Arg 50 55 60 Arg Glu Val Glu Leu His Trp Arg Ala Ser Gln Cys
Pro His Ile Val 65 70 75 80 Arg Ile Val Asp Val Tyr Glu Asn Leu Tyr
Ala Gly Arg Lys Cys Leu 85 90 95 Leu Ile Val Met Glu Cys Leu Asp
Gly Gly Glu Leu Phe Ser Arg Ile 100 105 110 Gln Asp Arg Gly Asp Gln
Ala Phe Thr Glu Arg Glu Ala Ser Glu Ile 115 120 125 Met Lys Ser Ile
Gly Glu Ala Ile Gln Tyr Leu His Ser Ile Asn Ile 130 135 140 Ala His
Arg Asp Val Lys Pro Glu Asn Leu Leu Tyr Thr Ser Lys Arg 145 150 155
160 Pro Asn Ala Ile Leu Lys Leu Thr Asp Phe Gly Phe Ala Lys Glu Thr
165 170 175 Thr Ser His Asn Ser Leu Thr Thr Pro Cys Tyr Thr Pro Tyr
Tyr Val 180 185 190 Ala Pro Glu Val Leu Gly Pro Glu Lys Tyr Asp Lys
Ser Cys Asp Met 195 200 205 Trp Ser Leu Gly Val Ile Met Tyr Ile Leu
Leu Cys Gly Tyr Pro Pro 210 215 220 Phe Tyr Ser Asn His Gly Leu Ala
Ile Ser Pro Gly Met Lys Thr Arg 225 230 235 240 Ile Arg Met Gly Gln
Tyr Glu Phe Pro Asn Pro Glu Trp Ser Glu Val 245 250 255 Ser Glu Glu
Val Lys Met Leu Ile Arg Asn Leu Leu Lys Thr Glu Pro 260 265 270 Thr
Gln Arg Met Thr Ile Thr Glu Phe Met Asn His Pro Trp Ile Met 275 280
285 Gln Ser Thr Lys Val Pro Gln Thr Pro Leu His Thr Ser Arg Val Leu
290 295 300 Lys Glu Asp Lys Glu Arg Trp Glu Asp Val Lys Glu Glu Met
Thr Ser 305 310 315 320 Ala Leu Ala Thr Met Arg 325 5 981 DNA Homo
sapiens 5 atgggtcagc agttcccgca gttccacgtc aagtccggcc tgcagatcaa
gaagaacgcc 60 atcatcgatg actacaaggt caccagccag gtcctggggc
tgggcatcaa cggcaaagtt 120 ttgcagatct tcaacaagag gacccaggag
aaattcgccc tcaaaatgct tcaggactgc 180 cccaaggccc gcagggaggt
ggagctgcac tggcgggcct cccagtgccc gcacatcgta 240 cggatcgtgg
atgtgtacga gaatctgtac gcagggagga agtgcctgct gattgtcatg 300
gaatgtttgg acggtggaga actctttagc cgaatccagg atcgaggaga ccaggcattc
360 acagaaagag aagcatccga aatcatgaag agcatcggtg aggccatcca
gtatctgcat 420 tcaatcaaca ttgcccatcg ggatgtcaag cctgagaatc
tcttatacac ctccaaaagg 480 cccaacgcca tcctgaaact cactgacttt
ggctttgcca aggaaaccac cagccacaac 540 tctttgacca ctccttgtta
tacaccgtac tatgtggctc cagaagtgct gggtccagag 600 aagtatgaca
agtcctgtga catgtggtcc ctgggtgtca tcatgtacat cctgctgtgt 660
gggtatcccc ccttctactc caaccacggc cttgccatct ctccgggcat gaagactcgc
720 atccgaatgg gccagtatga atttcccaac ccagaatggt cagaagtatc
agaggaagtg 780 aagatgctca ttcggaatct gctgaaaaca gagcccaccc
agagaatgac catcaccgag 840 tttatgaacc acccttggat catgcaatca
acaaaggtcc ctcaaacccc actgcacacc 900 agccgggtcc tgaaggagga
caaggagcgg tgggaggatg tcaaggagga gatgaccagt 960 gccttggcca
caatgcgctg a 981 6 6 PRT Homo sapiens MISC_FEATURE X = any amino
acid; ATP binding site motiff misc_feature (2)..(2) Xaa can be any
naturally occurring amino acid misc_feature (4)..(5) Xaa can be any
naturally occurring amino acid 6 Gly Xaa Gly Xaa Xaa Gly 1 5 7 18
PRT Homo sapiens MISC_FEATURE MK2 bipartite nuclear localization
signal 7 Lys Lys Ile Glu Asp Asp Ala Ser Asn Pro Leu Leu Leu Lys
Arg Arg 1 5 10 15 Lys Lys 8 6 PRT Homo sapiens 8 Gly Lys Gly Ile
Asn Gly 1 5 9 4 PRT Homo sapiens MISC_FEATURE MK2 flexible hinge 9
Asp Gly Gly Glu 1 10 16 PRT Salmonella typhosa MISC_FEATURE LSP-1
peptide 10 Arg Thr Pro Lys Leu Ala Arg Gln Ala Ser Ile Glu Leu Pro
Ser Met 1 5 10 15
* * * * *