Báo cáo Y học: Solution structure of the HIV gp120 C5 Domain pptx

Solution structure of the HIV gp120 C5 DomainLaure Guilhaudis, Amy Jacobs and Michael Caffrey Department of Biochemistry and Molecular Biology, University of Illinois at Chicago, IL, USA

Solution structure of the HIV gp120 C5 Domain

Laure Guilhaudis, Amy Jacobs and Michael Caffrey

Department of Biochemistry and Molecular Biology, University of Illinois at Chicago, IL, USA

In HIV the viral envelope protein is processed by a host cell

protease to form gp120 and gp41 The C1 and C5 domains of

gp120 are thought to directly interact with gp41 but are

largely missing from the available X-ray structure

Bio-physical studies of the HIV gp120 C5 domain (residues 489–

511 of HIV-1 strain HXB2), which corresponds to the

carboxy terminal region of gp120, have been undertaken

CD studies of the C5 domain suggest that it is unstructured

in aqueous solutions but partially helical in trifluoroethanol/

aqueous and hexafluoroisopropanol/aqueous buffers The

solution structure of the C5 peptide in 40% trifluoroethanol/

aqueous buffer was determined by NMR spectroscopy The

resulting structure is a turn helix structural motif, consistent

with the CD results Fluorescence titration experiments

suggest that HIV C5 forms a 1 : 1 complex with the HIV gp41 ectodomain in the presence of cosolvent with an apparent Kdof
1.0 lM The absence of complex forma-tion in the absence of cosolvent indicates that formaforma-tion of the turn-helix structural motif of C5 is necessary for complex formation Examination of the C5 structure provides insight into the interaction between gp120 and gp41 and provides a possible target site for future drug therapies designed to disrupt the gp120/gp41 complex In addition, the C5 struc-ture lends insight into the site of HIV envelope protein maturation by the host enzymes furin and PC7, which provides other possible targets for drug therapies

Keywords: AIDS; gp41; gp120; HIV; NMR

Envelope proteins are known to play roles in directing viral

particles to the appropriate target cell and initiating the

fusion of the viral and cellular membranes in diverse viruses

including retroviridae, herpesviridae, filoviridaie and

para-myxoviridae [1] In the retrovirus HIV, which is the

causative agent of AIDS, the envelope proteins exist as a

noncovalent complex of a surface subunit (gp120) and a

transmembrane subunit (gp41), which are proteolytic

products of the env gene HIV infection, as well as SIV

(simian immunodeficiency virus) infection, is thought to

occur in three discreet steps [2] During the initial step, the

gp41/gp120 complex associates with the CD4 receptor In

the second step, gp120 associates with a chemokine receptor

that is specific to the cell type and dissociates from gp41 In

the final step, gp41 mediates fusion of the viral and the

cellular membranes by a poorly understood mechanism,

which is thought to involve the N-terminal hydrophobic

region of gp41, termed the fusion peptide gp120 is known

to be well exposed on the HIV surface and play an initial

and critical role in HIV infection Thus, gp120 is an

attractive target for vaccines and structure-based drugs

designed for the prevention and treatment of AIDS The

search for newAIDS therapies is of critical importance in

light of recent reports that current highly active

antiretro-viral treatments are ineffective in 20–50% of treated patients

[3] Moreover, therapies designed to disrupt gp120 function

are especially attractive because of their potential

prophy-lactic properties, as well as being alternative and

comple-mentary therapies to the protease and reverse transcriptase inhibitors currently utilized

In HIV, the precursor envelope protein, gp160, is proteo-lytically cleaved to form gp120 and gp41 by the host specific proteases furin and PC7 [4] Cleavage of the precursor viral envelope protein at the carboxyl side of the amino acid sequence REKR is critical to HIV function, as shown by mutagenesis studies [5] Moreover, inhibitors of furin and PC7 have been shown to block gp160 processing and HIV infection [6] The mature gp120 consists of 5 conserved regions (C1-C5) and five variable regions (V1–V5) Recently, the structure of HIV gp120 in complex with regions of the CD4 receptor and a monoclonal antibody that blocks gp120 binding to the chemokine receptor has been determined by X-ray crystallography [7] However, for technical reasons, large regions of the conserved and variable domains of gp120 are missing from the X-ray structure Mutagenesis and deletion studies have suggested that the C1 and C5 domains

of gp120, which are largely missing in the crystal structure but would be expected to be proximal to one another, are involved in the interaction with gp41 [8,9] Furthermore, it has recently been shown that the introduction of non-native intermolecular disulfide bonds between the gp41 loop region and the C1 or C5 domains of gp120 stabilizes the gp41/gp120 complex and mimics the antigenic properties of the native envelope complex [10] Taken together, the mutagenesis studies suggest that there is a direct interaction between the gp120 C1 and C5 domains and the gp41 loop region In an effort to further characterize the gp41/gp120 interaction, the solution structure of the C5 domain of HIV gp120 has been determined by NMR spectroscopy

E X P E R I M E N T A L P R O C E D U R E S The HIV gp120 C5 domain was prepared by solid phase peptide synthesis (Biological Resource Center, University of Illinois at Chicago) The peptide was modified by the

Correspondence to M Caffrey, Department of Biochemistry and

Molecular Biology, University of Illinois at Chicago, 1819 W Polk St.,

Chicago, IL 60612, USA.

Fax: + 1 312 413 0364, Tel.: + 1 312 996 4959.

E-mail: caffrey@uic.edu.

(Received 12 June 2002, revised 9 August 2002,

accepted 19 August 2002)

additions of acetyl and amido groups to the amino and

carboxy termini, respectively The identity of the peptide

was verified by mass spectrometry (mass¼ 2700.9) The

wild-type HIV gp41 ectodomain was prepared as previously

described [11] CD spectra were recorded on a Jasco J-710

spectropolarimeter with a 0.1-cm path length at 23C

Fluorescence titrations were performed with a PTI

Fluo-rescence System using a 0.1-cm path length at 25C Before

performing the titrations, the HIV gp41 ectodomain and

gp120 C5 were equilibrated in 50 mM sodium formate

(pH 3.0), 40% trifluoroethanol (v/v) Data were analyzed

withKALEIDAGRAPH3.08

For the NMR experiments, the conditions were 1 mM

C5, 50 mMPO4(pH 6.0), 40% trifluoroethanol-d3

(Cam-bridge Isotopes), 5% D2O NMR experiments were

per-formed on a Bruker DRX 600 equipped with a triple

resonance probe TOCSY and NOESY experiments were

acquired at 285, 290, 295, 300 and 305 K For the TOCSY

experiments the isotropic mixing time was 72 ms; for the

NOESY experiments, the mixing times were 75, 150, and

250 ms.13C-edited HSQC were acquired at 300 and 305 K

The HIV gp120 C5 structure was calculated with the

programCNS[12] The protocol consisted of four steps: (a)

high temperature torsion angle molecular dynamics

start-ing from an extended conformation; (b) slow-coolstart-ing

torsion angle molecular dynamics; (c) slow-cooling

Carte-sian dynamics; and (d) conjugate gradient minimization

For the first step, the temperature was set to 50 000 K for

2000 steps of 15 fs In this step, the force constants for NOE,

dihedral angles, and van der Waals forces were 150 kcalÆ

mol)1ÆA˚)2, 100 kcalÆmol)1Ærad)2 and 0.1 kcalÆmol)1ÆA˚)4,

respectively For the second step, the temperature was set

to 50 000 K and cooled to 0 K over 1000 steps of 15 fs In

this step, the force constants for NOE, dihedral angles, and

van der Waals forces were 150 kcalÆmol)1ÆA˚)2, 200 kcalÆ

mol)1Ærad)2 and 0.1–1.0 kcalÆmol)1ÆA˚)4, respectively For

the third step, the starting temperature was set to 2000 K

and cooled to 0 K in 3000 steps of 5 fs In this step, the force

constants for NOE, dihedral angles, and van der Waals

forces were 150 kcalÆmol)1ÆA˚)2, 200 kcalÆmol)1Ærad)2 and

1.0–4.0 kcalÆmol)1ÆA˚)4, respectively In the final step, 200

steps of conjugate gradient minimization were performed

In this step, the force constants for NOE, dihedral angles,

and van der Waals forces were 75 kcalÆmol)1ÆA˚)2, 400 kcalÆ

mol)1Ærad)2 and 1.0 kcalÆmol)1ÆA˚)4, respectively In all

steps, an energy term for a conformational database was

included with a force constant of 1.0 [13] The / and u

dihedral angles were set to the values derived from1H and

13C chemical shifts using the programTALOS[14] For the

structure calculation, errors of ± 30 were used for the

dihedral restraints NOEs were classified as strong

(<2.7 A˚), medium (<3.3 A˚ or <3.5 A˚ for amide protons),

weak (<5.0 A˚) and very w eak (<6.0 A˚) A correction

of 0.5 A˚ was added to proton distances involving

methyl groups The electrostatic map and structural

figures were generated by the molecular graphics program

MOLMOL[15]

R E S U L T S

To characterize the HIV gp120 C5 domain, a 23 residue

peptide was synthesized, corresponding to residues 489–511

of HIV-1 strain HXB2 (Fig 1A) The synthetic C5 peptide

was found to be soluble under all experimental conditions that were examined As shown in Fig 1B, the CD spectra of C5 in aqueous solution suggest the absence of regular secondary structure The absence of secondary structure was found to be independent of temperature, pH and salt concentration (data not shown) In contrast, the CD spectra

of C5 in trifluoroethanol/H2O or hexafluoroisopropanol/

H2O mixtures suggest the presence of helix In trifluoro-ethanol/H2O mixtures, the maximal amount of helical structure is observed in 40% trifluoroethanol (Fig 1B) A similar amount of helical structure is observed in 60% hexafluoroisopropanol (v/v, data not shown) In the presence of 40% trifluoroethanol, the molar ellipticity at

222 nm suggested that the helical content of C5 is
40% (i.e.
10 residues) Importantly, the use of trifluoroethanol

or hexafluoroisopropanol poses no technical difficulties for high-resolution NMR studies due to the availability of deuterated compounds

We found that the NMR spectra of HIV C5 in 40% trifluoroethanol were superior to those of C5 in 60% hexafluoroisopropanol Consequently, we chose to further characterize the 40% trifluoroethanol sample The 1H resonances of C5 in 40% trifluoroethanol were assigned by

a series of TOCSY and NOESY experiments at different temperatures (285–305 K) The13Caand13Cbwere assigned

by13C-edited HSQC experiments on a natural abundance sample Due to the small degree of spectral dispersion, it was critical to make assignments at various temperatures The NOESY spectra of C5 at lower temperatures are superior to those at higher temperatures, due to an increased correlation time (and thus an increased NOE) An example

of the NOESY spectrum at 285 K is given in Fig 2A The

Fig 1 (A) Amino acid sequence and (B) CD spectra of the HIVgp120 C5 peptide (A) Numbering corresponds to that of HIV-1 strain HXB2 [16] (B) Dotted lines represent the spectrum in aqueous solution (25 l M C5, 100 m M Tris/HCl/pH 8.0); solid lines represent the spec-trum in the presence of an organic solvent (25 l M C5, 40% trifluoro-ethanol, 100 m Tris/HCl/pH 8.0).

presence of sequential HN-HNcrosspeaks is characteristic of

helical regions Indeed, the crosspeak pattern shown in

Fig 2A suggests the presence of a continuous helix between

residues 499 and 509 The secondary structure was further

characterized by1Haand13Cachemical shifts As shown by

Fig 2B, the1Haand13Cachemical shifts of residues 499–

509 suggest the presence of a helix The prolines at positions

493 and 498 are apparently in the trans conformation, based

on the presence of strong a/d NOEs Moreover, the absence

of peak splitting in the TOCSY spectra of C5 indicates that

the trans isomer is the major isomer Interestingly, the HN

and Haline-widths of the region encompassing the prolines

and preceding the helix (residues 489–498) are similar to

those of the helix Accordingly, the relaxation properties of

this region indicate that it is not appreciably more mobile

than the helix

The tertiary structure of HIV gp120 C5 was determined

by an iterative method of torsion angle molecular

dynam-ics followed by simulated annealing [18] A summary of

the NOEs used in the calculation is presented in Fig 3

From Fig 3, it is apparent that there are numerous

long-range contacts between residues in the N- and C-terminal

regions of C5 (e.g V489 to V505 and I491 to V506) An

ensemble of 20 lowenergy structures is presented in

Fig 4A and the structural statistics of the ensemble are

summarized in Table 1 The overall backbone RMSD of

the ensemble to the mean is 0.53 A˚ and the overall heavy atom RMSD (i.e nonhydrogen) of the ensemble to the mean is 1.25 A˚ In Fig 4B, the backbone RMSD of the C5 ensemble is plotted as a function of residue number

to illustrate the relative uncertainties of the backbone Figure 5A shows the ribbon diagram of the energy minimized mean structure of HIV gp120 C5 The structural motif of C5 is that of a turn-helix with the amino and carboxy termini in close proximity As shown

by Fig 5A, the amino terminal end of the C5 domain would be attached to remaining domains of gp120 in the native structure The carboxy terminal end of the C5 domain would be attached to the amino terminus of gp41

in the unprocessed form (termed gp160) Note that the last four residues of the C5 domain form the furin/PC7 recognition site (amino acid sequence ¼ REKR) and processing at this site by furin liberates the gp41 amino terminus, which contains the fusion peptide In Fig 5B, the electrostatic map of C5 is presented Pertinent features include a relatively uncharged hydrophobic

elbow in the turn (residues 493–499, amino acid sequence ¼ PLGVAPT), the presence of two negative charges (E492 and E509), and the presence of numerous positive charges along the exterior of the helix (e.g K490, K500, R504, R508 and R511) Interestingly, the hydrophobic elbow is absolutely conserved among

Fig 2 (A) NOESY spectrum and (B) secondary chemical shifts of1H a and13C a of HIVgp120 C5 in 40% trifluoroethanol (A) Experimental conditions were 1 m M C5, 40% trifluoroethanol, 50 m M PO 4 (pH 6.0), 5% D 2 O at 285 K with a mixing time of 150 ms (B) Secondary chemical shifts of1H a (a) and13C a (b) of the HIV gp120 C5 in 40% trifluoroethanol Experimental conditions were 1 m M C5, 40% trifluoroethanol, 50 m M

PO 4 (pH 6.0) at 300 K Random coil values of a protein in trifluoroethanol were taken from Merutka et al [17].

Fig 3 (A) Summary of the number and type of NOEs observed for each residue and (B) NOE contact map for HIVgp120 C5 in 40% tri-fluoroethanol (A) The NOE classification is given in Table 1.

HIV species [16] Moreover, the charged residues at

positions 490, 500, 504, 508, 509, 510 and 511 are highly

conserved among HIV species [16]

The next logical step is to assay the ability of HIV C5 to

bind to HIV gp41 As discussed in the introduction, the loop

region of gp41 is the most likely binding site for C5 The

gp41 loop region contains four highly conserved tryptophan

residues (W85, W99, W103, and W112), which could

possibly serve as fluorescent reporters of a gp41/C5

interaction Importantly, the C5 domain employed in the

present study does not possess any tryptophan residues

Therefore, we performed fluorescence titrations of the HIV

gp41 ectodomain in the presence of trifluoroethanol as

cosolvent As shown by Fig 6A, the tryptophan

fluores-cence of HIV gp41 increases as HIV C5 is added The

increased tryptophan fluorescence at 340 nm suggests that

at least some of the gp41 tryptophan sidechains are being shielded from solvent by the addition of HIV C5 (Fig 6B) Based on the fluorescence changes shown by Fig 6A, HIV C5 forms a 1 : 1 complex with the HIV gp41 ectodomain with an apparent Kd of
1.0 lM Interestingly, titrations performed in the absence of cosolvent did not exhibit measurable changes in tryptophan fluorescence, which suggests that the turn-helix structural motif present of C5

is critical to complex formation

D I S C U S S I O N The CD results suggest that HIV gp120 C5 is in a random coil conformation in aqueous solutions, which is in agree-ment with previous studies of similar C5 peptides [20,21] In contrast the presence of helix is clearly indicated by CD

Fig 4 (A) Ensemble of 20 low-energy

struc-tures of HIVgp120 C5 in 40% trifluoroethanol

and (B) plot of the backbone RMSD of the final

C5 ensemble as a function of residue number.

(A) For clarity the amino and carboxy termini

have been denoted by their residue number.

(B) For this calculation, the backbone was

defined as the N, C a and C atoms.

Table 1 Structural statistics for the final 20 simulated annealing structures (ÆSAæ) and the minimized mean structure (ÆSAæ r ) None of the structures exhibited distance violations greater than 0.5 A˚ or dihedral violations greater than 6.

RMS deviations from dihedral restraints () (47) a

Measures of structure quality b

Coordinate precisionc

a

Torsion angle restraints consisted of 21 /, 21 w, 3 v 1 , and 2 v 2 bThe overall quality of the structure was assessed by the program

PROCHECK 3.5 [19].cDefined as average RMS difference between the final 20 simulated annealing structures and the mean coordinates.

when C5 is in the presence of the cosolvents trifluoroethanol

or hexafluoroisopropanol The high-resolution solution

structure of HIV gp120 C5 in 40% trifluoroethanol was

determined by NMR spectroscopy The presence of a

carboxy terminal helix (residues 499–510) was consistent

with the CD experiments, which predicted a helical content

of
10 residues We recognize that from a physiological

standpoint the structural and dynamic characterization of

C5 in the presence of a cosolvent is less desirable than under

strictly aqueous conditions However, it is increasingly

apparent that trifluoroethanol only stabilizes helical

struc-tures in protein domains with inherent propensity for helix

formation [22–24] Moreover, trifluoroethanol/aqueous

mixtures are thought to mimic the hydrophobic

environ-ment of protein interiors [22,25–28] In the present case, C5

in isolation is missing the other domains of gp120 as well as

its putative hydrophobic interaction site on gp41 [10,18,29]

Consequently, the trifluoroethanol/aqueous mixture may

very well mimic the hydrophobic environment of the gp120

C5 domain in vivo Interestingly, the presence of helix at residues 500–511 was predicted for HIV gp120 by various methods of secondary structure prediction [30] and thus the C5 propensity for helical structure is not surprising Importantly, we have shown by fluorescence titration that HIV C5 directly interacts with the HIV gp41 ectodomain in the presence of cosolvent, suggesting that the C5 structure presented herein is physiologically relevant

The HIV gp120 C5 structure provides insight into the functional properties of the C5 domain First, the C5 structure encompasses the site of envelope protein matur-ation Specifically, the furin/PC7 recognition site REKR is present at the C terminal helix of the present C5 construct (Fig 5) Mutations at this cleavage site inhibit fusion and hence HIV infection [5] and thus this site is a valid target for drug therapies against HIV However, it is important to note that the presence of helix in this site is unknown in the precursor envelope protein and further study is clearly warranted Second, the C5 structure provides insight into the structure and action of a peptide that displays antiviral activity against HIV-1 and HIV-2 [31] This peptide, which

is called CLIV, corresponds to four linked copies of residues 499–511 The peptide has been shown to disrupt the fusion process after binding of CD4, which indicates the CLIV peptide does not function by disrupting gp120 maturation [32] Third, the C5 structure can be used to interpret the results of mutagenesis studies of the gp120–gp41 interaction For example substitutions of C5 residues inhibit association

of gp120 with gp41 [8] Moreover, substitution of I491, P493, G495, A497, P498, T499 or A501 by cysteines resulted

in intermolecular crosslinks to non-native cysteines intro-duced into the gp41 loop region [10] Together the mutagenesis, structural studies, and fluorescence titration experiments [7,18,29,33] (and the present work) provide compelling evidence that the hydrophobic loop of gp41 interacts with the hydrophobic turn region of C5

The HIV gp120 C5 structure can be used to model the gp41/gp120 complex In Fig 7, we present a model of the gp41 and gp120 components, which were determined by

Fig 5 (A) Ribbon diagram of the minimized mean structure of HIV

gp120 C5 in 40% trifluoroethanol and (B) electrostatic map of the

minimized mean structure of HIVgp120 C5 (A) The location of the

furin/PC7 site is shown The locations of the missing gp120 domains

and gp41 are denoted (B) In this orientation, the conserved

hydro-phobic elbow of C5 is located at the upper left corner of the figure.

Fig 6 (A) Fluorescence titration of HIVgp41 ectodomain by HIVgp120 C5 and (B) location of tryptophan residues in HIVgp41 ectodomain (coordinates taken from reference [33]) (A) Experimental conditions were 20 l M HIV gp41 ectodomain in 50 m M sodium formate (pH 3.0), 40% trifluoroethanol at 25 C Titrations of a buffer blank by HIV C5 exhibited no change in the 340 nm fluorescence The solid line represents the least squares fit of the data w ith K d ¼ 1.0 ± 0.8 l M and n ¼ 0.98 ± 0.13 (B) The exposed tryptophans found in the gp41 loop are labeled The unlabeled tryptophans, which include W60, W117 and W120, are found in the protein interior and not exposed to solvent [18,33].

NMR spectroscopy and X-ray crystallography [7,18,33]

and the present work As discussed above, knowledge

of the gp41/gp120 structure is limited by the absence of

structural information for the C1 and C5 domains of

gp120, which are indicated to interact directly with gp41

Accordingly, we have modeled the complex by first

placing C5 in the context of the gp120 core structure

determined by X-ray crystallography [7] The first three

residues of the C5 domain used in the present study

(amino acids VKI) are observed in the gp120 X-ray

structure; thus, the C5 domain can be overlaid with the C

terminus of the gp120 X-ray structure (the RMSD of the

NMR VKI backbone to the X-ray VKI backbone is

0.4 A˚) The extended conformation of these residues in

solution is consistent with being part of a b sheet observed

in the gp120 X-ray structure (Fig 7) The location of C5

with respect to the gp120 core is also consistent with

immunological studies that suggest that the C5 domain

interacts directly with the C1 and C2 domains [34] In the

second step, we have used mutagenesis studies of gp41

and gp120 to reveal residues that are in close contact in

the gp41/gp120 complex As discussed above, a number of

non-native cysteines placed within the gp41 loop form

intermolecular crosslinks to non-native cysteines placed in

the C5 domain [10] Interestingly, the most reactive

non-native cysteines occur at position 94 of gp41 (denoted by

the blue sphere in Fig 7) and at position 501 of gp120

(denoted by the red sphere in Fig 7), which can be placed

in close proximity in the present model The C5/gp41

interaction depicted in Fig 7 w ould be expected to

partially shield W85, W99 and W103 of gp41 from

solvent (cf Figure 6B), which is consistent with the

fluorescence titration experiments discussed above Finally,

the placement of the viral and target cell membranes is

facilitated by the structures of gp41 and CD4 receptor

Specifically, gp41 contains a transmembrane domain that

is C-terminal to the ectodomain and thus located at the

left of Fig 7 The CD4 receptor contains a C-terminal

membrane anchor and thus is located at the right of

Fig 7 In addition, the location of the target cell

chemokine receptor can be inferred by the binding site

of the anitbody 17b, which blocks binding of gp120 to the chemokine receptor [7] Note that the model is consistent with immunological studies that indicate that residues 501–511 are well exposed and immunodominant in the native complex [35], consistent with its being charged and thus hydrophilic (Fig 5B) Furthermore, antibody acces-sibility studies have suggested that the amino terminal region of the gp120 C5 domain (residues 489–500) is not exposed in the gp120/gp41 complex but exposed in the monomeric form when gp120 dissociates from gp41 [36], consistent with the notion that the hydrophobic elbow of C5 (Fig 5B) interacts with the hydrophobic loop region

of gp41 [18,33] In total, the resulting model is consistent with a large number of biochemical and structural studies

It is important to note that the gp41 helical regions are generally thought to undergo large-scale structural chan-ges during the binding of gp120 to CD4 and the subsequent fusion event (cf [37] but see [18,38] for an alternative viewpoint) Accordingly, the known structures for gp41 [18,33] may not represent the gp41 conformation bound to gp120 However, we are unaware of any evidence for structural changes in the gp41 loop connect-ing the helical regions

In conclusion, we feel that the HIV gp120 C5 structure is relevant to biomedical research on AIDS in general and vaccine development in particular Firstly, a full understanding of gp120 function requires high-resolution structural and dynamic information for all regions of gp120 The present work presents the first high-resolution struc-tural information for C5, albeit in 40% trifluoroethanol Secondly, based on the immunological studies [35,36] and the model of the gp41/gp120 complex presented in Fig 7, the C5 domain is expected to be partially accessible to solvent and thus accessible to the immune system Conse-quently, the gp120 C5 domain, which is highly conserved among HIV and SIV isolates, may be an attractive target for vaccine development Finally, as discussed above, the C5 domain represents the most likely site for gp41 binding Disruption of this binding is expected to inhibit HIV infection and thus the C5 domain is an attractive target for structure-based drug therapies

Fig 7 Model for the gp41–gp120 complex

upon association with the target cell receptors.

The HIV gp41 ectodomain is shown in blue

and is a taken from the model structure [33],

which is based on the NMR structure of the

SIV gp41 ectodomain [18] The gp120 C5

structure is show n in red and is taken from the

present work The structure of the core

gp120-CD4 receptor-antibody 17b complex is taken

from Kwong et al [7] The gp120 core is

shown in green; the CD4 receptor is shown in

cyan, and the antibody 17b is shown in violet.

The sites of the most reactive non-native

cysteines in gp41 (at position 94) and gp120

(at position 501) are shown as spheres and

are taken from Binley et al [10].

A C K N O W L E D G E M E N T S

The coordinates of the minimized mean structure of the HIV gp120 C5

have been deposited in the Protein Data Bank (ID 1MEQ) This work

was supported by RO1 grant AI47674.

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