Structural and functional analysis of interhelical interactions in the human immunodeficiency virus type 1 gp41 envelope glycoprotein by alanine-scanning mutagenesis

Membrane fusion by human immunodeficiency virus type 1 (HIV-1) is promoted by the refolding of the viral envelope glycoprotein into a fusion-active conformation. The structure of the gp41 ectodomain core in its fusion-active state is a trimer of hairpins in which three antiparallel carboxyl-terminal helices pack into hydrophobic grooves on the surface of an amino-terminal trimeric coiled coil. In an effort to identify amino acid residues in these grooves that are critical for gp41 activation, we have used alanine-scanning mutagenesis to investigate the importance of individual side chains in determining the biophysical properties of the gp41 core and the membrane fusion activity of the gp120-gp41 complex. Alanine substitutions at Leu-556, Leu-565, Val-570, Gly-572, and Arg-579 positions severely impaired membrane fusion activity in envelope glycoproteins that were for the most part normally expressed. Whereas alanine mutations at Leu-565 and Val-570 destabilized the trimer-of-hairpins structure, mutations at Gly-572 and Arg-579 led to the formation of a stable gp41 core. Our results suggest that the Leu-565 and Val-570 residues are important determinants of conserved packing interactions between the amino- and carboxyl-terminal helices of gp41. We propose that the high degree of sequence conservation at Gly-572 and Arg-579 may result from selective pressures imposed by prefusogenic conformations of the HIV-1 envelope glycoprotein. Further analysis of the gp41 activation process may elucidate targets for antiviral intervention.     

Mutations that destabilize the gp41 core are determinants for stabilizing the simian immunodeficiency virus-CPmac envelope glycoprotein complex

The human and simian immunodeficiency viruses (HIV and SIV) envelope glycoprotein consists of a trimer of two noncovalently and weakly associated subunits, gp120 and gp41. Upon binding of gp120 to cellular receptors, this labile native envelope complex undergoes conformational changes, resulting in a stable trimer-of-hairpins structure in gp41. Formation of the hairpin structure is thought to mediate membrane fusion by placing the viral and cellular membranes in close proximity. An in vitro-derived variant of SIVmac251, denoted CPmac, has acquired an unusually stable virion-associated gp120-gp41 complex. This unique phenotype is conferred by five amino acid substitutions in the gp41 ectodomain. Here we characterize the structural and physicochemical properties of the N40(L6)C38 model of the CPmac gp41 core. The 1.7-A resolution crystal structure of N40(L6)C38 is very similar to the six-helix bundle structure present in the parent SIVmac251 gp41. In both structures, three N40 peptides form a central three-stranded coiled coil, and three C38 peptides pack in an antiparallel orientation into hydrophobic grooves on the coiled-coil surface. Thermal unfolding studies show that the CPmac mutations destabilize the SIVmac251 six-helix bundle by 15 kJ/mol. Our results suggest that the formation of the gp41 trimer-of-hairpins structure is thermodynamically coupled to the conformational stability of the native envelope glycoprotein and raise the intriguing possibility that introduction of mutations to destabilize the six-helix bundle may lead to the stabilization of the trimeric gp120-gp41 complex. This study suggests a potential strategy for the production of stably folded envelope protein immunogens for HIV vaccine development.     

Structural and functional analysis of the HIV gp41 core containing an Ile573 to Thr substitution: implications for membrane fusion

The envelope glycoprotein of HIV-1 consists of the surface subunit gp120 and the transmembrane subunit gp41. Binding of gp120 to target cell receptors induces a conformational change in gp41, which then mediates the fusion of viral and cellular membranes. A buried isoleucine (Ile573) in a central trimeric coiled coil within the fusion-active gp41 ectodomain core is thought to favor this conformational activation. The role of Ile573 in determining the structure and function of the gp120-gp41 complex was investigated by mutating this residue to threonine, a nonconservative substitution in HIV-1 that occurs naturally in SIV. While the introduction of Thr573 markedly destabilized the gp41 core, the three-dimensional structure of the mutant trimer of hairpins was very similar to that of the wild-type molecule. A new hydrogen-bonding interaction between the buried Thr573 and Thr569 residues appears to allow formation of the trimer-of-hairpins structure at physiological temperature. The mutant envelope glycoprotein expressed in 293T cells and incorporated within pseudotyped virions displayed only a moderate reduction in syncytium-inducing capacity and virus infectivity, respectively. Our results demonstrate that the proper folding of the gp41 core underlies the membrane fusion properties of the gp120-gp41 complex. An understanding of the gp41 activation process may suggest novel strategies for vaccine and antiviral drug development.     

The fusion activity of HIV-1 gp41 depends on interhelical interactions

Infection by human immunodeficiency virus type I requires the fusogenic activity of gp41, the transmembrane subunit of the viral envelope protein. Crystallographic studies have revealed that fusion-active gp41 is a "trimer-of-hairpins" in which three central N-terminal helices form a trimeric coiled coil surrounded by three antiparallel C-terminal helices. This structure is stabilized primarily by hydrophobic, interhelical interactions, and several critical contacts are made between residues that form a deep cavity in the N-terminal trimer and the C-helix residues that pack into this cavity. In addition, the trimer-of-hairpins structure has an extensive network of hydrogen bonds within a conserved glutamine-rich layer of poorly understood function. Formation of the trimer-of-hairpins structure is thought to directly force the viral and target membranes together, resulting in membrane fusion and viral entry. We test this hypothesis by constructing four series of gp41 mutants with disrupted interactions between the N- and C-helices. Notably, in the three series containing mutations within the cavity, gp41 activity correlates well with the stability of the N-C interhelical interaction. In contrast, a fourth series of mutants involving the glutamine layer residue Gln-653 show fusion defects even though the stability of the hairpin is close to wild-type. These results provide evidence that gp41 hairpin stability is critical for mediating fusion and suggest a novel role for the glutamine layer in gp41 function.     

Helical interactions in the HIV-1 gp41 core reveal structural basis for the inhibitory activity of gp41 peptides

The HIV-1 gp41 envelope protein mediates membrane fusion that leads to virus entry into the cell. The core structure of fusion-active gp41 is a six-helix bundle in which an N-terminal three-stranded coiled coil is surrounded by a sheath of antiparallel C-terminal helices. A conserved glutamine (Gln 652) buried in this helical interface replaced by leucine increases HIV-1 infectivity. To define the basis for this enhanced membrane fusion activity, we investigate the role of the Gln 652 to Leu substitution on the conformation, stability, and biological activity of the N34(L6)C28 model of the gp41 ectodomain core. The 2.0 A resolution crystal structure of the mutant molecule shows that the Leu 652 side chains make prominent contacts with hydrophobic grooves on the surface of the central coiled coil. The Gln 652 to Leu mutation leads to a marginal stabilization of the six-helix bundle by -0.8 kcal/mol, evaluated from thermal unfolding experiments. Strikingly, the mutant N34(L6)C28 peptide is a potent inhibitor of HIV-1 infection, with 10-fold greater activity than the wild-type molecule. This inhibitory potency can be traced to the corresponding C-terminal mutant peptide that likely has greater potential to interact with the coiled-coil trimer. These results provide strong evidence that conserved interhelical packing interactions in the gp41 core are important determinants of HIV-1 entry and its inhibition. These interactions also offer a test-bed for the development of more potent analogues of gp41 peptide inhibitors.     

Interactions between HIV-1 gp41 core and detergents and their implications for membrane fusion

The gp41 envelope protein mediates entry of human immunodeficiency virus type 1 (HIV-1) into the cell by promoting membrane fusion. The crystal structure of a gp41 ectodomain core in its fusion-active state is a six-helix bundle in which a N-terminal trimeric coiled coil is surrounded by three C-terminal outer helices in an antiparallel orientation. Here we demonstrate that the N34(L6)C28 model of the gp41 core is stabilized by interaction with the ionic detergent sodium dodecyl sulfate (SDS) or the nonionic detergent n-octyl-beta-D-glucopyranoside (betaOG). The high resolution x-ray structures of N34(L6)C28 crystallized from two different detergent micellar media reveal a six-helix bundle conformation very similar to that of the molecule in water. Moreover, N34(L6)C28 adopts a highly alpha-helical conformation in lipid vesicles. Taken together, these results suggest that the six-helix bundle of the gp41 core displays substantial affinity for lipid bilayers rather than unfolding in the membrane environment. This characteristic may be important for formation of the fusion-active gp41 core structure and close apposition of the viral and cellular membranes for fusion.     

Design of a novel peptide inhibitor of HIV fusion that disrupts the internal trimeric coiled-coil of gp41

The pre-hairpin intermediate of gp41 from the human immunodeficiency virus (HIV) is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. Using rational design, two 36-residue peptides, N36(Mut(e,g)) and N36(Mut(a,d)), were derived from the parent N36 peptide comprising the N-terminal helix of the gp41 ectodomain (residues 546-581 of HIV-1 envelope), characterized by analytical ultracentrifugation and CD, and assessed for their ability to inhibit HIV fusion using a quantitative vaccinia virus-based fusion assay. N36(Mut(e,g)) contains nine amino acid substitutions designed to disrupt interactions with the C-terminal region of gp41 while preserving contacts governing the formation of the trimeric coiled-coil. N36(Mut(a,d)) contains nine substitutions designed to block formation of the trimeric coiled-coil but retains residues that interact with the C-terminal region of gp41. N36(Mut(a,d)) is monomeric, is largely random coil, does not interact with the C34 peptide derived from the C-terminal region of gp41 (residues 628-661), and does not inhibit fusion. The trimeric coiled-coil structure is therefore a prerequisite for interaction with the C-terminal region of gp41. N36(Mut(e,g)) forms a monodisperse, helical trimer in solution, does not interact with C34, and yet inhibits fusion about 50-fold more effectively than the parent N36 peptide (IC(50) approximately 308 nm versus approximately 16 microm). These results indicate that N36(Mut(e,g)) acts by disrupting the homotrimeric coiled-coil of N-terminal helices in the pre-hairpin intermediate to form heterotrimers. Thus N36(Mut(e,g)) represents a novel third class of gp41-targeted HIV fusion inhibitor. A quantitative model describing the interaction of N36(Mut(e,g)) with the pre-hairpin intermediate is presented.     

Mutational Analysis of Residues in the Coiled-Coil Domain of Human Immunodeficiency Virus Type 1 Transmembrane Protein gp41

The envelope glycoprotein (Env) of human immunodeficiency virus mediates virus entry into cells by undergoing conformational changes that lead to fusion between viral and cellular membranes. A six-helix bundle in gp41, consisting of an interior trimeric coiled-coil core with three exterior helices packed in the grooves (core structure), has been proposed to be part of a fusion-active structure of Env (D. C. Chan, D. Fass, J. M. Berger, and P. S. Kim, Cell 89:263–273, 1997; W. Weissenhorn, A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley, Nature 387:426–430, 1997; and K. Tan, J. Liu, J. Wang, S. Shen, and M. Lu, Proc. Natl. Acad. Sci. USA 94:12303, 1997). We analyzed the effects of amino acid substitutions of arginine or glutamic acid in residues in the coiled-coil (heptad repeat) domain that line the interface between the helices in the gp41 core structure. We found that mutations of leucine to arginine or glutamic acid in position 556 and of alanine to arginine in position 558 resulted in undetectable levels of Env expression. Seven other mutations in six positions completely abolished fusion activity despite incorporation of the mutant Env into virions and normal gp160 processing. Single-residue substitutions of glutamic acid at position 570 or 577 resulted in the only viable mutants among the 16 mutants studied, although both viable mutants exhibited impaired fusion activity compared to that of the wild type. The glutamic acid 577 mutant was more sensitive than the wild type to inhibition by a gp41 coiled-coil peptide (DP-107) but not to that by another peptide corresponding to the C helix in the gp41 core structure (DP-178). These results provide insight into the gp41 fusion mechanism and suggest that the DP-107 peptide may inhibit fusion by binding to the homologous region in gp41, probably by forming a peptide-gp41 coiled-coil structure.     

A fluorescence assay for rapid detection of ligand binding affinity to HIV-1 gp41

The fusion-active conformation of the envelope protein gp41 of HIV-1 consists of an N-terminal trimeric alpha-helical coiled-coil domain and three anti-parallel C-terminal helices that fold down the grooves of the coiled-coil to form a six-helix bundle. Disruption of the six-helix bundle is considered to be a key component of an effective non-peptide fusion inhibitor. In the current study, a fluorescence resonance energy transfer (FRET) experiment for the detection of inhibitor binding to the gp41 N-peptide coiled-coil of HIV-1 was performed, utilizing peptide inhibitors derived from the gp41 C-terminal helical region. The FRET acceptor is a 31-residue N-peptide containing a known deep hydrophobic pocket, stabilized into a trimer by ferrous ion ligation. The FRET donor is a 16-18-residue fluorophore-labeled C-peptide, designed to test the specificity of the N-C interaction. Low microM dissociation constants were observed, correlated to the correct sequence and helical propensity of the C-peptides. Competitive inhibition was demonstrated using the assay, allowing for rank ordering of peptide inhibitors according to their affinity in the 1-20 microM range. The assay was conducted by measuring fluorescence intensity in 384-well plates. The rapid detection of inhibitor binding may permit identification of novel drug classes from a library.     

Design and properties of N(CCG)-gp41, a chimeric gp41 molecule with nanomolar HIV fusion inhibitory activity.

The design and characterization of a chimeric protein, termed N(CCG)-gp41, derived from the ectodomain of human immunodeficiency virus (HIV), type I gp41 is described. N(CCG)-gp41 features an exposed trimeric coiled-coil comprising the N-terminal helices of the gp41 ectodomain. The trimeric coiled-coil is stabilized both by fusion to a minimal thermostable ectodomain of gp41 and by engineered intersubunit disulfide bonds. N(CCG)-gp41 is shown to inhibit HIV envelope-mediated cell fusion at nanomolar concentrations with an IC(50) of 16.1 +/- 2.8 nm. It is proposed that N(CCG)-gp41 targets the exposed C-terminal region of the gp41 ectodomain in its pre-hairpin intermediate state, thereby preventing the formation of the fusogenic form of the gp41 ectodomain, which comprises a highly stable trimer of hairpins arranged in a six-helix bundle. N(CCG)-gp41 has potential as a therapeutic agent for the direct inhibition of HIV cell entry, as an anti-HIV vaccine, and as a component of a rapid throughput assay for screening for small molecule inhibitors of HIV envelope-mediated cell fusion. It is anticipated that antibodies raised against N(CCG)-gp41 may target the trimeric coiled-coil of N-terminal helices of the gp41 ectodomain that is exposed in the pre-hairpin intermediate state in a manner analogous to peptides derived from the C-terminal helix of gp41 that are currently in clinical trials.     

Thermodynamics of peptide inhibitor binding to HIV-1 gp41

The gp41 subunit of the human immunodeficiency virus type 1 envelope glycoprotein mediates fusion of the cellular and viral membranes. The gp41 ectodomain is a trimer of alpha-helical hairpins, where N-terminal helices form a parallel three-stranded coiled-coil core and C-terminal helices pack around the core. A deep hydrophobic pocket on the N-terminal core represents an attractive target for antiviral therapeutics. We have employed a soluble derivative of the gp41 core ectodomain and small cyclic disulfide D-peptide inhibitors to define the stoichiometry, affinity, and thermodynamics of ligand binding to this pocket using isothermal titration calorimetry. These inhibitors bind with micromolar affinity to the pocket with the expected stoichiometry of three peptides per gp41 core trimer. There are no cooperative interactions among the three binding sites. Linear eight- or nine-residue D-peptides derived from the pocket-binding domain of the cyclic molecules also bind specifically. A negative heat capacity change is observed and is consistent with burial of hydrophobic surface upon binding. Contrary to expectations for a reaction dominated by the classical hydrophobic effect, peptide binding is enthalpically driven and is opposed by an unfavorable negative entropy change. The calorimetry data support models whereby dominant negative inhibitors bind to a transiently exposed surface on the prefusion intermediate state of gp41 and disrupt subsequent resolution to the fusion-active six-stranded hairpin conformation.     

Identification of a critical motif for the human immunodeficiency virus type 1 (HIV-1) gp41 core structure: implications for designing novel anti-HIV fusion inhibitors

Human immunodeficiency virus type 1 (HIV-1) entry into the host cell involves a cascade of events and currently represents one of most attractive targets in the search for new antiviral drugs. The fusion-active gp41 core structure is a stable six-helix bundle (6-HB) folded by its trimeric N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR). Peptides derived from the CHR region of HIV-1 gp41 are potent fusion inhibitors that target the NHR to block viral and cellular membrane fusion in a dominant negative fashion. However, all CHR peptides reported to date are derived primarily from residues 628 to 673 of gp41; little attention has been paid to the upstream sequence of the pocket binding domain (PBD) in the CHR. Here, we have identified a motif ((621)QIWNNMT(627)) located at the upstream region of the gp41 CHR, immediately adjacent to the PBD ((628)WMEWEREI(635)). Biophysical characterization demonstrated that this motif is critical for the stabilization of the gp41 6-HB core. The peptide CP621-652, containing the (621)QIWNNMT(627) motif, was able to interact with T21, a counterpart peptide derived from the NHR, to form a typical 6-HB structure with a high thermostability (thermal unfolding transition [T(m)] value of 82 degrees C). In contrast, the 6-HB formed by the peptides N36 and C34, which has been considered to be a core structure of the fusion-active gp41, had a T(m) of 64 degrees C. Different from T-20 (brand name Fuseon), which is the first and only HIV-1 fusion inhibitor approved for clinical use, CP621-652 could efficiently block 6-HB formation in a dose-dependent manner. Significantly, CP621-652 had potent inhibitory activity against HIV-1-mediated cell-cell fusion and infection, especially against T-20- and C34-resistant virus. Therefore, our works provide important information for understanding the core structure of the fusion-active gp41 and for designing novel anti-HIV peptides.     

Selection of gp41-mediated HIV-1 cell entry inhibitors from biased combinatorial libraries of non-natural binding elements.

The trimeric, alpha-helical coiled-coil core of the HIV-1 gp41 ectodomain is thought to be part of a transient, receptor-triggered intermediate in the refolding of the envelope glycoprotein into a fusion-active conformation. In an effort to discover small organic inhibitors that block gp41 activation, we have generated a biased combinatorial chemical library of non-natural binding elements targeted to the gp41 core. From this library of 61,275 potential ligands, we have identified elements that, when covalently attached to a peptide derived from the gp41 outer-layer alpha-helix, contribute to the formation of a stable complex with the inner core and to inhibition of gp41-mediated cell fusion.     

Buried polar interactions and conformational stability in the simian immunodeficiency virus (SIV) gp41 core

For human (HIV) and simian (SIV) immunodeficiency viruses, the gp41 envelope protein undergoes a receptor-activated conformational change from a labile native structure to an energetically more stable fusogenic conformation, which then mediates viral-cell membrane fusion. The core structure of fusion-active gp41 is a six-helix bundle in which three antiparallel carboxyl-terminal helices are packed against an amino-terminal trimeric coiled coil. Here we show that a recombinant model of the SIV gp41 core, designated N36(L6)C34, forms an alpha-helical trimer that exhibits a cooperative two-state folding-unfolding transition. We investigate the importance of buried polar interactions in determining the overall fold of the gp41 core. We have replaced each of four polar amino acids at the heptad a and d positions of the coiled coil in N36(L6)C34 with a representative hydrophobic amino acid, isoleucine. The Q565I, T582I, and T586I variants form six-helix bundle structures that are significantly more stable than that of the wild-type peptide, whereas the Q575I variant misfolds into an insoluble aggregate under physiological conditions. Thus, the buried polar residues within the amino-terminal heptad repeat are important determinants of the structural specificity and stability of the gp41 core. We suggest that these conserved buried polar interactions play a role in governing the conformational state of the gp41 molecule.     

Fast folding of the HIV-1 and SIV gp41 six-helix bundles

Human (HIV-1) and simian (SIV) immunodeficiency virus fusion with the host cell is promoted by the receptor-triggered refolding of the gp41 envelope protein into a stable trimer-of-hairpins structure that brings viral and cellular membranes into close proximity. The core of this hairpin structure is a six-helix bundle in which an inner homotrimeric coiled coil is buttressed by three antiparallel outer HR2 helices. We have used stopped-flow circular dichroism spectroscopy to characterize the unfolding and refolding kinetics of the six-helix bundle using the HIV-1 and SIV N34(L6)C28 polypeptides. In each case, the time-course of ellipticity changes in refolding experiments is well described by a simple two-state model involving the native trimer and the unfolded monomers. The unfolding free energy of the HIV-1 and SIV trimers and their urea dependence calculated from kinetic data are in very good agreement with data measured directly by isothermal unfolding experiments. Thus, formation of the gp41 six-helix bundle structure involves no detectable population of stable, partly folded intermediates. Folding of HIV-1 N34(L6)C28 is five orders of magnitudes faster than folding of its SIV counterpart in aqueous buffer: k(on),(HIV-1)=1.3 x 10(15)M(-2)s(-1) versus k(on),(SIV)=1.1 x 10(10)M(-2)s(-1). The unfolding rates are similar: k(off),(HIV-1)=1.1 x 10(-5)s(-1) versus k(off),(SIV=)5.7 x 10(-4)s(-1). Kinetic m-values indicate that the transition state for folding of the HIV-1 protein is significantly more compact than the transition state of the SIV protein. Replacement of a single SIV threonine by isoleucine corresponding to position 573 in the HIV-1 sequence significantly stabilizes the protein and renders the folding rate close to that of the HIV-1 protein yet without making the transition state of the mutant as compact as that of the HIV-1 protein. Therefore, the overall reduction of surface exposure in the high-energy transition state seems not to account for different folding rates. While the available biological evidence suggests that refolding of the gp41 protein is slow, our study implies that structural elements outside the trimer-of-hairpins limit the rate of HIV-1 fusion kinetics.     

A Conformation-Specific Monoclonal Antibody Reacting with Fusion-Active gp41 from the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein plays a major role in the membrane fusion step of viral infection. The ectodomain of gp41 contains a six-helix structural domain that likely represents the core of the fusion-active conformation of the molecule. A monoclonal antibody (MAb), designated NC-1, was generated and cloned from a mouse immunized with the model polypeptide N36(L6)C34, which folds into a stable six-helix bundle. NC-1 binds specifically to both the α-helical core domain and the oligomeric forms of gp41. This conformation-dependent reactivity is dramatically reduced by point mutations within the N-terminal coiled-coil region of gp41 which impede formation of the gp41 core. NC-1 binds to the surfaces of HIV-1-infected cells only in the presence of soluble CD4. These results indicate that NC-1 is capable of reacting with fusion-active gp41 in a conformation-specific manner and can be used as a valuable biological reagent for studying the receptor-induced conformational changes in gp41 required for membrane fusion and HIV-1 infection.     

Genetic evidence that interhelical packing interactions in the gp41 core are critical for transition of the human immunodeficiency virus type 1 envelope glycoprotein to the fusion-active state

The envelope glycoprotein complex (gp120-gp41) of human immunodeficiency virus type 1 (HIV-1) promotes the fusion of viral and cellular membranes through formation of the fusion-active six-helix bundle in the gp41 ectodomain. This gp41 core structure consists of three C-terminal helices packed in an antiparallel manner into hydrophobic grooves on the surface of the N-terminal trimeric coiled coil. Alanine mutations that destabilize the N- and C-terminal interhelical packing interactions also reduce viral infectivity. Here we show that viruses bearing these mutations exhibit a marked potentiation of inhibition by peptides that make up the gp41 core. By contrast, these viruses are unchanged in their sensitivities to soluble CD4, the CXCR4 coreceptor ligand SDF-1alpha, and human anti-HIV immunoglobulin, reagents that impact the initial, receptor-induced conformational changes in the envelope glycoprotein. Our results support the notion that these alanine mutations specifically affect the conformational transition to the fusion-active gp41 structure. The mutations also increase viral sensitivity to the gp41-directed monoclonal antibody 2F5, suggesting that this broadly neutralizing antibody may also interfere with this transition. The conformational activation of the HIV-1 envelope glycoprotein likely represents a viable target for vaccine and antiviral drug development.     

Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41.

The solution structure of the ectodomain of simian immunodeficiency virus (SIV) gp41 (e-gp41), consisting of residues 27-149, has been determined by multidimensional heteronuclear NMR spectroscopy. SIV e-gp41 is a symmetric 44 kDa trimer with each subunit consisting of antiparallel N-terminal (residues 30-80) and C-terminal (residues 107-147) helices connected by a 26 residue loop (residues 81-106). The N-terminal helices of each subunit form a parallel coiled-coil structure in the interior of the complex which is surrounded by the C-terminal helices located on the exterior of the complex. The loop region is ordered and displays numerous intermolecular and non-sequential intramolecular contacts. The helical core of SIV e-gp41 is similar to recent X-ray structures of truncated constructs of the helical core of HIV-1 e-gp41. The present structure establishes unambiguously the connectivity of the N- and C-terminal helices in the trimer, and characterizes the conformation of the intervening loop, which has been implicated by mutagenesis and antibody epitope mapping to play a key role in gp120 association. In conjunction with previous studies, the solution structure of the SIV e-gp41 ectodomain provides insight into the binding site of gp120 and the mechanism of cell fusion. The present structure of SIV e-gp41 represents one of the largest protein structures determined by NMR to date.     

A monoclonal Fab derived from a human nonimmune phage library reveals a new epitope on gp41 and neutralizes diverse human immunodeficiency virus type 1 strains

A monoclonal Fab (Fab 3674) selected from a human nonimmune phage library by panning against the chimeric construct N_CCG-gp41 (which comprises an exposed coiled-coil trimer of gp41 N helices fused in the helical phase onto the minimal thermostable ectodomain of gp41) is described. Fab 3674 is shown to neutralize diverse laboratory-adapted B strains of human immunodeficiency virus type 1 (HIV-1) and primary isolates of subtypes A, B, and C in an Env-pseudotyped-virus neutralization assay, albeit with reduced potency (approximately 25-fold) compared to that of 2F5 and 4E10. Alanine scanning mutagenesis maps a novel epitope to a shallow groove on the N helices of gp41 that is exposed between two C helices in the fusogenic six-helix bundle conformation of gp41. Bivalent Fab 3674 and the C34 peptide (a potent fusion inhibitor derived from the C helix of gp41) are shown to act at similar stages of the fusion reaction and to neutralize HIV-1 synergistically, providing additional evidence that the epitope of Fab 3674 is new and distinct from the binding site of C34.     

Interhelical interactions in the gp41 core: implications for activation of HIV-1 membrane fusion

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein complex (gp120-gp41) promotes viral entry by mediating the fusion of viral and cellular membranes. Formation of a stable trimer-of-hairpins structure in the gp41 ectodomain brings the two membranes into proximity, leading to membrane fusion. The core of this hairpin structure is a six-helix bundle in which three carboxyl-terminal outer helices pack against an inner trimeric coiled coil. Here we investigate the role of these conserved interhelical interactions on the structure and function of both the envelope glycoprotein and the gp41 core. We have replaced each of the eight amino acids at the buried face of the carboxyl-terminal helix with a representative amino acid, alanine. Structural and physicochemical characterization of the alanine mutants shows that hydrophobic interactions are a dominant factor in the stabilization of the six-helix bundle. Alanine substitutions at the Trp628, Trp631, Ile635, and Ile642 residues also affected envelope processing and/or gp120-gp41 association and abrogated the ability of the envelope glycoprotein to mediate cell-cell fusion. These results suggest that the amino-terminal region of the gp41 outer-layer alpha-helix plays a key role in the sequence of events associated with HIV-1 entry and have implications for the development of antibodies and small-molecule inhibitors of this conserved element.