Structural and dynamical characterization of tubular HIV‐1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy

The wild‐type HIV‐1 capsid protein (CA) self‐assembles in vitro into tubular structures at high ionic strength. We report solid state nuclear magnetic resonance (NMR) and electron microscopy measurements on these tubular CA assemblies, which are believed to contain a triangular lattice of hexameric CA proteins that is similar or identical to the lattice of capsids in intact HIV‐1. Mass‐per‐length values of CA assemblies determined by dark‐field transmission electron microscopy indicate a variety of structures, ranging from single‐wall tubes to multiwall tubes that approximate solid rods. Two‐dimensional (2D) solid state ¹³C? ¹³C and ¹⁵N? ¹³C NMR spectra of uniformly ¹⁵N,¹³C‐labeled CA assemblies are highly congested, as expected for a 25.6 kDa protein in which nearly the entire amino acid sequence is immobilized. Solid state NMR spectra of partially labeled CA assemblies, expressed in 1,3‐¹³C₂‐glycerol medium, are better resolved, allowing the identification of individual signals with line widths below 1 ppm. Comparison of crosspeak patterns in the experimental 2D spectra with simulated patterns based on solution NMR chemical shifts of the individual N‐terminal (NTD) and C‐terminal (CTD) domains indicates that NTD and CTD retain their individual structures upon self‐assembly of full‐length CA into tubes. 2D ¹H‐¹³C NMR spectra of CA assemblies recorded under solution NMR conditions show relatively few signals, primarily from segments that link the α‐helices of NTD and CTD and from the N‐ and C‐terminal ends. Taken together, the data support the idea that CA assemblies contain a highly ordered 2D protein lattice in which the NTD and CTD structures are retained and largely immobilized.     

Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR

Recent studies of noncrystalline HIV-1 capsid protein (CA) assemblies by our laboratory and by Polenova and coworkers (Protein Sci 19:716–730, 2010; J Mol Biol 426:1109–1127, 2014; J Biol Chem 291:13098–13112, 2016; J Am Chem Soc 138:8538–8546, 2016; J Am Chem Soc 138:12029–12032, 2016; J Am Chem Soc 134:6455–6466, 2012; J Am Chem Soc 132:1976–1987, 2010; J Am Chem Soc 135:17793–17803, 2013; Proc Natl Acad Sci USA 112:14617–14622, 2015; J Am Chem Soc 138:14066–14075, 2016) have established the capability of solid state nuclear magnetic resonance (NMR) measurements to provide site-specific structural and dynamical information that is not available from other types of measurements. Nonetheless, the relatively high molecular weight of HIV-1 CA leads to congestion of solid state NMR spectra of fully isotopically labeled assemblies that has been an impediment to further progress. Here we describe an efficient protocol for production of segmentally labeled HIV-1 CA samples in which either the N-terminal domain (NTD) or the C-terminal domain (CTD) is uniformly ¹⁵N,¹³C-labeled. Segmental labeling is achieved by trans-splicing, using the DnaE split intein. Comparisons of two-dimensional solid state NMR spectra of fully labeled and segmentally labeled tubular CA assemblies show substantial improvements in spectral resolution. The molecular structure of HIV-1 assemblies is not significantly perturbed by the single Ser-to-Cys substitution that we introduce between NTD and CTD segments, as required for trans-splicing.     

Retroviral Capsid Assembly: A Role for the CA Dimer in Initiation

In maturing retroviral virions, CA protein assembles to form a capsid shell that is essential for infectivity. The structure of the two folded domains [N-terminal domain (NTD) and C-terminal domain (CTD)] of CA is highly conserved among various retroviruses, and the capsid assembly pathway, although poorly understood, is thought to be conserved as well. In vitro assembly reactions with purified CA proteins of the Rous sarcoma virus (RSV) were used to define factors that influence the kinetics of capsid assembly and provide insights into underlying mechanisms. CA multimerization was triggered by multivalent anions providing evidence that in vitro assembly is an electrostatically controlled process. In the case of RSV, in vitro assembly was a well-behaved nucleation-driven process that led to the formation of structures with morphologies similar to those found in virions. Isolated RSV dimers, when mixed with monomeric protein, acted as efficient seeds for assembly, eliminating the lag phase characteristic of a monomer-only reaction. This demonstrates for the first time the purification of an intermediate on the assembly pathway. Differences in the intrinsic tryptophan fluorescence of monomeric protein and the assembly-competent dimer fraction suggest the involvement of the NTD in the formation of the functional dimer. Furthermore, in vitro analysis of well-characterized CTD mutants provides evidence for assembly dependence on the second domain and suggests that the establishment of an NTD-CTD interface is a critical step in capsid assembly initiation. Overall, the data provide clear support for a model whereby capsid assembly within the maturing virion is dependent on the formation of a specific nucleating complex that involves a CA dimer and is directed by additional virion constituents.     

1H, 13C, and 15N resonance assignment of the N-terminal domain of Mason-Pfizer monkey virus capsid protein, CA 1-140

Mason-Pfizer monkey virus (M-PMV) belongs to the family of betaretroviruses characterized by the assembly of immature particles within cytoplasm of infected cells in contrast to other retroviruses (e.g. HIV, RSV) that assemble their immature particles at a plasma membrane. Simultaneously with or shortly after budding a virus-encoded protease is activated and the Gag polyprotein is cleaved into three major structural proteins: matrix (MA), capsid (CA), and nucleocapsid (NC) protein. Mature retroviral CA proteins consist of two independently folded structural domains: N-terminal domain (NTD) and C-terminal dimerization domain (CTD), separated by a flexible linker. As a first step toward the solution structure elucidation, we present nearly complete backbone and side-chain ¹H, ¹⁵N and ¹³C resonance assignment of the M-PMV NTD CA.     

1H, 15N and 13C assignments of the dimeric C-terminal domain of HIV-1 capsid protein

HIV-1 capsid protein (CA) encloses the viral RNA genome and forms a conical-shaped particle in the mature HIV-1 virion, with orderly capsid assembly and disassembly critically important for viral infectivity. The 231 residue CA is composed of two helical domains, connected by a short linker sequence. In solution, CA exhibits concentration dependent dimerization which is mediated by the C-terminal domain (CTD). Here, we present nearly complete ¹H, ¹⁵N and ¹³C assignments for the 20 kDa homodimeric CA–CTD, a prerequisite for structural characterization of the CA–CTD dimer.     

Association Equilibrium of the HIV-1 Capsid Protein in a Crowded Medium Reveals that Hexamerization during Capsid Assembly Requires a Functional C-Domain Dimerization Interface

Polymerization of the intact capsid protein (CA) of HIV-1 into mature capsidlike particles at physiological ionic strength in vitro requires macromolecularly crowded conditions that approach those inside the virion, where the mature capsid is assembled in vivo. The capsid is organized as a hexameric lattice. CA subunits in each hexamer are connected through interfaces that involve the CA N-terminal domain (NTD); pairs of CA subunits belonging to different hexamers are connected through a different interface that involves the C-terminal domain (CTD). At physiological ionic strength in noncrowded conditions, CA subunits homodimerize through this CTD-CTD interface, but do not hexamerize through the other interfaces (those involving the NTD). Here we have investigated whether macromolecular crowding conditions are able to promote hexamerization of the isolated NTD and/or full-length CA (with an inactive CTD-CTD interface to prevent polymerization). The oligomerization state of the proteins was determined using analytical ultracentrifugation in the absence or presence of high concentrations of an inert macromolecular crowding agent. Under the same conditions that promoted efficient assembly of intact CA dimers, neither NTD nor CA with an inactive CTD-CTD interface showed any tendency to form hexamers or any other oligomer. This inability to hexamerize was observed even in macromolecularly crowded conditions. The results indicate that a functional CTD-CTD interface is strictly required for hexamerization of HIV-1 CA through the other interfaces. Together with previous results, these observations suggest that establishment of NTD-CTD interactions involved in CA hexamerization during mature HIV-1 capsid assembly requires a homodimerization-dependent conformational switching of CTD.     

The core of Ure2p prion fibrils is formed by the N-terminal segment in a parallel cross-β structure: evidence from solid-state NMR

Intracellular fibril formation by Ure2p produces the non-Mendelian genetic element [URE3] in Saccharomyces cerevisiae, making Ure2p a prion protein. We show that solid-state NMR spectra of full-length Ure2p fibrils, seeded with infectious prions from a specific [URE3] strain and labeled with uniformly ¹⁵N-¹³C-enriched Ile, include strong, sharp signals from Ile residues in the globular C-terminal domain (CTD) with both helical and nonhelical ¹³C chemical shifts. Treatment with proteinase K eliminates these CTD signals, leaving only nonhelical signals from the Gln-rich and Asn-rich N-terminal segment, which are also observed in the solid-state NMR spectra of Ile-labeled fibrils formed by residues 1-89 of Ure2p. Thus, the N-terminal segment, or “prion domain” (PD), forms the fibril core, while CTD units are located outside the core. We additionally show that, after proteinase K treatment, Ile-labeled Ure2p fibrils formed without prion seeding exhibit a broader set of solid-state NMR signals than do prion-seeded fibrils, consistent with the idea that structural variations within the PD core account for prion strains. Measurements of ¹³C-¹³C magnetic dipole-dipole couplings among ¹³C-labeled Ile carbonyl sites in full-length Ure2p fibrils support an in-register parallel β-sheet structure for the PD core of Ure2p fibrils. Finally, we show that a model in which CTD units are attached rigidly to the parallel β-sheet core is consistent with steric constraints.     

Structure of a monomeric mutant of the HIV-1 capsid protein

The capsid protein (CA) of HIV-1 plays a significant role in the assembly of the immature virion and is the critical building block of its mature capsid. Thus, there has been significant interest in the CA protein as a target in the design of inhibitors of early and late stage events in the HIV-1 replication cycle. However, because of its inherent flexibility from the interdomain linker and the monomer-dimer equilibrium in solution, the HIV-1 wild-type CA monomer has defied structural determinations by X-ray crystallography and nuclear magnetic resonance spectroscopy. Here we report the detailed solution structure of full-length HIV-1 CA using a monomeric mutant that, though noninfective, preserves many of the critical properties of the wild-type protein. The structure shows independently folded N-terminal (NTD) and C-terminal domains (CTD) joined by a flexible linker. The CTD shows some differences from that of the dimeric wild-type CTD structures. This study provides insights into the molecular mechanism of the wild-type CA dimerization critical for capsid assembly. The monomeric mutant allows investigation of interactions of CA with human cellular proteins exploited by HIV-1, directly in solution without the complications associated with the monomer-dimer equilibrium of the wild-type protein. This structure also permits the design of inhibitors directed at a novel target, viz., interdomain flexibility, as well as inhibitors that target multiple interdomain interactions critical for assembly and interactions of CA with host cellular proteins that play significant roles within the replication cycle of HIV-1.     

Solution structure of a double mutant of the carboxy-terminal dimerization domain of the HIV-1 capsid protein

As in other retroviruses, the HIV-1 capsid (CA) protein is composed of two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), joined by a flexible linker. The dimerization of the CTD is thought to be a critical step in the assembly of the immature and mature viral capsids. The precise nature of the functional form of CTD dimerization interface has been a subject of considerable interest. Previously, the CTD dimer was thought to involve a face-to-face dimerization observed in the early crystallographic studies. Recently, the crystallographic structure for a domain-swapped CTD dimer has been determined. This dimer, with an entirely different interface that includes the major homology region (MHR) has been suggested as the functional form during the Gag assembly. The structure determination of the monomeric wt CTD of HIV-1 has not been possible because of the monomer-dimer equilibrium in solution. We report the NMR structure of the [W184A/M185A]-CTD mutant in its monomeric form. These mutations interfere with dimerization without abrogating the assembly activity of Gag and CA. The NMR structure shows some important differences compared to the CTD structure in the face-to-face dimer. Notably, the helix-2 is much shorter, and the kink seen in the crystal structure of the wt CTD in the face-to-face dimer is absent. These NMR studies suggest that dimerization-induced conformational changes may be present in the two crystal structures of the CTD dimers and also suggest a mechanism that can simultaneously accommodate both of the distinctly different dimer models playing functional roles during the Gag assembly of the immature capsids.     

Protease Cleavage Leads to Formation of Mature Trimer Interface in HIV-1 Capsid

During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles.     

Homodimerization propensity of the intrinsically disordered N-terminal domain of Ultraspiracle from Aedes aegypti

The mosquito Aedes aegypti is the principal vector of dengue, one of the most devastating arthropod-borne viral infections in humans. The isoform specific A/B region, called the N-terminal domain (NTD), is hypervariable in sequence and length and is poorly conserved within the Ultraspiracle (Usp) family. The Usp protein together with ecdysteroid receptor (EcR) forms a heterodimeric complex. Up until now, there has been little data on the molecular properties of the isolated Usp-NTD. Here, we describe the biochemical and biophysical properties of the recombinant NTD of the Usp isoform B (aaUsp-NTD) from A. aegypti. These results, in combination with in silico bioinformatics approaches, indicate that aaUsp-NTD exhibits properties of an intrinsically disordered protein (IDP). We also present the first experimental evidence describing the dimerization propensity of the isolated NTD of Usp. These characteristics also appear for other members of the Usp family in different species, for example, in the Usp-NTD from Drosophila melanogaster and Bombyx mori. However, aaUsp-NTD exhibits the strongest homodimerization potential. We postulate that the unique dimerization of the NTD might be important for Usp function by providing an additional platform for interactions, in addition to the nuclear receptor superfamily dimerization via DNA binding domains and ligand binding domains that has already been extensively documented. Furthermore, the unique NTD–NTD interaction that was observed might contribute new insight into the dimerization propensities of nuclear receptors.     

Second-site compensatory mutations of HIV-1 capsid mutations

The human immunodeficiency virus (HIV) capsid (CA) protein assembles into a hexameric lattice that forms the mature virus core. Contacts between the CA N-terminal domain (NTD) of one monomer and the C-terminal domain (CTD) of the adjacent monomer are important for the assembly of this core. In this study, we have examined the effects of mutations in the NTD region associated with this interaction. We have found that such mutations yielded modest reductions of virus release but major effects on viral infectivity. Cell culture and in vitro assays indicate that the infectivity defects relate to abnormalities in the viral cores. We have selected second-site compensatory mutations that partially restored HIV infectivity. These mutations map to the CA CTD and to spacer peptide 1 (SP1), the portion of the precursor Gag protein immediately C terminal to the CTD. The compensatory mutations do not locate to the molecularly modeled intermolecular NTD-CTD interface. Rather, the compensatory mutations appear to act indirectly, possibly by realignment of the C-terminal helix of the CA CTD, which participates in the NTD-CTD interface and has been shown to serve an important role in the assembly of infectious virus.     

Solution Structure of Mouse Hepatitis Virus (MHV) nsp3a and Determinants of the Interaction with MHV Nucleocapsid (N) Protein

Coronaviruses (CoVs) are positive-sense, single-stranded, enveloped RNA viruses that infect a variety of vertebrate hosts. The CoV nucleocapsid (N) protein contains two structurally independent RNA binding domains, designated the N-terminal domain (NTD) and the dimeric C-terminal domain (CTD), joined by a charged linker region rich in serine and arginine residues (SR-rich linker). An important goal in unraveling N function is to molecularly characterize N-protein interactions. Recent genetic evidence suggests that N interacts with nsp3a, a component of the viral replicase. Here we present the solution nuclear magnetic resonance (NMR) structure of mouse hepatitis virus (MHV) nsp3a and show, using isothermal titration calorimetry, that MHV N219, an N construct that extends into the SR-rich linker (residues 60 to 219), binds cognate nsp3a with high affinity (equilibrium association constant [K_a], [1.4 ± 0.3] × 10⁶ M⁻¹). In contrast, neither N197, an N construct containing only the folded NTD (residues 60 to 197), nor the CTD dimer (residues 260 to 380) binds nsp3a with detectable affinity. This indicates that the key nsp3a binding determinants localize to the SR-rich linker, a finding consistent with those of reverse genetics studies. NMR chemical shift perturbation analysis reveals that the N-terminal region of an MHV N SR-rich linker peptide (residues 198 to 230) binds to the acidic face of MHV nsp3a containing the acidic α2 helix with an affinity (expressed as K_a) of 8.1 × 10³ M⁻¹. These studies reveal that the SR-rich linker of MHV N is necessary but not sufficient to maintain this high-affinity binding to N.     

Retrovirus capsid protein assembly arrangements

During retrovirus particle assembly and morphogenesis, the retrovirus structural (Gag) proteins organize into two different arrangements: an immature form assembled by precursor Gag (PrGag) proteins; and a mature form, composed of proteins processed from PrGag. Central to both Gag protein arrangements is the capsid (CA) protein, a domain of PrGag, which is cleaved from the precursor to yield a mature Gag protein composed of an N-terminal domain (NTD), a flexible linker region, and a C-terminal domain (CTD). Because Gag interactions have proven difficult to examine in virions, a number of investigations have focused on the analysis of structures assembled in vitro. We have used electron microscope (EM) image reconstruction techniques to examine assembly products formed by two different CA variants of both human immunodeficiency virus type 1 (HIV-1) and the Moloney murine leukemia virus (M-MuLV). Interestingly, two types of hexameric protein arrangements were observed for each virus type. One organizational scheme featured hexamers composed of putative NTD dimer subunits, with sharing of subunits between neighbor hexamers. The second arrangement used apparent NTD monomers to coordinate hexamers, involved no subunit sharing, and employed putative CTD interactions to connect hexamers. Conversion between the two assembly forms may be achieved by making or breaking the proposed symmetric NTD dimer contacts in a process that appears to mimic viral morphogenesis.     

Structure and plasticity of the peptidyl-prolyl isomerase Par27 of Bordetella pertussis revealed by X-ray diffraction and small-angle X-ray scattering

Par27 from Bordetella pertussis belongs to a newly discovered class of dimeric peptidyl-prolyl isomerase (PPIase)/chaperones from the parvulin family. It is a tripartite protein with a central PPIase domain surrounded by N- and C-terminal sub-domains (NTD and CTD). Here, the Par27 structure was characterized by X-ray crystallography, small-angle X-ray scattering and template-based modeling. In the crystal lattice, Par27 consists of alternating well ordered and poorly ordered domains. The PPIase domains gave rise to diffuse scattering and could not be solved, whereas a 2.2 Å resolution crystal structure was obtained for the NTD and CTD, revealing a cradle-shaped dimeric platform. Despite a lack of sequence similarity with corresponding sub-domains, the topology of the peptide chain in the NTD/CTD core is similar to that of other monomeric PPIase/chaperones such as SurA and trigger factor from Escherichia coli. In Par27, dimerization occurs by sub-domain swapping. Because of the strong amino acid sequence similarity to other parvulin domains, a model for the Par27 PPIase domain was built by template-based modeling and validated against small-angle X-ray scattering (SAXS) data. A model of the full-length dimeric Par27 structure was built by rigid-body modeling and filtering against SAXS data using the partial crystal structure of the NTD/CTD core and the template-based PPIase model. The flexibility of protein was accounted for by representing the structure as an ensemble of different conformations that collectively reproduce the scattering data. The refined models exhibit a cradle-like shape reminiscent of other PPIase/chaperones, and the variability in the orientation of the PPIase domains relative to the NTD/CTD core platform observed in the different models suggests inter-domain flexibility that could be important for the biological activity of this protein.     

Carboxyl terminal domain basic amino acids of mycobacterial topoisomerase I bind DNA to promote strand passage

Bacterial DNA topoisomerase I (topoI) carries out relaxation of negatively supercoiled DNA through a series of orchestrated steps, DNA binding, cleavage, strand passage and religation. The N-terminal domain (NTD) of the type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn²⁺ finger motifs in the CTD. The Zn²⁺ finger motifs were found to be essential in Escherichia coli topoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial topoI lacks Zn²⁺ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. We also show that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the catalytic step. Although the NTD binds to DNA in a site-specific fashion to carry out DNA cleavage and religation, the basic residues in CTD bind to non-scissile DNA in a sequence-independent manner to promote the crucial strand passage step during DNA relaxation. The loss of Zn²⁺ fingers from the mycobacterial topoI could be associated with Zn²⁺ export and homeostasis.     

Pleomorphic configuration of the trimeric capsid proteins of Rice dwarf virus that allows formation of both the outer capsid and tubular crystals

In the double-shelled capsid of Phytoreovirus, the outer capsid attaches firmly to the 3-fold axes of the T = 1 core. It then forms a T = 13 lattice via lateral interactions among the P8 trimers (Wu et al., 2000, Virology 271, 18-25). Purified P8 molecules also assemble into hexagonal monolayers as well as tubular crystals. To explore the mechanisms of formation of these structures, the configurations of P8 trimers were compared and verified in particles of Rice dwarf virus and in tubular crystals (tubes) whose structure was determined by cryoelectron microscopy using helical reconstruction technique. Remarkable variations in intertrimer contacts were observed in the tubes and in the surface lattice of Rice dwarf virus capsid. Superposition of the atomic structure of P8 trimers in the structures analyzed by cryoelectron microscopy allowed us to identify groups of specific and stable interactions, some of which were preserved in the tubes and the quasi-equivalent T = 13 icosahedral lattice of the virion's shell. The flexible nature of the binding between P8 trimers, created via electrostatic interactions that hold radially inward, appears to allow the outer-capsid P8 trimers to envelop the ragged surface of the core, forming the double shell of an intact viral particle.     

Global fold and backbone dynamics of the hepatitis C virus E2 glycoprotein transmembrane domain determined by NMR

E1 and E2 are two hepatitis C viral envelope glycoproteins that assemble into a heterodimer that is essential for membrane fusion and penetration into the target cell. Both extracellular and transmembrane (TM) glycoprotein domains contribute to this interaction, but study of TM–TM interactions has been limited because synthesis and structural characterization of these highly hydrophobic segments present significant challenges. In this NMR study, by successful expression and purification of the E2 transmembrane domain as a fusion construct we have determined the global fold and characterized backbone motions for this peptide incorporated in phospholipid micelles. Backbone resonance frequencies, relaxation rates and solvent exposure measurements concur in showing this domain to adopt a helical conformation, with two helical segments spanning residues 717–726 and 732–746 connected by an unstructured linker containing the charged residues D728 and R730 involved in E1 binding. Although this linker exhibits increased local motions on the ps timescale, the dominating contribution to its relaxation is the global tumbling motion with an estimated correlation time of 12.3 ns. The positioning of the helix–linker–helix architecture within the mixed micelle was established by paramagnetic NMR spectroscopy and phospholipid-peptide cross relaxation measurements. These indicate that while the helices traverse the hydrophobic interior of the micelle, the linker lies closer to the micelle perimeter to accommodate its charged residues. These results lay the groundwork for structure determination of the E1/E2 complex and a molecular understanding of glycoprotein heterodimerization.