U.S. patent application number 10/200007 was filed with the patent office on 2003-05-29 for core structure of gp41 from the hiv envelope glycoprotein.
This patent application is currently assigned to Whitehead Institute for Biomedical Research. Invention is credited to Berger, James M., Chan, David C., Fass, Deborah, Kim, Peter S., Lu, Min.
Application Number | 20030099935 10/200007 |
Document ID | / |
Family ID | 26720235 |
Filed Date | 2003-05-29 |
United States Patent
Application |
20030099935 |
Kind Code |
A1 |
Chan, David C. ; et
al. |
May 29, 2003 |
Core structure of GP41 from the HIV envelope glycoprotein
Abstract
Described are the crystal structure of the .alpha.-helical
domain of the gp41 component of HIV-1 envelope glycoprotein which
represents the core of fusion-active gp41, methods of identifying
and designing drugs which inhibit gp41 function and drugs which do
so.
Inventors: |
Chan, David C.; (Brookline,
MA) ; Fass, Deborah; (Cambridge, MA) ; Lu,
Min; (New York, NY) ; Berger, James M.;
(Cambridge, MA) ; Kim, Peter S.; (Lexington,
MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Whitehead Institute for Biomedical
Research
Cambridge
MA
|
Family ID: |
26720235 |
Appl. No.: |
10/200007 |
Filed: |
July 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10200007 |
Jul 18, 2002 |
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09484925 |
Jan 18, 2000 |
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6506554 |
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09484925 |
Jan 18, 2000 |
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09062241 |
Apr 17, 1998 |
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6150088 |
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60043280 |
Apr 17, 1997 |
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Current U.S.
Class: |
435/5 ; 435/7.1;
530/324; 530/826 |
Current CPC
Class: |
A61K 38/00 20130101;
C12N 2740/16122 20130101; C07K 14/005 20130101 |
Class at
Publication: |
435/5 |
International
Class: |
C12Q 001/70 |
Goverment Interests
[0002] Work described herein was funded by the Howard Hughes
Medical Institute.
Claims
What is claimed is:
1. A method of identifying a drug that inhibits the HIV membrane
fusion machinery by inhibiting interactions between the N36 peptide
trimer and the C34 peptide trimer of HIV gp41, comprising: (a)
combining HIV gp41 N36 peptide trimer, HIVgp41 C34 peptide trimer
and a drug to be assessed for its ability to inhibit interaction
between the two trimers, to produce a combination; (b) maintaining
the combination under conditions appropriate for interactions to
occur between N36 peptide trimers and C34 peptide trimers; and (c)
assessing whether interactions occurred between N36 peptide trimers
and C34 peptide trimers, wherein if interactions between the N36
peptide trimer and the C34 peptide trimer did not occur in the
presence of the drug or occurred to a lesser in the presence of the
drug than in its absence, the drug is a drug that inhibits the HIV
membrane fusion machinery.
2. The method of claim 1 wherein in step (c) the interaction
assessed is packing of amino acid residues or peptides of C34
peptide trimers into highly conserved cavities on N36 peptide
trimers.
3. The method of claim 2 wherein the interaction assessed is
packing of amino acid residues or peptides of C34 into cavities on
N36 peptide trimers which are: (a) lined by Leu-566 of the left N36
helix and Leu-565 of the right N36 helix; (b) formed on the left
side by sidechains from the left N36 helix, including residues (top
to bottom) Val-570, Lys-574 (aliphatic portion) and Gln-577; (c)
formed on the right side by residues Leu-568, Trp-571 and Gly-572
of the right N36 helix; and (d) composed on its floor of Thr-569,
Ile-573 and Leu-576.
4. A method of producing a drug which inhibits interaction of two
components of the core of fusion-active HIV-1 envelope gp41,
wherein the two components are referred to as N36 peptide trimer
and C34 peptide trimer, respectively, comprising identifying a
compound or designing a compound which fits into a cavity on the
N36 peptide trimer which is: (a) lined by Leu-566 of the left N36
helix and Leu-565 of the right N36 helix; (b) formed on the left
side by sidechains from the left N36 helix, including residues (top
to bottom) Val-570, Lys-574 (aliphatic portion) and Gln-577; (c)
formed on the right side by residues Leu-568, Trp-571 and Gly-572
of the right N36 helix; and (d) composed on its floor of Thr-569,
Ile-573 and Leu-576.
5. The method of claim 4 wherein N36 peptide trimer and C34 peptide
trimer are recombinantly produced.
6. A method of producing a drug which inhibits interaction of N36
peptide trimer with C34 peptide trimer, wherein N36 peptide trimer
and C34 peptide trimer comprise the core of fusion-active HIV-1
envelope gp41, comprising identifying a compound or designing a
compound which: (a) fits into a cavity on the N36 peptide trimer:
(1) lined by Leu-566 of the left N36 helix and Leu-565 of the right
N36 helix; (2) formed on the left side by sidechains from the left
N36 helix, including residues (top to bottom) Val-570, Lys-574
(aliphatic portion) and Gln-577; (3) formed on the right side by
residues Leu-568, Trp-571 and Gly-572 of the right N36 helix; and
(4) composed on its floor of Thr-569, Ile-573 and Leu-576 and (b)
mimics the ability of Ile-635, Trp-631 and Trp-628 of C34 peptide
trimer to fit into the cavity of (a) and Asp-632 of C34 peptide
trimer to form a conserved salt bridge with Lys-574 of the N36
peptide trimer.
7. A compound which inhibits interaction of N36 peptide trimer of
the .alpha.-helical domain of HIV-1 gp41 which is the core or
fusion active gp41 with C34 peptide trimer of the x-helical
domain.
8. The compound of claim 8 wherein the compound fits into a cavity
on the N36 peptide trimer: (a) lined by Leu-566 of the left N36
helix and Leu-565 of the right N36 helix; (b) formed on the left
side by sidechains from the left N36 helix, including residues (top
to bottom) Val-570, Lys-574 (aliphatic portion) and Gln-577; (c)
formed on the right side by residues Leu-568, Trp-571 and Gly-572
of the right N36 helix; and (d) composed on its floor of Thr-569,
Ile-573 and Leu-576.
Description
RELATED APPLICATION(S)
[0001] This application is a continuation of U.S. application Ser.
No. 09/484,925, entitled, "Core Structure of gp41 From the HIV
Envelope Glycoprotein", by David C. Chan, Deborah Fass, Min Lu,
James M. Berger and Peter S. Kim, filed Jan. 18, 2000, which is a
Divisional application Ser. No. 09/062,241, entitled, "Core
Structure of gp41 From the HIV Envelope Glycoprotein", by David C.
Chan, Deborah Fass, Min Lu, James M. Berger and Peter S. Kim,
(filed on Apr. 17, 1998) now U.S. Pat. No. 6,150,088 (issued Nov.
21, 2000), which claims the benefit of U.S. Provisional Application
No. 60/043,280, entitled "Core Structure of gp41 from the HIV
Envelope Glycoprotein", by David C. Chan, Deborah Fass, Min Lu,
James M. Berger and Peter S. Kim (filed Apr. 17, 1997). The entire
teachings of the above applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] The surface glycoproteins of enveloped viruses play critical
roles in the initial events of viral infection, mediating virion
attachment to cells and fusion of the viral and immune response in
infected hosts. Envelope glycoproteins are also major targets for
the anti-viral immune response in infected hosts. The human
immunodeficiency virus type 1 (HIV-1) envelope glycoprotein
consists of two noncovalently associated subunits, gp120 and gp41,
that are generated by proteolytic cleavage of a precursor
polypeptide, gp160. Luciw, P. A., In Fields Virology, Third
Edition, B. N. Fields et al., eds., Lippincott-Raven Publishers,
Philadelphia, pp. 1881-1952 (1996); Freed, E. O. et al., J. Biol.
Chem. 270: 23883-23886 (1995). gp120 directs target-cell
recognition and viral tropism through interaction with the
cell-surface receptor CD4 and one of several co-receptors that are
members of the chemokine receptor family. Broder, C. C. et al.,
Pathobiology 64:171-179 (1996); D'Souza, M. P. et al., Nature Med.
2:1293-1300 (1996); Wilkinson, D., Current Biology 6:1051-1053
(1996). The membrane-spanning gp41 subunit then promotes fusion of
the viral and cellular membranes, a process that results in the
release of viral contents into the host cell. It has not yet been
possible to obtain a detailed structure for gp41, either alone or
in complex with gp120.
SUMMARY OF THE INVENTION
[0004] Described herein is the crystal structure of the
.alpha.-helical domain of the gp41 component of HIV-1 envelope
glycoprotein which represents the core of fusion-active gp41. Also
described herein is Applicants' determination, with reference to
the crystal structure, that certain amino acid residues within the
core are essential for interaction of the component peptides and,
thus, for gp41 activity. The core of fusion-active gp41 is composed
of a trimer of two interacting peptides, referred to here as N36
and C34. The minimal stable envelope subdomain has been shown to
consist of a 36-residue peptide (N-36) and a 34-residue peptide
(C-34) whose amino acid sequences are presented below. The crystal
structure of the N36/C34 complex is a six-helix bundle in which
three N36 helices form an interior, parallel coiled coil and three
C34 helices pack in an oblique, anti-parallel manner into highly
conserved, hydrophobic grooves on the surface of the N36 trimer. It
shows striking similarity to the low-pH induced conformation of
influenza hemagglutinin (HA).
[0005] Applicants have determined the structural basis for
interaction between two peptide fragments of HIV gp41: one peptide
fragment derived from the N-terminal region of the ectodomain of
gp41 and one peptide fragment derived from the C-terminal region of
the gp41 ectodomain. The N-terminal peptide fragment, N36, includes
amino acid residue 546 through and including amino acid residue
581, numbered according to their position in HIV-1 gp160; it
includes amino acid residues which comprise a region of the
ectodomain which encompasses the 4-3 hydrophobic repeat. The amino
acid sequence of the N36 peptide is:
[0006] SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQRIL (SEQ ID NO.: 1).
[0007] The C-terminal region peptide fragment C34 includes amino
acid residue 628 through and including amino acid residue 661,
numbered according to their position in HIV-1 gp160; it is derived
from the region prior to the transmembrane segment. The amino acid
sequence of the C34 peptide is:
[0008] WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL (SEQ ID NO.: 2). The
three-dimension coordinates for the atoms in the N36/C34 gp41
complex are presented herein. They can be used to display the
structure of the complex and to design molecules (drugs) which
interact with gp41 and inhibit its activity, such as those which
prevent interaction of key components (amino acid residues) of the
.alpha.-helical domain which represents the core of fusion-active
gp41.
[0009] Work described herein provides, for the first time, an
understanding of how the N-terminal peptide and the C-terminal
peptide interact. The crystal structure and information regarding
the interactions of these two peptides provide the basis for
development of drugs which inhibit HIV infection, such as
peptidomimetic or small-molecule inhibitors, using such methods as
combinatorial chemistry or rational drug design. Drugs developed or
identified with reference to the information provided herein are
also the subject of the present invention. Drugs which fit into or
line the N-peptide cavity, prevent the N-peptide cavity from
accommodating amino acid residues or peptides from the C-terminal
region of gp41 and, thus, prevent or inhibit gp41 activity are the
subject of this invention. Such drugs can be identified with
reference to the information about the structure of the complex and
the cavity shown to be present in the N36 trimer, provided herein,
or with reference to information about the structure of the complex
and the three dimensional coordinates of the cavity, also provided
herein, and known methods. In a particular embodiment of
identifying or designing a molecule which inhibits the fusion
active form of gp41 and, thus, inhibit HIV, in which combinatorial
chemistry is used, a library biased to include an increased number
of indole rings, hydrophobic moieties and/or negatively charged
molecules is used. An antibody which binds these key areas of
fusion-active gp41 is also the subject of the invention. For
example, an immunogen which is or includes a molecule with the
coordinates described herein or the N-peptide core can be used to
immunize an individual, resulting in production of antibodies that
bind the cavity or pocket on the N-terminal peptide and, thus,
render it unavailable for its normal interactions and prevent or
inhibit gp41 activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a schematic view of gp41 showing important
functional regions, including the 4-3 hydrophobic repeat, the
fusion peptide (fp), a disulfide linkage (S-S), and the
transmembrane region (tm). The ectodomain is drawn approximately to
scale. The peptides identified by protein dissection are shown
above, along with the sequences of N36 and C34. The residues are
numbered according to their position in gp160.
[0011] FIG. 2 is a representative portion of the initial electron
density map calculated using experimental structure-factor
amplitudes and solvent-flattened MAD phases, shown with the refined
molecular model. The map is contoured at 1.5 standard deviations
above the mean density. The figure was generated with the program O
(Jones, T. A., and Kjeldgaard, M., O--The Manual, Uppsala, Sweden:
http://kaktus.kemi.aau.dk (1992)).
[0012] FIGS. 3A and 3B present overall views of the N36/C34
complex. FIG. 3A shows an end-on view of the N36/C34 complex
looking down the three-fold axis of the trimer. FIG. 3B shows a
side view with one N36 and one C34 helix labeled. The amino termini
of the N36 helices (grey) point towards the top of the page, while
those of the C34 helices (black) point towards the bottom. Diagrams
were prepared using the program MOLSCRIPT (Kraulis, P., J. Appl.
Cryst. 24:924-950 (1991)).
[0013] FIG. 4 shows a helical wheel representation of N36 and C34;
three N36 helices and one C34 helix are represented as helical
wheel projections. The view is from the top of the complex, as in
FIG. 3A. The residues at each position are represented by the
single-letter codes for amino acids. The N36 helices interact
through "knobs-into-holes" packing interactions at the a and d
positions. Positions of the N36 and C34 helices that occupy the
interhelical space between two N36 helices and a C34 helix are
shown (arrows). The helical wheel positions in C34 are indicated by
ellipses to represent the oblique orientation of this helix
relative to N36. At the top of the complex, C34 is slightly tilted
towards the left N36 helix, while at the bottom of the complex, it
is slightly tilted towards the right N36 helix.
[0014] FIGS. 5A-5P present the three-dimension coordinates for the
atoms in the N36/C34 gp41 complex; the atom types (column 3) in
each amino acid (column 4) are listed, along with their coordinates
(columns 6, 7, 8) in space. The three-dimension coordinates can be
used to display the structure of the N36/C34 complex. The
coordinates are available from the Protein Data Bank at the
Brookhaven National Laboratory.
[0015] FIG. 6 represents the distances (in .ANG.) between the atoms
in the four amino acid residues of C34 that dock into the cavity on
the N36 trimer surface. The two tryptophan residues, and the
isoleucine residue and the aspartic acid residue are indicated in
green.
DETAILED DESCRIPTION OF THE INVENTION
[0016] For the first time, a high-resolution picture of the protein
fragment that enables HIV to invade human cells has been produced.
As described, Applicants have determined the crystal structure of a
key fragment of the HIV envelope protein. The envelope protein
resides on the surface of the virus and plays a crucial role in HIV
infection. One part of the protein, known as gp120, allows the
virus to bind to human cells. Another subunit, gp41, mediates
fusion of the viral membrane and the cell membrane--it initiates
entry of the virus into the cell. The core structure of gp41 has
been determined using X-ray crystallography.
[0017] The images of the protein fragment reveal a compact,
six-helix bundle punctuated by deep cavities which are key targets
for the development of new antiviral drugs. The existence of the
cavities could not have been determined without the images.
[0018] Despite its importance, there are no antiviral drugs that
target the envelope protein of HIV, in part because the virus is
extraordinarily clever at changing the pieces of the protein it
presents to the outside world. Work presented herein shows that the
cavity structure may not be so amenable to change; therefore, drugs
directed towards this region are useful against many HIV
strains.
[0019] The HIV fusion protein has characteristics similar to those
of the fusion structure of influenza virus. Surprisingly, the HIV
fusion protein has a deep cavity or pocket at the base of each
groove in the N36 coiled coil. In the active structure, each cavity
is filled by a knob-like protrusion from C34. This ball-and-socket
arrangement of C34 and N36 is a target for drug design or
discovery. The structure, combined with data from other
laboratories, supports the idea that a small molecule constructed
specifically to block this interaction will stop fusion and prevent
the virus from entering cells.
[0020] There are at least three reasons why such a molecule would
be effective in preventing HIV from entering cells. First, test
tube studies have shown that fragments, or peptides, of gp41
encompassing or overlapping with N36 or C34 have potent anti-viral
activity. However, peptides generally make poor drugs because they
are poorly absorbed and the body breaks them down almost
immediately. A small molecule targeting just the cavity structure
could escape this fate.
[0021] Second, the inhibitors derived from the C and N peptides are
effective in the test tube against a wide range of HIV strains,
including patient isolates and laboratory-adapted strains. By
contrast, neutralizing antibodies and drug candidates designed to
block the binding activity of the envelope protein are typically
effective against only a limited subset of HIV strains.
[0022] Third, alteration of the walls of the N36 cavity can block
the fusion reaction, indicating that the ball-and-socket
arrangement of N36 and C34 must be preserved to obtain viral
infection. In addition, the protein building blocks that make up
the walls are highly conserved among HIV strains and between HIV
and SIV, the virus responsible for AIDS in monkeys. This suggests
that the virus cannot tolerate much change in this region and that
HIV may have more difficulty developing resistance to a
cavity-blocking drug than to many other compounds.
[0023] Applicants have analyzed the crystal structure of the
.alpha.-helical domain of the HIV-1 transmembrane protein gp41 by
means of assessment of a complex, referred to herein as the N36/C34
complex, which is composed of two interacting peptides: N36, which
is derived from the N-terminal region of the gp41 ectodomain and
C34, which is derived from the C-terminal region of the gp41
ectodomain. As described herein, Applicants have shown that the
N36/C34 complex is a six-helix bundle (FIG. 3), in which the center
consists of a parallel, trimeric coiled-coil of three N36 helices
wrapped in a gradual left-handed superhelix. Three C34 helices wrap
antiparallel to the N36 helices in a left-handed direction around
the outside of the central coiled-coil N36 trimer. The N36/C34
complex is a cylinder which is approximately 35 .ANG. in diameter
and approximately 55 .ANG. in height. FIG. 4 is a helical wheel
representations of N36 and C34 in which three N36 helices and one
C34 helix are represented as helical wheel projections. As can be
seen, the interior amino acid residues at the a and d positions of
the N36 heptad repeat are predominately hydrophobic (isoleucine,
leucine). The characteristic "knobs-into-holes" packing of coiled
coils occurs in the N36 trimer. That is, the amino acid residues
(knobs) at the a and d layers pack into cavities (holes) between
four residues of an adjacent helix. Crick, F. H. C., Acta. Cryst.,
6: 689-697 (1953); O'Shea, E. K., et al., Science, 254:539-544
(1991). Further description of the N36 trimer is presented in
Example 2.
[0024] An electrostatic potential map of the cylindrical N36
superhelix shows that the surface of the superhelix is largely
uncharged. The grooves that are the sites for C34 interaction have
been determined to be lined with predominantly hydrophobic amino
acid residues. The surface of the N36/C34 complex is much more
highly charged than the isolated N-peptides, due to the acidic
residues on the outside of the C34 helices. This explains why the
heterodimeric complex exhibits greater solubility than the isolated
peptides.
[0025] Three C34 helices pack obliquely against the outside of the
N36 coiled-coil trimer in an antiparallel orientation. Interaction
between the C34 helices and N36 occurs mainly through hydrophobic
residues in three grooves on the surface of the central coiled-coil
trimer. The amino acid residues which line these grooves are highly
conserved between HIV and SIV gp41. In contrast, the N36 residues
which flank the C34 helices are divergent. The pattern of sequence
conservation is also apparent on the helical wheel representation
of three N36 helices and one C34 helix of FIG. 4. (See Example 3.)
Each of the grooves on the surface of the N36 trimer has a
particularly deep cavity. The cavity is approximately 16 .ANG.
long, approximately 7 .ANG. wide and approximately 5-6 .ANG. deep.
It accommodates three hydrophobic amino acid residues from the
abutting C34 helix: isoleucine-635 (I.sub.635), tryptophan-631
(W.sub.631) and tryptophan-628 (W.sub.628). The top of the N36
cavity is lined by leucine-566 (Leu-566) of the left N36 helix and
leucine-565 (Leu-565) of the right N36 helix. The left side of the
cavity is formed by side chains from the left N36 helix, including
amino acid residues (top to bottom): valine-570 (Val-570),
lysine-574 (Lys-574, aliphatic portion) and glutamine-577
(Gln-577). The right wall of the cavity is formed by amino acid
residues leucine-568 (Leu-568), tryptophan-571 (Trp-571) and
glycine-572 (Gly-572) of the right N36 helix. The cavity floor is
composed of threonine-569 (Thr-569), isoleucine-573 (Ile-573) and
leucine-576 (Leu-576). Thus, interactions within the cavity are
predominantly hydrophobic. In addition, aspartic acid-632 (Asp-632)
of C34 forms a conserved salt bridge with lysine-574 (Lys-574) of
N36 immediately to the left of the cavity.
[0026] As a result of the work described, a region of the HIV-1
transmembrane protein gp41 which is a target for HIV inhibitors has
been defined and is available for designing and/or developing new
drugs and identifying existing drugs which inhibit HIV. A
particularly valuable target for an HIV inhibitor are the highly
conserved, deep cavities on the N-peptide coiled-coil trimer that
accommodate C-peptide amino acid residues. The amino acid residues
which form the cavity have been defined. Thus, a drug (e.g., a
peptide, peptidomimetic, small molecule or other agent) which fits
into or lines the N-peptide cavity or socket, prevents the
N-peptide cavity from accommodating peptides from the C-terminal
region of gp41 and, thus, prevents or inhibits gp41 activity, can
be identified or designed. For example, a drug which fits into or
lines the cavity can be identified or designed, using known
methods. One such drug is a molecule or compound which fits into or
lines a cavity:
[0027] a) lined by Leu-566 of the left N36 helix and Leu-565 of the
right N36 helix;
[0028] b) formed on the left side by sidechains from the left N36
helix, including residues (top to bottom) Val-570, Lys-574
(aliphatic portion) and Gln-577;
[0029] c) formed on the right side by residues Leu-568, Trp-571 and
Gly-572 of the right N36 helix; and
[0030] d) composed on its floor of Thr-569, Ile-573 and
Leu-576.
[0031] The cavities present on the N-peptide coiled-coil trimer
each accommodate three hydrophobic amino acid residues from the
abutting C34 helix: Ile-635, Trp-631 and Trp-628 and a negatively
charged amino acid residue from C34: Asp-632, which forms a
conserved salt bridge with Lys-574 of N36 immediately to the left
of the cavity. A drug which mimics the ability of these three
residues (Trp-Trp-Ile) to fit into or line N36 cavities can also be
developed. Such a drug can be developed, for example, with
reference to the three-dimension coordinates provided (FIGS. 5A-5P)
and the information provided (FIG. 6, for example) regarding the
distances between the atoms in the four amino acid residues of C34
that dock into the cavity on the N36 trimer surface.
[0032] For example, a structure-based approach can be used, along
with available computer-based design programs, to identify or
design a drug which will fit into, line or bind a cavity or pocket
on N36 (or block C34 from doing so) and inhibit or prevent the
activity of gp41 and, as a result, reduce (partially or totally)
the ability of HIV-1 to infect cells. In one embodiment of the
present invention, the following method is carried out to design or
identify a molecule or drug which inhibits gp41 activity (and
reduces HIV-1 infection of cells) by fitting into or lining the N36
cavity. In a computer processor having a digital processor, a
method of designing or identifying a drug or molecule which
inhibits (totally or partially) the interaction of N36 and C34 or
fits into or lines a cavity on N36, comprises the steps of: (a)
providing a library of molecules, compounds or drugs whose crystal
structures, coordinates, chemical configurations or structures are
known; (b) providing a crystal structure of a target molecule,
which is the .alpha.-helical domain of the gp41 component of HIV-1
envelope glycoprotein which represents the core of fusion-active
gp41 (referred to for convenience as the N36/C34 complex or
N36/C34); and (c) comparing coordinates, crystal structure
components, chemical configurations or structures of members of the
library of molecules with those of the target molecule, such as by
using a processor routine executed by the digital processor to
search the library to find a molecule or a molecule component which
fits into or lines the cavity on N36, the processor routine
providing design or identification of a member or members of the
library which fit into or line the cavity on N36 or a member or
members which comprise a component moiety or component moieties
which fit into or line the cavity on N36. For example, this method
can be carried out by comparing the members of the library with the
crystal structure of gp41 N36/C34 presented herein using computer
programs known to those of skill in the art (e.g., Dock, Kuntz, I.
D. et al., Science, 257:1078-1082 (1992); Kuntz, I. D. et al., J.
Mol. Biol., 161.269 (1982); Meng, E. C., et al., J. Comp. Chem.,
13:505-524 (1992) or CAVEAT).
[0033] In the method, the library of molecules to be searched in
(a) can be any library, such as a database (i.e., online, offline,
internal, external) which comprises crystal structures,
coordinates, chemical configurations or structures of molecules,
compounds or drugs (referred to collectively as to be assessed or
screened for their ability inhibit N36/C34 interaction candidate
N36 ligands). For example, databases for drug design, such as the
Cambridge Structural Database (CSD), which includes about 100,000
molecules whose crystal structures have been determined or the Fine
Chemical Director (FCD) distributed by Molecular Design Limited
(San Leandro, Calif.) can be used. [CSD: Allen, F. H., et al., Acta
Crystallogr. Section B, 35:2331 (1979)] In addition, a library,
such as a database, biased to include an increased number of
members which comprise indole rings, hydrophobic moieties and/or
negatively-charged molecules can be used.
[0034] Coordinates of the molecules in the library can be compared
in the method to coordinates of the cavity on N36 or to coordinates
of C36 and its components which fit into or line an N36 cavity or
pocket. The cavity on N36 is described in detail herein, as are key
components of C34 which are accommodated by cavities on the
N-peptide. Upon finding a match to coordinates of at least one
molecule in the library, at least one member is, thus, determined
or identified as an N36 ligand (at least one member is determined
to be a member which will inhibit N36/C34 interaction).
[0035] Additional steps in the searching process can include
combining certain library members or components of library members
to form collective coordinates or molecules which combine features
or coordinates of two or more library members; comparing the
resulting collective coordinates or molecules with the crystal
structure of the target molecule and identifying those which will
interact with an N36 cavity (or cavities).
[0036] Upon identification of an existing drug or design of a novel
molecule as described herein, its ability to line or fit into a
cavity on N36 or block N36/C34 interaction can be assessed using
known methods, such as by expressing N36 and C34 in an appropriate
host cell (e.g., a bacterial cell containing and expressing DNA
encoding N36 and C34), combining the expressed products with the
drug to be assessed and determining whether it interferes with the
interaction of N36 and C34, lines a cavity on N36 and C34. Drugs
which are found to do so can be assessed in additional assays, both
in vitro and in vivo (e.g., an appropriate animal model challenged
by HIV infection). Once a drug has been identified or designed, it
may be desirable to refine or reconfigure it in such a manner that
a drug which binds better (e.g., with greater specificity and/or
affinity) is produced. In this case, the processor routine further
determines the quality of matches and calculates a goodness of fit,
making it possible to do so.
[0037] A drug or molecule which binds or fits into a cavity or
pocket on the surface of N36, can be used alone or in combination
with other drugs (as part of a drug cocktail) to prevent or reduce
HIV infection of humans. A drug designed or formed by a method
described herein is also the subject of this invention.
[0038] Also the subject of this invention is a method of treating
an individual infected with HIV or at risk of being infected with
HIV, in order to reduce the extent of infection or to prevent
infection. In the method, a drug which fits into, lines or binds a
cavity or cavities on N36 is administered to the individual, alone
or in combination with other drugs.
[0039] A further subject of this invention is an immunogen based on
a molecule with coordinates as described herein which is used to
produce antibodies that bind the N36 cavity or pocket and, thus,
prevent N36/C34 interaction and inhibit gp41 activity. For example,
the N-peptide core can be used, in known methods, to produce
polyclonal or monoclonal antibodies, which can be administered to
an individual. Alternatively, an individual (e.g., a human infected
with HIV or at risk or being infected) can be immunized with the
N-peptide core. The individual will, as a result, produce
antibodies which will bind the N36 pocket or cavity and prevent or
reduce gp41 activity. Thus, this invention also relates to a
vaccine to reduce or prevent gp41 function (and, as a result, HIV
infection).
[0040] As described above, Applicants have provided the identity of
amino acid residues which form the cavity into which amino acid
residues of the gp41 C-peptides fit. Thus, they have defined target
amino acid residues which can be mutated or modified, individually
or jointly, to further assess the structural basis for interaction
between the two peptides, identify amino acid residues essential
for the two to fit together and design or identify molecules or
compounds which inhibit/prevent the two helices from fitting
together and, thus, inhibit or prevent gp41 membrane--fusion
activity.
[0041] Numerous studies have led to the proposal that there are
native (nonfusogenic) and fusion-active (fusogenic) states of viral
membrane fusion proteins. Extensive conformational changes in the
HIV envelope complex are thought to be involved in the transition
from the native to the fusogenic state. Binding of CD4 to gp120
exposes the V3 loop of gp120, which likely interacts with the
co-receptors. Choe, H. et al., Cell 85:1135-1148 (1996); Trkola, A.
et al., Nature 384:184-187 (1996); Wu, L. et al., Nature
384:179-183 (1996). For some laboratory-adapted isolates of HIV-1,
the conformational changes in gp120 upon CD4 binding are sufficient
to cause gp120 to physically dissociate or "shed" from the viral
surface, leaving the membrane-anchored gp41 subunit behind. Hart,
T. K. et al., Proc. Natl. Acad. Sci., USA 88:2189-2193 (1991);
Moore, J. P. et al., Science 250:1139-1142 (1990). Primary isolates
of the virus generally do not shed gp120 readily in the presence of
CD4 alone, although CD4 binding still induces conformational
changes in gp120. (Sattentau, Q. J. et al., Phil. Trans. Royal Soc.
B 342:59-66 (1993); Sattentau, Q. J. et al., J. Virol. 67:7383-7393
(1993); Sullivan, N. et al., J. Virol. 69:4413-4422 (1995),
Stamatatos, L. et al., J. Virol. 69:6191-6198 (1995)).
[0042] CD4 binding also induces conformational changes in gp41, as
inferred from changes in antibody binding and sensitivity to
limited proteolysis (Sattentau, Q. J. et al., Phil. Trans. Royal
Soc. B 342:59-66 (1993); Sattentau, Q. J. et al., J. Virol.
67:7383-7393 (1993)). Moreover, addition of low levels of soluble
CD4 enhances the infectivity of some viral isolates, suggesting
that the gp120/gp41 conformational changes induced by CD4 play a
role in membrane fusion (Allan, J. S. et al., Science 247:1084-1088
(1990); Sullivan, N. et al., J. Virol. 69:4413-4422 (1995)). These
conformational changes are thought to expose the hydrophobic,
glycine-rich fusion-peptide region of gp41 that is essential for
membrane-fusion activity.
[0043] To obtain a detailed structure for gp41, a
protein-dissection approach, in which key substructures of a
protein are identified and studied was applied. See, for example,
Oas, T. G. et al., Nature 336:42-48 (1988). Limited proteolysis of
a fragment corresponding to the ectodomain of gp41 generated a
stable, soluble complex composed of two peptide fragments denoted
N51 and C43 (FIG. 1) that are derived from the N- and C-terminal
regions of the ectodomain, respectively (Lu, M. et al., Nature
Struct. Biol. 2:1075-1082 (1995)). In gp41, the region following
the fusion peptide has a high .alpha.-helical propensity and a 4-3
heptad repeat of hydrophobic residues, a sequence feature
characteristic of coiled coils. Chambers, P. et al., J. Gen. Virol.
71:3075-3080 (1990); Delwart, E. L. et al., AIDS Res. Hum.
Retroviruses 6:703-706 (1990); Gallaher, W. R. et al., AIDS Res.
Hum. Retroviruses 5:431-440 (1989). The N51 peptide corresponds to
the 4-3 hydrophobic repeat region adjacent to the fusion peptide,
while the C43 peptide is derived from the region prior to the
transmembrane segment (FIG. 1).
[0044] Interestingly, isolated peptides that overlap, or are
derived from, the N51 and C43 regions of gp41 can have potent
anti-viral activity (Wild, C. T. et al., Proc. Natl. Acad. Sci.,
USA 89:10537-10541 (1992); Wild, C. T. et al., Proc. Natl. Acad.
Sci., USA 91:9770-9774 (1994); Jiang, S. et al., Nature 365:113
(1993)). Peptides from the C-terminal region of the ectodomain have
the highest activity. Consistent with these studies, both N51 and
C43 are capable of inhibiting HIV envelope-mediated cell fusion;
the C43 peptide exhibits 10-fold greater activity than N51 (Lu, M.
et al., Nature Struct. Biol. 2:1075-1082 (1995)). The inhibitory
activity of the C43 peptide, however, is markedly reduced when
stoichiometric amounts of N51 are present, suggesting that the C43
peptide inhibits membrane fusion in a dominant-negative manner, by
associating with an N51 region within intact gp41 (Lu, M. et al.,
Nature Struct. Biol. 2:1075-1082 (1995)). Thus, in addition to
providing insights into the mechanism of membrane fusion,
determining the structural basis for interaction between the N51
and C43 regions will assist anti-viral drug-development
efforts.
[0045] Biophysical studies showed that the N51 and C43 peptides
associate to form a highly thermostable, helical, trimeric complex
of heterodimers, in which the N51 and C43 helices are oriented in
an antiparallel manner. Lu, M. et al., Nature Struct. Biol.
2:1075-1082 (1995). Analogous experiments with the gp41 ectodomain
from simian immunodeficiency virus (SIV) gave almost identical
results, indicating that the gp41 core identified in these
protein-dissection studies is conserved among lentiviruses.
Blacklow, S. C. et al., Biochemistry 34:14955-14962 (1995). On the
basis of these results and other considerations, we proposed that
the gp41 core consists of an interior coiled-coil trimer formed by
the N51 region, against which three C43 helices pack. Lu, M. et
al., Nature Struct. Biol. 2:1075-1082 (1995); Blacklow, S. C. et
al., Biochemistry 34:14955-14962 (1995).
[0046] The thermal denaturation of the N51/C43 complexes from HIV-1
or SIV gp41 is irreversible, probably as a result of aggregation of
the unfolded peptides at high temperature. Lu, M. et al., Nature
Struct. Biol. 2:1075-1082 (1995); Blacklow, S. C. et al.,
Biochemistry 34:14955-14962 (1995). With a view towards
crystallographic studies, further protein dissection experiments
were used to define a smaller subdomain with more favorable
thermodynamic properties. These studies led to the identification
of the peptides N36 and C34 (FIG. 1). Like the longer peptides, N36
and C34 form a stable, trimeric complex of heterodimers with 100%
.alpha.-helix content. Unlike the larger complex, however, the
N36/C34 complex has a reversible thermal unfolding transition.
Presented herein is the crystal structure of the N36/C34 complex
solved to 2.0 .ANG. resolution, as well as a discussion of the
implications of this structure for HIV viral membrane fusion and
its inhibition.
[0047] The work described herein provides good evidence that the
structure of gp41 obtained is found in the fusion-active state of
HIV envelope. That this is the core of gp41 in the fusogenic state
is supported by several considerations.
[0048] First, the N36/C34 complex folds in the absence of gp120.
The fusogenic state of gp41 is expected to be stable in the absence
of gp120, since dissociation of gp120 from the envelope
glycoprotein is thought to accompany the conversion from a native
to a fusogenic state. Cohen, J., Science 274:502 (1996); Wilkinson,
D., Current Biology 6:1051-1053, (1996). Similarly, the conversion
of influenza HA2 to the fusogenic state is accompanied by loss of
most of its contacts with HA1. Proteolysis of the low-pH converted
form of HA prior to crystallization removes most of the
receptor-binding HA1 subunit. Bullough, P. A. et al., Nature
371:37-43 (1994). Moreover, the structural features of the
fusogenic state are preserved in fragments of HA2 that fold
cooperatively in the complete absence of the HA1 subunit. Carr, C.
M. et al., Cell 73:823-832 (1993); Chen, J. et al., Proc. Natl.
Acad. Sci., USA 92:12205-12209 (1995).
[0049] Second, the isolated gp41 core is exceedingly stable to
thermal denaturation. The N51/C43 complex has an apparent melting
temperature of approximately 90.degree. C. Lu, M. et al., Nature
Struct. Biol. 2:1075-1082 (1995). In contrast, the native state of
the HIV envelope glycoprotein is not particularly stable, as
evidenced by the ease with which gp120 is shed in preparations of
virus particles. Helseth, E. et al., J. Virol. 65:2119-2123 (1991);
Kalyanaraman, V. S. et al., AIDS Res. Hum. Retroviruses 6, 371-380
(1990).
[0050] Third, mutations in gp41 that abolish infectivity and
membrane fusion often map to residues that are expected to
stabilize the gp41 core structure determined here. Numerous studies
show that mutations in the 4-3 hydrophobic repeat region abolish
membrane fusion, although these mutants tend to have additional
defects. Dubay, J. W. et al., J. Virol. 66:4748-4756 (1992); Chen,
S. S., J. Virol. 68:2002-2010 (1994); Chen, S. S. et al., J. Virol.
67, 3615-3619 (1993); Wild, C. et al., Proc. Natl. Acad. Sci., USA
91:12676-12680 (1994); Poumbourios, P., J. Virol. 71:2041-2049
(1997). The Leu-568.fwdarw.Ala, Trp-571.fwdarw.Arg, and
Asn-656.fwdarw.Leu mutations are particularly noteworthy because
cells expressing mutant envelope glycoproteins with one of these
point mutations are completely defective in membrane fusion, as
judged by an inability to form syncytia with CD4-positive human
lymphocyte lines, even though the mutant proteins exhibit
substantial cell-surface expression, CD4 binding, gp120/gp41
association, gp160 precursor processing, and soluble CD4-induced
shedding. Cao, J. et al., J. Virol. 67:2747-2755 (1993). Leu-568
and Trp-571 are N36 residues that line the right wall of the
cavity. Asn-656 is in an a position of the C34 peptide and packs
against the central N36 coiled-coil trimer. The locations of these
key mutations suggest that interactions between the N36 and C34
helices are critical for membrane fusion.
[0051] Fourth, that the N36/C34 structure corresponds to the core
of the fusogenic state of gp41 is consistent with a large body of
data on the inhibition of HIV-1 infection and syncytia formation by
derivatives of the peptides that make up this core. This issue is
discussed in more detail below. Finally, the structural similarity
of the N36/C34 complex to the low-pH induced conformation of
influenza HA2 (Bullough, P. A. et al., Nature 371:37-43 (1994)) and
to the structure of Mo-MLV TM (Fass, D. et al., Nature Struct.
Biol. 3:465-469 (1996)), each of which has been proposed to
represent fusion-active conformations, supports the idea that
N36/C34 is the core of the fusogenic conformation of gp41. For all
three structures, the hydrophobic fusion peptide would be
immediately amino terminal to a central, three-stranded coiled
coil. In influenza HA2 and HIV-1 gp41, the central three-stranded
coiled coils are each stabilized by three helices that pack
obliquely against the coiled-coil trimer in an antiparallel manner.
In the TM subunit of Mo-MLV, these obliquely packed helices are
replaced by a short helix and an extended region that serve a
similar structural role.
[0052] Work described herein also relates to inhibitors of HIV-1
infection and targets for developing new peptidomimetic or
small-molecule inhibitors of HIV infection. Synthetic peptides
containing approximately 40 residues from gp41 that overlap, or
include all of, the residues in N36 or C34 can be effective
inhibitors, at micromolar to nanomolar concentrations, of HIV
infection and syncytia formation. Lu, M. et al., Nature Struct.
Biol. 2:1075-1082 (1995); Jiang, S. et al., Nature 365:113 (1993);
Wild, C. T. et al., Proc. Natl. Acad. Sci., USA 89:10537-10541
(1992); Wild, C. T. et al., Proc. Natl. Acad. Sci. USA,
91:9770-9774 (1994). Assessment previously of the inhibitory
properties of the N51 and C43 peptides implied that these peptides
work in a dominant negative manner (Herskowitz, I., Nature
329:219-222 (1987)) by binding to viral gp41 (Lu, M. et al., Nature
Struct. Biol. 2:1075-1082 (1995)), a conclusion that was also
reached through studies of a gp41 ectodomain chimeric protein
(Chen, C. H. et al., J. Virol. 69:3771-3777 (1995)). Further
evidence in support of a dominant-negative mechanism is provided by
the finding that mutations in C-peptide derivatives that disrupt
their interactions with N-peptide correlate with decreased potency
as inhibitors. Wild, C. et al., AIDS Res. Hum. Retroviruses
11:323-325 (1995).
[0053] The gp41 core crystal structure is fully consistent with
this dominant-negative mechanism of inhibition (FIG. 3). The
C-peptide derivatives could act as dominant-negative inhibitors by
binding to the endogenous N-peptide coiled-coil trimer within viral
gp41. The N-peptides might inhibit fusion by interfering with
formation of the central, coiled-coil trimer within viral gp41,
and/or by binding to endogenous viral C-peptide regions.
[0054] Both the N- and C-peptide classes of inhibitors are
effective against a wide range of HIV strains, including
laboratory-adapted strains and primary isolates. Wild, C. T. et
al., Proc. Natl. Acad. Sci., USA 89:10537-10541 (1992); Jiang, S.
et al., Nature 365:113 (1993); Wild, C. T. et al., Proc. Natl.
Acad. Sci., USA 91:9770-9774 (1994). In contrast, soluble CD4 and
many neutralizing antibodies are typically effective only on a
limited subset of HIV strains (e.g., Daar, E. S. et al., Proc.
Natl. Acad. Sci., USA 87:6574-6578 (1990); Palker, T. J. et al.,
Proc. Natl. Acad. Sci., USA 85:1932-1936 (1988); Nara, P. L. et
al., J. Virol. 62:2622-2628 (1988); Moore, J. P. et al., J.
Virology 69:101-109 (1995). There is a striking conservation of
residues involved in interactions between the N-peptide and
C-peptide, comparing gp41 from HIV-1 and SIV. The broad
neutralizing effects of the N- and C-peptides derive from the
strong sequence conservation of these residues.
[0055] The highly conserved, deep cavities on the N-peptide
coiled-coil trimer that accommodate conserved C-peptide residues
are useful targets for the development of new peptidomimetic or
small-molecule inhibitors of HIV infection. The two indole rings
and neighboring sidechains that occupy the prominent cavity in N36
are a particularly attractive target for the design and/or
development of new drugs or identification of existing drugs which
inhibit HIV infection. Not only is this cavity deep and highly
conserved, but two of the three key mutations that disrupt membrane
fusion, discussed above, map to one wall of this cavity. Because
some of the known potent peptide inhibitors (Wild, C. T. et al.,
Proc. Natl. Acad. Sci., USA 91:9770-9774 (1994)) extend beyond N36
and C34 and do not involve this cavity region, it is likely that
other distinctive surface features exist in the interface between
the N- and C-helices of longer peptides such as N51 and C43. Lu, M.
et al., Nature Struct. Biol. 2:1075-1082 (1995). The importance of
identifying drugs that target the HIV membrane-fusion machinery is
emphasized by the success of combination drug regimens for the
treatment of AIDS. As yet, these combination therapies do not
target the HIV envelope. A method of identifying a drug which is an
inhibitor of N36/C34 peptide interaction (and, thus, is an
inhibitor of the HIV membrane fusion machinery and, as a result,
reduces or prevents HIV entry into (infection of) cells is the
subject of this invention. In the method, N36 and C34 are combined
with a drug to be assessed, under conditions suitable for N36 and
C34 to interact (suitable for cavities on the N-peptide coiled-coil
trimer to accommodate C-peptide amino acid residues). The resulting
combination is maintained under these conditions for sufficient
time to permit N36 and C34 to interact (e.g., for sufficient time
for N36 and C34 to interact in the absence of the drug being
assessed). Whether interaction occurs and/or the extent to which
N36 and, C34 interact is assessed, using known methods. If N36 and
C34 do not interact or interact to a lesser extent in the presence
of the drug being assessed than in the absence of the drug, the
drug to be assessed is an inhibitor of N36/34 interaction. Such a
drug is an inhibitor of the HIV membrane fusion machinery. Such an
inhibitor can be further assessed, using in vitro or in vivo
methods, for its ability to reduce or prevent HIV entry into
cells.
[0056] Results of the work described have implications for gp41
function and viral membrane fusion. The structures of the cores of
the membrane-fusion subunits from HIV, Mo-MLV and influenza virus
are remarkably similar. It appears that these diverse viruses
present fusion peptides to target cells via a common scaffold, in
which the fusion peptides are atop a central, three-stranded coiled
coil that is supported by additional, carboxy-terminal structures.
This scaffold is likely to be a common feature of viral
membrane-fusion proteins since many of these proteins contain
coiled-coil signature sequences, with 4-3 heptad repeats of
hydrophobic amino acids, adjacent to an amino-terminal
fusion-peptide region. Delwart, E. L. et al, AIDS Res. Hum.
Retroviruses 6:703-706 (1990); Chambers, P. et al., J. Gen. Virol.
71:3075-3080 (1990); Gallaher, W. R. et al., AIDS Res. Hum.
Retroviruses 5:431-440 (1989). Moreover, studies of the fusion
proteins of several paramyoviruses have identified regions with
similarity to the N- and C-peptide regions of HIV and SIV gp41
(Lambert, D. M. et al., Proc. Natl. Acad. Sci., USA 93:2186-2191
(1996)). These common structural features suggest that the rich
body of work investigating the mechanism of membrane fusion for
many other viruses, including influenza, is relevant for
understanding the mechanism of HIV-mediated membrane fusion.
[0057] Given the similarity in structure between the HIV gp41 core
and the low-pH converted conformation of HA2, it is worth
considering whether the structural rearrangements that occur during
the transition of HA2 to the fusogenic state are analogous to those
in gp41. In the native, non-fusogenic conformation of influenza HA,
part of the N-terminal coiled-coil trimer seen in the fusogenic
state (Bullough, P. A. et al., Nature 371:37-43 (1994)) is held in
a non-helical, hairpin structure, as a result of extensive
interactions with the receptor-binding HA1 subunit (Wilson, I. A.
et al., Nature 289:366-373 (1981)). Thus, the receptor-binding HA1
subunit acts as a "clamp" that binds this N-terminal region of HA2,
holding it in the non-coiled coil conformation. The
receptor-binding domains dissociate in the fusogenic conformation
of HA, as in HIV, although in the case of influenza, the HA1
subunits are still tethered via a disulfide bond to HA2. Upon
release of the HA1 clamp, a dramatic conformational change in HA2
occurs, including coiled-coil formation by this N-terminal region
(Bullough, P. A. et al., Nature 371:37-43 (1994); Carr, C. M. et
al., Cell 73:823-832 (1993)).
[0058] A substantial conformational change in the envelope
glycoprotein complex also appears to be critical during HIV
infection, although few details are understood. It remains to be
determined whether the HIV envelope complex also utilizes
coiled-coil formation as part of a spring-loaded mechanism, or if
the gp41 core structure determined here is present in the native as
well as the fusogenic state. It is possible that the N36/C34
structure is the core structure of gp41 even when it is bound to
gp120, and that release of gp120 simply exposes the fusion-peptide
region of gp41. Alternatively, HIV gp120, like influenza HA1, may
serve as a clamp that represses formation of the N36/C34 structure
presented here, with gp120 shedding allowing its formation. This
gp41 core structure serves as the starting point for addressing
this and other essential structural questions about the mechanism
of HIV entry into cells.
[0059] The present invention is illustrated by the following
examples, which are not intended to be limiting in any way.
EXAMPLES
[0060] The materials and methods described below were used in the
examples which follow.
[0061] Materials and Methods
[0062] Peptide Purification and Crystallization
[0063] Peptides N36 and C34 were synthesized by standard FMOC
peptide chemistry and have an acetylated N-terminus and a
C-terminal amide. N36 corresponds to residues 546 to 581 of gp160,
while C34 corresponds to residues 628 to 661. After cleavage from
the resin, the peptides were desalted on a Sephadex G-25 column
(Pharmacia) and lyophilized. Peptides were then purified by
reverse-phase high performance liquid chromatography (Waters, Inc.)
on a Vydac C18 preparative column. The identity of the peptides was
verified by mass spectrometry. Peptide concentration was determined
by tyrosine and tryptophan absorbance in 6 M GuHCl. Edelhoch, H.,
Biochemistry 6:1948-1954 (1967).
[0064] To grow crystals, a 10 mg/ml stock of the N36/C34 complex
was diluted 1:1 in a sitting drop with 80 mM NH.sub.4Cl, 20%
PEG200, and 50% isopropanol and allowed to equilibrate against a
reservoir of 80 mM NH.sub.4Cl, 20% PEG200, and 30% isopropanol.
Crystals grew as hexagonal prisms and belonged to the space group
P321 (a=b=49.5 .ANG., c=55.3 .ANG.). For native data sets and heavy
atom screens, crystals were flash-frozen in a MSC cryogenic crystal
cooler (X-stream), and data was collected on a Rigaku RU-200
rotating-anode X-ray generator with an R-axis IIc detector.
[0065] Heavy Atom Screen and Phase Determination
[0066] Multiwavelength anomalous diffraction (MAD) data were
collected at the Howard Hughes Medical Institute beamline X4A of
the National Synchrotron Light Source at Brookhaven National
Laboratory. Fluorescence spectra (1.1459 to 1.1354 .ANG.) were
obtained from a single flash-frozen crystal soaked in 0.04%
OsO.sub.4 in harvest buffer (80 mM NH.sub.4Cl, 20% PEG200, 30%
isopropanol) for 4 hours. Based on the fluorescence profile,
individual data sets were collected on Fuji imaging plates at four
wavelengths (1.sub.1=1.1396 .ANG., 1.sub.2=1.1398 .ANG.,
1.sub.3=1.1402 .ANG., and 1.sub.4=1.1344 .ANG.). Reflections were
integrated and scaled with DENZO and SCALEPACK. (Otwinowski, Z.,
Daresbury Study Weekend Proceedings, 1993.)
[0067] Data merging, phase determination and map generation were
all performed using the CCP4 suite of programs. CCP4, Acta Cryst.
D50:760-763 (1994). Anomalous and dispersive difference Patterson
maps from MAD data sets all showed a single clear peak
corresponding to the osmium binding site. The position of the site
was calculated from the single z=0 Harker section and from cross
peaks found at z=0.28 and z=0.71. Phases generated with the program
MLPHARE (Otwinowski, Z., Daresbury Study Weekend Proceedings, 1991)
gave an overall figure of merit of 0.89 (Table) and produced an
interpretable electron density map with a clear solvent boundary.
Density modification was subsequently performed using DM (Cowtan,
K. D., Newsletter on Protein Crystallography 31:34-38 (1994)),
resulting in maps of high quality in which electron density for the
entire main chain and all side chains was evident.
[0068] Model Refinement
[0069] The polypeptide chain was traced and the side chains readily
positioned into a 2.7 .ANG. density-modified map using the program
O (Jones, T. A., and Kjeldgaard, M., O--The Manual, Uppsala,
Sweden: http://kaktus.kemi.aau.dk, 1992). The initial model of
N36/C34 was refined with the program XPLOR (Brunger, A. T., A
system for X-ray crystallography and NMR. X-PLOR Version 3.1, Yale
University Press, New Haven, Conn., 1992) against data to 2.0 .ANG.
from a native crystal. An anisotropic B-factor was applied to the
native structure factors using XPLOR, and a free R set (Brunger, A.
T., Nature 355:472-475 (1992)) was taken from the data prior to
refinement (Table). The model was refined by iterative cycles of
grouped B-factor, positional, and individual B-factor refinement.
As the refinement proceeded, 43 waters were added and a bulk
solvent correction was applied. At no time during the refinement
did the molecule differ enough from the original model so as to
require manual rebuilding, though main chain and side chain
geometries were optimized in 0 between cycles of refinement. The
quality of the structure was verified by PROCHECK (Laskowski, R. A.
et al., J. Appl. Cryst. 26:283-291 (1993)), with all residues but
one (Ile-580) occupying most-preferred regions of Ramachandran
space. Ile-580 lies in the additionally allowed region of
Ramachandran space and is the second residue from the C-terminus of
the N36 peptide; inspection of the solvent-flattened MAD-phased
maps confirmed its position.
Example 1
Production of Crystals of N36/C34
[0070] Crystals of N36/C34 were grown by sitting-drop vapor
diffusion (see Methods). An initial model of the complex was built
into an electron density map generated by multi-wavelength
anomalous dispersion (MAD) analysis (Hendrickson, W. A., Science
254:51-58 (1991)) of an osmium-derivatized crystal. Details of data
collection and MAD phasing statistics are listed in the Table. A
representative portion of the solvent-flattened electron density
map used for building the initial model is shown in FIG. 2. The
structure was refined against data to 2.0 .ANG. from a native
crystal to yield an R.sub.free of 0.266 and an R.sub.cryst of 0.238
(Table).
1TABLE Crystallographic and refinement statistics Data collection
Crystal .lambda.(.ANG.) % complete R.sub.sym.sup.1(%) Resol.(.ANG.)
Native 1.5418 96.5 5.5 2.0 OsO.sub.4 .lambda.1 1.1398 96.4 4.3 2.7
OsO.sub.4 .lambda.2 1.1396 96.4 4.3 2.7 OsO.sub.4 .lambda.3 1.1344
96.8 4.5 2.7 OsO.sub.4 .lambda.4 1.1406 93.4 4.5 2.7 Phasing
statistics (12-2.7 .ANG.) R.sub.diff.sup.3(%) R.sub.cullis.sup.4
R.sub.cullis.sup.4 R.sub.cullis.sup.4 Ph. power.sup.5 Ph.
power.sup.5 Anom. Derivative R.sub.iso.sup.2(%) (weight) Acentric
Centric Anom. Acentric Centric Occ..sup.6 Occ..sup.6 OsO.sub.4
.lambda.1 vs. .lambda.4 4.4 6.7 0.46 0.53 0.21 2.46 1.53 0.075
2.165 OsO.sub.4 .lambda.2 vs. .lambda.4 6.6 9.3 0.37 0.37 0.22 3.34
2.38 0.132 1.784 OsO.sub.4 .lambda.3 vs. .lambda.4 5.4 7.4 0.42
0.44 0.35 2.94 2.12 0.105 1.005 Overall figure of merit (before
solvent flattening): 0.89 Refinement statistics (12-2.0 .ANG.)
R.m.s. Non-hydrogen Number of reflections deviations protein atoms
Waters working free R.sub.cryst.sup.7 R.sub.free.sup.7 bonds
(.ANG.) angles (.degree.) 596 43 5212 371 (7.12%) 0.238 0.266 0.014
2.742 .sup.1R.sub.sym =
.SIGMA..SIGMA..sub.j.vertline..vertline..sub.j-
<.vertline.>.vert-
line./.SIGMA..vertline.<.vertline.>.vertline., where
.vertline..sub.j is the recorded intensity of the reflection j and
<.vertline.> is the mean recorded intensity over multiple
recordings. .sup.2R.sub.iso = .SIGMA..vertline..vertline.F.sub..-
lambda.i .+-. F.sub..lambda.4.vertline. -
.vertline.F.sub..lambda.i.vertli-
ne..vertline./.SIGMA..vertline.F.sub..lambda.4.vertline., where
F.sub..lambda.i is the structure factor at wavelength .lambda.i and
F.sub..lambda.4 is the structure factor at the reference wavelenth
.lambda.4. .sup.3R.sub.diff = [.SIGMA..vertline.(F.sup.2.sub.(.l-
ambda.4) - .PHI..sub.mean)/.phi.F.sup.2.sub.(.lambda.4).vertline. +
.vertline.F.sup.2.sub.(.lambda.i)
-.PHI..sub.mean)/.phi.F.sup.2.sub.(.lam-
bda.i).vertline.]/[.SIGMA.[(F.sup.2.sub.(.lambda.4)/.phi.F.sup.2.sub.(.lam-
bda.4) + (F.sup.2.sub.(.lambda.i)/.phi.F.sup.2.sub.(.lambda.i))]],
where .sup..PHI.mean =
[(F.sup.2.sub.(.lambda.4)/.phi.F.sup.2.sub.(.lambda.4)) +
(F.sup.2.sub.(.lambda.i))/.phi.F.sup.2.sub.(.lambda.i))]/
#[(1/.phi.F.sup.2.sub.(.lambda.4)) +
(1/.phi.F.sup.2.sub.(.lambda.i))]and .phi.F.sup.2.sub.(n) =
[Variance (F.sup.2.sub.(n))]4F.sup.2.sub.(n). .sup.4R.sub.cullis =
.SIGMA..vertline..vertline.F.sub..lambda.i .+-. F.sub.80
4.vertline. - .vertline.F.sub.h(.lambda.i),c.vertline..vertline.
/.SIGMA..vertline.F.sub.80 .vertline. .+-.
F.sub..lambda.4.vertline., where F.sub.h(.lambda.i),c is the
calculated heavy atom structure factor. .sup.5Phase power =
<F.sub.h(.lambda.i)>/E, where <F.sub.h(.lambda.i)> is
the root-mean-square heavy atom structure factor and E is the
residual lack of closure error. .sup.6Occupancies are values output
from MLPHARE. .sup.7R.sub.cryst, free =
.SIGMA..vertline..vertline.F.sub.obs.vertline. -
.vertline.F.sub.calc.vertline..vertline./
.vertline.F.sub.obs.vertline.- , where the crystallographic and
free R factors are calculated using the working and free reflection
sets, respectively.
Example 2
Assessment of the Structure of the N36/C34 Complex
[0071] The N36/C34 complex is a six-stranded helical bundle (FIG.
3). The center of this bundle consists of a parallel, trimeric
coiled coil of three N36 helices wrapped in a gradual left-handed
superhelix. Three C34 helices wrap antiparallel to the N36 helices
in a left-handed direction around the outside of the central
coiled-coil trimer. The complex is a cylinder measuring .about.35
.ANG. in diameter and .about.55 .ANG. in height.
[0072] As in other naturally-occurring coiled coils (Cohen, C. et
al., Proteins 7:1-15 (1990)), the interior residues at the a and d
positions of the N36 heptad repeat are predominantly hydrophobic,
although occasional buried polar interactions are also present in
the central three-stranded coiled coil (FIG. 4). A sequence
comparison of HIV-1 (HXB2 strain) and SIV (Mac239 strain) gp41
shows that the residues at these two heptad-repeat positions are
highly conserved (FIG. 4). The characteristic "knobs-into-holes"
packing of coiled coils is utilized, whereby the residues (knobs)
at the a and d layers pack into cavities (holes) between four
residues of an adjacent helix (Crick, F. H. C., Acta Cryst.
6:689-697 (1953); O'Shea, E. K. et al., Science 254:539-544
(1991)). Of the three types of knobs-into-holes packing geometry
observed in coiled-coil structures (Harbury, P. B. et al., Science
262:1401-1407 (1993); Harbury, P. et al., Nature 371:80-83 (1994)),
the N36 trimer demonstrates exclusively "acute" packing geometry,
similar to that found in the crystal structure of an
isoleucine-zipper trimer (Harbury, P. et al., Nature 371:80-83
(1994)). This type of packing arrangement in the interior of the
coiled coil is characteristic of trimers because it allows P
branched residues (e.g., isoleucine) to pack favorably at both the
a and d positions (Harbury, P. et al., Nature 371:80-83 (1994)).
Trimeric coiled coils, like the N36 trimer (FIG. 4), tend to have
.beta. branched residues at both the a and d positions.
[0073] Although complexes of the N- and C-peptides are clearly
trimeric (Lu, M. et al., Nature Struct. Biol. 2:1075-1082 (1995);
Blacklow, S. C. et al., Biochemistry 34:14955-14962 (1995)),
isolated N-peptides corresponding to the 4-3 hydrophobic repeat
from gp41 have been reported to form tetramers, leading to
conflicting conclusions regarding the oligomeric state of gp41
(Lawless, M. et al., Biochemistry 35:13697-13708 (1996);
Rabenstein, M. et al., Biochemistry 34:13390-13397 (1995);
Rabenstein, M. D. et al., Biochemistry 35:13922-13928 (1996);
Shugars, D. C. et al., J. Virol. 70:2982-2991 (1996)). An
electrostatic potential map of the N36 coiled-coil trimer shows
that its surface is largely uncharged. The grooves that are the
sites for C34 interaction are lined with predominantly hydrophobic
residues (see below) that would be expected to lead to aggregation
upon exposure to solvent. Indeed, previous studies have shown that
the isolated N-peptides tend to aggregate (Blacklow, S. C. et al.,
Biochemistry 34:14955-14962 (1995); Lu, M. et al., Nature Struct.
Biol. 2:1075-1082 (11995)). Thus, conclusions regarding the
oligomerization state of gp41 based on studies of isolated
N-peptides are probably misleading. The N36/C34 complex shows a
much more highly charged surface due to acidic residues on the
outside of the C34 helices, explaining the greater solubility of
the heterodimeric complex.
Example 3
Determination of Interactions between the - and C-Peptide
Helices
[0074] Three C34 helices pack obliquely against the outside of the
N36 coiled-coil trimer in an antiparallel orientation. These C34
helices interact with N36 mainly through hydrophobic residues in
three grooves on the surface of the central coiled-coil trimer.
Sequence comparisons between HIV and SIV gp41 shows that the
residues lining these grooves are highly conserved. In contrast,
the N36 residues flanking the C34 helices are divergent between HIV
and SIV.
[0075] This pattern of sequence conservation is also apparent on a
helical wheel representation of three N36 helices and one C34 helix
(FIG. 4). In this diagram, the residue positions in C34 are
depicted as ellipses to indicate the oblique tilt of the C34 helix
relative to the N36 superhelix and to emphasize that C34 is not
part of a coiled coil. Residues at the e and g positions of the N36
helices lie on the outside of the central coiled coil and point
into the triangular interhelical space between two N36 helices and
a buttressing C34 helix. In general, residues at positions a and d
of C34 pack against residues at the e and g positions of the N36
helices (FIG. 4), although contacts at other positions are often
observed. Comparing HIV and SIV gp41, no nonconservative changes
exist at the e and g positions of the N36 helix, and only two such
changes occur at the a and d positions of C34. In contrast, 8 of
the 9 nonconservative changes in the N36 helix occur at the outside
f, b, and c positions, while 13 of the 15 nonconservative changes
in the C34 helix occur at positions other than a and d. The
sequence of the N-peptide region of gp41 is among the most highly
conserved within the HIV envelope glycoprotein. Our results show
that the high sequence conservation in this region results from
selective pressure on the e and g positions to retain C34 peptide
interactions, as well as pressure on the a and d positions to
maintain trimeric coiled-coil interactions.
[0076] Each of the grooves on the surface of the N36 trimer has a
particularly deep cavity. This cavity is large (16 .ANG. long,
.about.7 .ANG. wide, and 5-6 .ANG. deep) and accommodates three
hydrophobic residues from the abutting C34 helix: Ile-635, Trp-631
and Trp-628. The top of the cavity is lined by Leu-566 of the left
N36 helix and Leu-565 of the right N36 helix. Side chains from the
left N36 helix form the left side of the cavity, including residues
(top to bottom) Val-570, Lys-574 (aliphatic portion), and Gln-577.
The right wall is formed by residues Leu-568, Trp-571, and Gly-572
of the right N36 helix. The floor of the cavity is composed of
Thr-569 and Leu-576 of the right N36 helix, and also Ile-573 of
both N36 helices. With the exception of Ile-573 (which is replaced
by Thr), all the residues forming the cavity are identical between
HIV-1 and SIV. In addition to these predominately hydrophobic
interactions within the cavity, Asp-632 of C34 forms a conserved
salt bridge with Lys-574 of N36 immediately to the left of the
cavity.
Example 4
Comparison of the Structure of the N36/C34 Complex with the Low-pH
Induced Conformation of HA
[0077] The N36/C34 complex shows striking structural similarity to
the low-pH induced conformation of the influenza HA.sub.2 subunit
(TBHA.sub.2) (Bullough, P. A. et al., Nature 3 71:37-43 (1994)) and
to the TM subunit of Mo-MLV (Fass, D. et al., Nature Struct. Biol.
3:465-469 (1996)), each of which has been proposed to be a
fusogenic conformation. Remarkably, the core of each of the three
structures contains a three-stranded coiled coil that would be
adjacent to the amino-terminal fusion peptide. The trimeric coiled
coil of gp41 is very similar to that of the Mo-MLV TM, both having
a similar superhelical pitch (.about.175 .ANG.) and a regular 4-3
periodicity. In contrast, the TBHA.sub.2 coiled coil is a typical
because it contains two regions with skips in the 4-3 periodicity,
resulting in an underwound superhelix (pitch of 300-400 .ANG.). As
in the gp41 core structure, TBHA.sub.2 contains three antiparallel
helices that are packed, with a left-handed tilt, against the
central trimeric coiled coil.
EQUIVALENTS
[0078] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims. Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the claims.
Sequence CWU 1
1
2 1 36 PRT Human Immunodeficiency Virus 1 Ser Gly Ile Val Gln Gln
Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala 1 5 10 15 Gln Gln His Leu
Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln 20 25 30 Ala Arg
Ile Leu 35 2 34 PRT Human Immunodeficiency Virus 2 Trp Met Glu Trp
Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His 1 5 10 15 Ser Leu
Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu 20 25 30
Leu Leu
* * * * *
References