Structures of brain-derived 42-residue amyloid-ß fibril polymorphs with unusual molecular conformations and intermolecular interactions.

Fibrils formed by the 42-residue amyloid-ß peptide (Aß42), a main component of amyloid deposits in Alzheimer's disease (AD), are known to be polymorphic, i.e., to contain multiple possible molecular structures. Previous studies of Aß42 fibrils, including fibrils prepared entirely in vitro or extracted from brain tissue and using solid-state NMR (ssNMR) or cryogenic electron microscopy (cryo-EM) methods, have found polymorphs with differences in amino acid sidechain orientations, lengths of structurally ordered segments, and contacts between cross-ß subunit pairs within a single filament. Despite these differences, Aß42 molecules adopt a common S-shaped conformation in all previously described high-resolution Aß42 fibril structures. Here we report two cryo-EM-based structures of Aß42 fibrils that are qualitatively different, in samples derived from AD brain tissue by seeded growth. In type A fibrils, residues 12 to 42 adopt a ν-shaped conformation, with both intra-subunit and intersubunit hydrophobic contacts to form a compact core. In type B fibrils, residues 2 to 42 adopt an υ-shaped conformation, with only intersubunit contacts and internal pores. Type A and type B fibrils have opposite helical handedness. Cryo-EM density maps and molecular dynamics simulations indicate intersubunit K16-A42 salt bridges in type B fibrils and partially occupied K28-A42 salt bridges in type A fibrils. The coexistence of two predominant polymorphs, with differences in N-terminal dynamics, is supported by ssNMR data, as is faithful propagation of structures from first-generation to second-generation brain-seeded Aß42 fibril samples. These results demonstrate that Aß42 fibrils can exhibit a greater range of structural variations than seen in previous studies.

A weakened interface in the P182L variant of HSP27 associated with severe Charcot-Marie-Tooth neuropathy causes aberrant binding to interacting proteins.

HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot-Marie-Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C-terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient-derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V-containing proteins. We identified multiple IxI/V-bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V-binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein-protein interactions.

Contribution of the catalytic dyad of SARS-CoV-2 main protease to binding covalent and noncovalent inhibitors.

The effect of mutations of the catalytic dyad residues of SARS-CoV-2 main protease (MProWT) on the thermodynamics of binding of covalent inhibitors comprising nitrile [nirmatrelvir (NMV), NBH2], aldehyde (GC373), and ketone (BBH1) warheads to MPro is examined together with room temperature X-ray crystallography. When lacking the nucleophilic C145, NMV binding is ∼400-fold weaker corresponding to 3.5 kcal/mol and 13.3 °C decrease in free energy (ΔG) and thermal stability (Tm), respectively, relative to MProWT. The H41A mutation results in a 20-fold increase in the dissociation constant (Kd), and 1.7 kcal/mol and 1.4 °C decreases in ΔG and Tm, respectively. Increasing the pH from 7.2 to 8.2 enhances NMV binding to MProH41A, whereas no significant change is observed in binding to MProWT. Structures of the four inhibitor complexes with MPro1-304/C145A show that the active site geometries of the complexes are nearly identical to that of MProWT with the nucleophilic sulfur of C145 positioned to react with the nitrile or the carbonyl carbon. These results support a two-step mechanism for the formation of the covalent complex involving an initial non-covalent binding followed by a nucleophilic attack by the thiolate anion of C145 on the warhead carbon. Noncovalent inhibitor ensitrelvir (ESV) exhibits a binding affinity to MProWT that is similar to NMV but differs in its thermodynamic signature from NMV. The binding of ESV to MProC145A also results in a significant, but smaller, increase in Kd and decrease in ΔG and Tm, relative to NMV.

Effects of an HIV-1 maturation inhibitor on the structure and dynamics of CA-SP1 junction helices in virus-like particles.

HIV-1 maturation involves conversion of the immature Gag polyprotein lattice, which lines the inner surface of the viral membrane, to the mature capsid protein (CA) lattice, which encloses the viral RNA. Maturation inhibitors such as bevirimat (BVM) bind within six-helix bundles, formed by a segment that spans the junction between the CA and spacer peptide 1 (SP1) subunits of Gag, and interfere with cleavage between CA and SP1 catalyzed by the HIV-1 protease (PR). We report solid-state NMR (ssNMR) measurements on spherical virus-like particles (VLPs), facilitated by segmental isotopic labeling, that provide information about effects of BVM on the structure and dynamics of CA-SP1 junction helices in the immature lattice. Although BVM strongly blocks PR-catalyzed CA-SP1 cleavage in VLPs and blocks conversion of VLPs to tubular CA assemblies, 15N and 13C ssNMR chemical shifts of segmentally labeled VLPs with and without BVM are very similar, indicating that interaction with BVM does not alter the six-helix bundle structure appreciably. Only the 15N chemical shift of A280 (the first residue of SP1) changes significantly, consistent with BVM binding to an internal ring of hydrophobic side chains of L279 residues. Measurements of transverse 15N spin relaxation rates reveal a reduction in the amplitudes and/or timescales of backbone N-H bond motions, corresponding to a rigidification of the six-helix bundles. Overall, our data show that inhibition of HIV-1 maturation by BVM involves changes in structure and dynamics that are surprisingly subtle, but still sufficient to produce a large effect on CA-SP1 cleavage.

Allosteric control of hemoglobin S fiber formation by oxygen and its relation to the pathophysiology of sickle cell disease.

The pathology of sickle cell disease is caused by polymerization of the abnormal hemoglobin S upon deoxygenation in the tissues to form fibers in red cells, causing them to deform and occlude the circulation. Drugs that allosterically shift the quaternary equilibrium from the polymerizing T quaternary structure to the nonpolymerizing R quaternary structure are now being developed. Here we update our understanding on the allosteric control of fiber formation at equilibrium by showing how the simplest extension of the classic quaternary two-state allosteric model of Monod, Wyman, and Changeux to include tertiary conformational changes provides a better quantitative description. We also show that if fiber formation is at equilibrium in vivo, the vast majority of cells in most tissues would contain fibers, indicating that it is unlikely that the disease would be survivable once the nonpolymerizing fetal hemoglobin has been replaced by adult hemoglobin S at about 1 y after birth. Calculations of sickling times, based on a recently discovered universal relation between the delay time prior to fiber formation and supersaturation, show that in vivo fiber formation is very far from equilibrium. Our analysis indicates that patients survive because the delay period allows the majority of cells to escape the small vessels of the tissues before fibers form. The enormous sensitivity of the duration of the delay period to intracellular hemoglobin composition also explains why sickle trait, the heterozygous condition, and the compound heterozygous condition of hemoglobin S with pancellular hereditary persistence of fetal hemoglobin are both relatively benign conditions.

MWC allosteric model explains unusual hemoglobin-oxygen binding curves from sickle cell drug binding.

An oxygen-affinity-modifying drug, voxelotor, has very recently been approved by the FDA for treatment of sickle cell disease. The proposed mechanism of action is by preferential binding of the drug to the R quaternary conformation, which cannot copolymerize with the T conformation to form sickle fibers. Here, we report widely different oxygen dissociation and oxygen association curves for normal blood in the presence of voxelotor and interpret the results in terms of the allosteric model of Monod, Wyman, and Changeux with the addition of drug binding. The model does remarkably well in quantitatively explaining a complex data set with just the addition of drug binding and dissociation rates for the R and T conformations. Whereas slow dissociation of the drug from R results in time-independent dissociation curves, the changing association curves result from slow dissociation of the drug from T, as well as extremely slow binding of the drug to T. By calculating true equilibrium curves from the model parameters, we show that there would be a smaller decrease in oxygen delivery from the left shift in the dissociation curve caused by drug binding if drug binding and dissociation for both R and T were rapid. Our application of the Monod, Wyman, and Changeux model demonstrates once more its enormous power in explaining many different kinds of experimental results for hemoglobin. It should also be helpful in analyzing oxygen binding and in vivo delivery in future investigations of oxygen-affinity-modifying drugs for sickle cell disease.

Constraints on the Structure of Fibrils Formed by a Racemic Mixture of Amyloid-ß Peptides from Solid-State NMR, Electron Microscopy, and Theory.

Previous studies have shown that racemic mixtures of 40- and 42-residue amyloid-ß peptides (d,l-Aß40 and d,l-Aß42) form amyloid fibrils with accelerated kinetics and enhanced stability relative to their homochiral counterparts (l-Aß40 and l-Aß42), suggesting a "chiral inactivation" approach to abrogating the neurotoxicity of Aß oligomers (Aß-CI). Here we report a structural study of d,l-Aß40 fibrils, using electron microscopy, solid-state nuclear magnetic resonance (NMR), and density functional theory (DFT) calculations. Two- and three-dimensional solid-state NMR spectra indicate molecular conformations in d,l-Aß40 fibrils that resemble those in known l-Aß40 fibril structures. However, quantitative measurements of 13C-13C and 15N-13C distances in selectively labeled d,l-Aß40 fibril samples indicate a qualitatively different supramolecular structure. While cross-ß structures in mature l-Aß40 fibrils are comprised of in-register, parallel ß-sheets, our data indicate antiparallel ß-sheets in d,l-Aß40 fibrils, with alternation of d and l molecules along the fibril growth direction, i.e., antiparallel "rippled sheet" structures. The solid-state NMR data suggest the coexistence of d,l-Aß40 fibril polymorphs with three different registries of intermolecular hydrogen bonds within the antiparallel rippled sheets. DFT calculations support an energetic preference for antiparallel alignments of the ß-strand segments identified by solid-state NMR. These results provide insight into the structural basis for Aß-CI and establish the importance of rippled sheets in self-assembly of full-length, naturally occurring amyloidogenic peptides.

Probing the mechanism of inhibition of amyloid-ß(1-42)-induced neurotoxicity by the chaperonin GroEL.

The human chaperonin Hsp60 is thought to play a role in the progression of Alzheimer's disease by mitigating against intracellular ß-amyloid stress. Here, we show that the bacterial homolog GroEL (51% sequence identity) reduces the neurotoxic effects of amyloid-ß(1-42) (Aß42) on human neural stem cell-derived neuronal cultures. To understand the mechanism of GroEL-mediated abrogation of neurotoxicity, we studied the interaction of Aß42 with GroEL using a variety of biophysical techniques. Aß42 binds to GroEL as a monomer with a lifetime of ∼1 ms, as determined from global analysis of multiple relaxation-based NMR experiments. Dynamic light scattering demonstrates that GroEL dissolves small amounts of high-molecular-weight polydisperse aggregates present in fresh soluble Aß42 preparations. The residue-specific transverse relaxation rate profile for GroEL-bound Aß42 reveals the presence of three anchor-binding regions (residues 16-21, 31-34, and 40-41) located within the hydrophobic GroEL-consensus binding sequences. Single-molecule FRET analysis of Aß42 binding to GroEL results in no significant change in the FRET efficiency of a doubly labeled Aß42 construct, indicating that Aß42 samples a random coil ensemble when bound to GroEL. Finally, GroEL substantially slows down the disappearance of NMR visible Aß42 species and the appearance of Aß42 protofibrils and fibrils as monitored by electron and atomic force microscopies. The latter observations correlate with the effect of GroEL on the time course of Aß42-induced neurotoxicity. These data provide a physical basis for understanding how Hsp60 may serve to slow down the progression of Alzheimer's disease.

Concentration-Dependent Structural Transition of the HIV-1 gp41 MPER Peptide into α-Helical Trimers.

The membrane proximal external region (MPER) of HIV-1 gp41 contains epitopes for at least four broadly neutralizing antibodies. Depending on solution conditions and construct design, different structures have been reported for this segment. We show that in aqueous solution the MPER fragment (gp160660-674 ) exists in a monomer-trimer equilibrium with an association constant in the micromolar range. Thermodynamic analysis reveals that the association is exothermic, more favorable in D2 O than H2 O, and increases with ionic strength, indicating hydrophobically driven intermolecular interactions. Circular dichroism, 13 Cα chemical shifts, NOE, and hydrogen exchange rates reveal that MPER undergoes a structural transition from predominately unfolded monomer at low concentrations to an α-helical trimer at high concentrations. This result has implications for antibody recognition of MPER prior to and during the process where gp41 switches from a pre-hairpin intermediate to its post-fusion 6-helical bundle state.

Probing the Interaction between HIV-1 Protease and the Homodimeric p66/p66' Reverse Transcriptase Precursor by Double Electron-Electron Resonance EPR Spectroscopy.

Following excision from the Gag-Pol polyprotein, HIV-1 reverse transcriptase is released as an asymmetric homodimer comprising two p66 subunits that are structurally dissimilar but identical in amino acid sequence. Subsequent cleavage of the RNase H domain from only one of the subunits, denoted p66', results in the formation of the mature p66/p51 enzyme in which catalytic activity resides in the p66 subunit, and the p51 subunit (derived from p66') provides a supporting structural scaffold. Here, we probe the interaction of the p66/p66' asymmetric reverse transcriptase precursor with HIV-1 protease by pulsed Q-band double electron-electron resonance EPR spectroscopy to measure distances between nitroxide labels introduced at surface-engineered cysteine residues. The data suggest that the flexible, exposed linker between the RNaseH and connection domains in the open state of the p66' subunit binds to the active site of protease in a configuration that is similar to that of extended peptide substrates.

Binding kinetics and substrate selectivity in HIV-1 protease-Gag interactions probed at atomic resolution by chemical exchange NMR.

The conversion of immature noninfectious HIV-1 particles to infectious virions is dependent upon the sequential cleavage of the precursor group-specific antigen (Gag) polyprotein by HIV-1 protease. The precise mechanism whereby protease recognizes distinct Gag cleavage sites, located in the intrinsically disordered linkers connecting the globular domains of Gag, remains unclear. Here, we probe the dynamics of the interaction of large fragments of Gag and various variants of protease (including a drug resistant construct) using Carr-Purcell-Meiboom-Gill relaxation dispersion and chemical exchange saturation transfer NMR experiments. We show that the conformational dynamics within the flaps of HIV-1 protease that form the lid over the catalytic cleft play a significant role in substrate specificity and ordered Gag processing. Rapid interconversion between closed and open protease flap conformations facilitates the formation of a transient, sparsely populated productive complex between protease and Gag substrates. Flap closure traps the Gag cleavage sites within the catalytic cleft of protease. Modulation of flap opening through protease-Gag interactions fine-tunes the lifetime of the productive complex and hence the likelihood of Gag proteolysis. A productive complex can also be formed in the presence of a noncognate substrate but is short-lived owing to lack of optimal complementarity between the active site cleft of protease and the substrate, resulting in rapid flap opening and substrate release, thereby allowing protease to differentiate between cognate and noncognate substrates.

Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET.

We describe a method that combines two- and three-color single-molecule FRET spectroscopy with 2D FRET efficiency-lifetime analysis to probe the oligomerization process of intrinsically disordered proteins. This method is applied to the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. TD exists as a monomer at subnanomolar concentrations and forms a dimer and a tetramer at higher concentrations. Because the dissociation constants of the dimer and tetramer are very close, as we determine in this paper, it is not possible to characterize different oligomeric species by ensemble methods, especially the dimer that cannot be readily separated. However, by using single-molecule FRET spectroscopy that includes measurements of fluorescence lifetime and two- and three-color FRET efficiencies with corrections for submillisecond acceptor blinking, we show that it is possible to obtain structural information for individual oligomers at equilibrium and to determine the dimerization kinetics. From these analyses, we show that the monomer is intrinsically disordered and that the dimer conformation is very similar to that of the tetramer but the C terminus of the dimer is more flexible.

Diverse Folding Pathways of HIV-1 Protease Monomer on a Rugged Energy Landscape.

The modern energy landscape theory of protein folding predicts multiple folding pathways connecting a myriad of unfolded conformations and a well-defined folded state. However, direct experimental observation of heterogeneous folding pathways is difficult. Naturally evolved proteins typically exhibit a smooth folding energy landscape for fast and efficient folding by avoiding unfavorable kinetic traps. In this case, rapid fluctuations between unfolded conformations result in apparent two-state behavior and make different pathways indistinguishable. However, the landscape roughness can be different, depending on the selection pressures during evolution. Here, we characterize the unusually rugged folding energy landscape of human immunodeficiency virus-1 protease monomer using single-molecule Förster resonance energy transfer spectroscopy. Our data show that fluctuations between unfolded conformations are slow, which enables the experimental observation of heterogeneous folding pathways as predicted by the landscape theory. Although the landscape ruggedness is sensitive to the mutations and fluorophore locations, the folding rate is similar for various protease constructs. The natural evolution of the protease to have a rugged energy landscape likely results from intrinsic pressures to maintain robust folding when human immunodeficiency virus-1 mutates frequently, which is essential for its survival.

Observation of ß-Amyloid Peptide Oligomerization by Pressure-Jump NMR Spectroscopy.

Brain tissue of Alzheimer's disease patients invariably contains deposits of insoluble, fibrillar aggregates of peptide fragments of the amyloid precursor protein (APP), typically 40 or 42 residues in length and referred to as Aß40 and Aß42. However, it remains unclear whether these fibrils or oligomers constitute the toxic species. Depending on sample conditions, oligomers can form in a few seconds or less. These oligomers are invisible to solution NMR spectroscopy, but they can be rapidly (<1 s) resolubilized and converted to their NMR-visible monomeric constituents by raising the hydrostatic pressure to a few kbar. Hence, utilizing pressure-jump NMR, the oligomeric state can be studied at residue-specific resolution by monitoring its signals in the monomeric state. Oligomeric states of Aß40 exhibit a high degree of order, reflected by slow longitudinal 15N relaxation (T1 > 5 s) for residues 18-21 and 31-34, whereas the N-terminal 10 residues relax much faster (T1 ≤ 1.5 s), indicative of extensive internal motions. Transverse relaxation rates rapidly increase to ca. 1000 s-1 after the oligomerization is initiated.

Inhibition of HIV Maturation via Selective Unfolding and Cross-Linking of Gag Polyprotein by a Mercaptobenzamide Acetylator.

For HIV to become infectious, any new virion produced from an infected cell must undergo a maturation process that involves the assembly of viral polyproteins Gag and Gag-Pol at the membrane surface. The self-assembly of these viral proteins drives formation of a new viral particle as well as the activation of HIV protease, which is needed to cleave the polyproteins so that the final core structure of the virus will properly form. Molecules that interfere with HIV maturation will prevent any new virions from infecting additional cells. In this manuscript, we characterize the unique mechanism by which a mercaptobenzamide thioester small molecule (SAMT-247) interferes with HIV maturation via a series of selective acetylations at highly conserved cysteine and lysine residues in Gag and Gag-Pol polyproteins. The results provide the first insights into how acetylation can be utilized to perturb the process of HIV maturation and reveal a new strategy to limit the infectivity of HIV.

Importance of time-ordered non-uniform sampling of multi-dimensional NMR spectra of Aß1-42 peptide under aggregating conditions.

Although the order of the time steps in which the non-uniform sampling (NUS) schedule is implemented when acquiring multi-dimensional NMR spectra is of limited importance when sample conditions remain unchanged over the course of the experiment, it is shown to have major impact when samples are unstable. In the latter case, time-ordering of the NUS data points by the normalized radial length yields a reduction of sampling artifacts, regardless of the spectral reconstruction algorithm. The disadvantage of time-ordered NUS sampling is that halting the experiment prior to its completion will result in lower spectral resolution, rather than a sparser data matrix. Alternatively, digitally correcting for sample decay prior to reconstruction of randomly ordered NUS data points can mitigate reconstruction artifacts, at the cost of somewhat lower sensitivity. Application of these sampling schemes to the Alzheimer's amyloid beta (Aß1-42) peptide at an elevated concentration, low temperature, and 3 kbar of pressure, where approximately 75% of the peptide reverts to an NMR-invisible state during the collection of a 3D 15N-separated NOESY spectrum, highlights the improvement in artifact suppression and reveals weak medium-range NOE contacts in several regions, including the C-terminal region of the peptide.

Transient HIV-1 Gag-protease interactions revealed by paramagnetic NMR suggest origins of compensatory drug resistance mutations.

Cleavage of the group-specific antigen (Gag) polyprotein by HIV-1 protease represents the critical first step in the conversion of immature noninfectious viral particles to mature infectious virions. Selective pressure exerted by HIV-1 protease inhibitors, a mainstay of current anti-HIV-1 therapies, results in the accumulation of drug resistance mutations in both protease and Gag. Surprisingly, a large number of these mutations (known as secondary or compensatory mutations) occur outside the active site of protease or the cleavage sites of Gag (located within intrinsically disordered linkers connecting the globular domains of Gag to one another), suggesting that transient encounter complexes involving the globular domains of Gag may play a role in guiding and facilitating access of the protease to the Gag cleavage sites. Here, using large fragments of Gag, as well as catalytically inactive and active variants of protease, we probe the nature of such rare encounter complexes using intermolecular paramagnetic relaxation enhancement, a highly sensitive technique for detecting sparsely populated states. We show that Gag-protease encounter complexes are primarily mediated by interactions between protease and the globular domains of Gag and that the sites of transient interactions are correlated with surface exposed regions that exhibit a high propensity to mutate in the presence of HIV-1 protease inhibitors.

Tilted, Uninterrupted, Monomeric HIV-1 gp41 Transmembrane Helix from Residual Dipolar Couplings.

Cryo-electron microscopy and X-ray crystallography have shown that the pre- and postfusion states of the HIV-1 gp41 viral coat protein, although very different from one another, each adopt C3 symmetric structures. A stable homotrimeric structure for the transmembrane domain (TM) also was modeled and supported by experimental data. For a C3 symmetric structure, alignment in an anisotropic medium must be axially symmetric, with the unique axis of the alignment tensor coinciding with the C3 axis. However, NMR residual dipolar couplings (RDCs) measured under three different alignment conditions were found to be incompatible with C3 symmetry. Subsequent measurements by paramagnetic relaxation enhancement, analytical ultracentrifugation, and DEER EPR, indicate that the transmembrane domain is monomeric. 15N NMR relaxation data and RDCs show that TM is highly ordered and uninterrupted for a total length of 32 residues, extending well into the membrane proximal external region.

Co-Evolutionary Fitness Landscapes for Sequence Design.

Efficient and accurate models to predict the fitness of a sequence would be extremely valuable in protein design. We have explored the use of statistical potentials for the coevolutionary fitness landscape, extracted from known protein sequences, in conjunction with Monte Carlo simulations, as a tool for design. As proof of principle, we created a series of predicted high-fitness sequences for three different protein folds, representative of different structural classes: the GA (all-α) and GB (α/ß) binding domains of streptococcal protein G, and an SH3 (all-ß) domain. We found that most of the designed proteins can fold stably to the target structure, and a structure for a representative of each for GA, GB and SH3 was determined. Several of our designed proteins were also able to bind to native ligands, in some cases with higher affinity than wild-type. Thus, a search using a statistical fitness landscape is a remarkably effective tool for finding novel stable protein sequences.

Dissociation of the trimeric gp41 ectodomain at the lipid-water interface suggests an active role in HIV-1 Env-mediated membrane fusion.

The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. The actual fusion process involves a switch from a homotrimeric prehairpin intermediate conformation, consisting of parallel coiled-coil helices, to a postfusion state where the ectodomains are arranged as a trimer of helical hairpins, adopting a six-helix bundle (6HB) state. Here, we show by solution NMR spectroscopy that a water-soluble 6HB gp41 ectodomain binds to zwitterionic detergents that contain phosphocholine or phosphatidylcholine head groups and phospholipid vesicles that mimic T-cell membrane composition. Binding results in the dissociation of the 6HB and the formation of a monomeric state, where its two α-helices, N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), become embedded in the lipid-water interface of the virus and host cell. The atomic structure of the gp41 ectodomain monomer, based on NOE distance restraints and residual dipolar couplings, shows that the NHR and CHR helices remain mostly intact, but they completely lose interhelical contacts. The high affinity of the ectodomain helices for phospholipid surfaces suggests that unzippering of the prehairpin intermediate leads to a state where the NHR and CHR helices become embedded in the host cell and viral membranes, respectively, thereby providing a physical force for bringing these membranes into close juxtaposition before actual fusion.