Characterization of the conformational state and flexibility of HIV-1 glycoprotein gp120 core domain

gp120 is key to the human immunodeficiency virus type 1 viral cell entry. Knowledge of the detailed conformational states of gp120 is crucial to intervention, yet the unbound form is still resistant to structural characterization probably because of its flexibility. Toward this goal, we performed molecular dynamics simulations on the wild type gp120 core domain extracted from its ternary crystal structure and on a modeled mutant, S375W, that experimentally has a significantly different phenotype from the wild type. Although the mutant retained a bound-like conformation, the wild type drifted to a different conformational state. The wild type beta strands 2 and 3 of the bridging sheet were very mobile and partially unfolded, and the organization among the inner and outer domains and beta strands 20 and 21 of the bridging sheet, near the mutation site, was more open than in the bound form, although the overall structure was maintained. These differences were apparently a result of the strengthening of the hydrophobic core in the mutant. This stabilization further explains the experimentally significantly different thermodynamic properties between the wild type and the mutant. Taken together, our results suggest that the free form, although different from the bound state, shares many of the bound structural features. The observed loss of freedom near the binding site, rather than the previously hypothesized more dramatic conformational transition from the unbound to the bound state, appears to be the major contributor to the large entropy cost for the CD4 binding to the wild type.     

Insight Derived from Molecular Dynamics Simulations into Molecular Motions,Thermodynamics and Kinetics of HIV-1 gp120

Although the crystal structures of the HIV-1 gp120 core bound and pre-bound by CD4 are known, the details of dynamics involved in conformational equilibrium and transition in relation to gp120 function have remained elusive. The homology models of gp120 comprising the N- and C-termini and loops V3 and V4 in the CD4-bound and CD4-unbound states were built and subjected to molecular dynamics (MD) simulations to investigate the differences in dynamic properties and molecular motions between them. The results indicate that the CD4-bound gp120 adopted a more compact and stable conformation than the unbound form during simulations. For both the unbound and bound gp120, the large concerted motions derived from essential dynamics (ED) analyses can influence the size/shape of the ligand-binding channel/cavity of gp120 and, therefore, were related to its functional properties. The differences in motion direction between certain structural components of these two forms of gp120 were related to the conformational interconversion between them. The free energy calculations based on the metadynamics simulations reveal a more rugged and complex free energy landscape (FEL) for the unbound than for the bound gp120, implying that gp120 has a richer conformational diversity in the unbound form. The estimated free energy difference of ∼−6.0 kJ/mol between the global minimum free energy states of the unbound and bound gp120 indicates that gp120 can transform spontaneously from the unbound to bound states, revealing that the bound state represents a high-probability “ground state” for gp120 and explaining why the unbound state resists crystallization. Our results provide insight into the dynamics-and-function relationship of gp120, and facilitate understandings of the thermodynamics, kinetics and conformational control mechanism of HIV-1 gp120.     

Atomic insight into the CD4 binding-induced conformational changes in HIV-1 gp120

The entry of HIV-1 into a target cell requires gp120 and receptor CD4 as well as coreceptor CCR5/CXCR4 recognition events associated with conformational changes of the involved proteins. The binding of CD4 to gp120 is the initiation step of the whole process involving structural rearrangements that are crucial for subsequent pathways. Despite the wealth of knowledge about the gp120/CD4 interactions, details of the conformational changes occurring at this stage remain elusive. We have performed molecular dynamics simulations in explicit solvent based on the gp120/CD4/CD4i crystal structure in conjunction with modeled V3 and V4 loops to gain insight into the dynamics of the binding process. Three differentiated interaction modes between CD4 and gp120 were found, which involve electrostatics, hydrogen bond and van der Waals networks. A "binding funnel" model is proposed based on the dynamical nature of the binding interface together with a CD4-attraction gradient centered in gp120 at the CD4-Phe43-binding cavity. Distinct dynamical behaviors of free and CD4-bound gp120 were monitored, which likely represent the ground and pre-fusogenic states, respectively. The transition between these states revealed concerted motions in gp120 leading to: i) loop contractions around the CD4-Phe43-insertion cavity; ii) stabilization of the four-stranded "bridging sheet" structure; and iii) translocation and clustering of the V3 loop and the bridging sheet leading to the formation of the coreceptor binding site. Our results provide new insight into the dynamic of the underlying molecular recognition mechanism that complements the biochemical and structural studies.     

A mechanism by which binding of the broadly neutralizing antibody b12 unfolds the inner domain α1 helix in an engineered HIV-1 gp120

Using all-atom simulations, we examine the role of the I109C/Q428C disulfide "stitch" in altering the conformational distribution of engineered HIV-1 gp120 core relevant for binding of the broadly neutralizing recombinant antibody b12. In particular, we propose that the I109C/Q428C stitch results in a conformational distribution favoring an unfolded inner-domain α1-helix upon binding of b12. Using targeted molecular dynamics, we show that folded α1 in the b12-bound conformation of gp120 is stable both with and without the stitch, but that with folded α1, the stitch requires an orientation of the β20/β21 sheet that is sterically incompatible with b12 binding. Forcing β20/β21 into the orientation displayed by the b12-bound conformation after folding α1 with the stitch intact results in partial unfolding of α1, whereas without the stitch, β20/β21 reorientation does not affect the conformation of α1. These findings collectively support the hypothesis that the disulfide stitch shifts the conformational distribution of α1 to the unfolded state, meaning an unfolded α1 is not a strict requirement of the b12-bound conformational ensemble of gp120's lacking the I109C/Q428C stitch.     

Homology models of the HIV‐1 attachment inhibitor BMS‐626529 bound to gp120 suggest a unique mechanism of action

HIV‐1 gp120 undergoes multiple conformational changes both before and after binding to the host CD4 receptor. BMS‐626529 is an attachment inhibitor (AI) in clinical development (administered as prodrug BMS‐663068) that binds to HIV‐1 gp120. To investigate the mechanism of action of this new class of antiretroviral compounds, we constructed homology models of unliganded HIV‐1 gp120 (UNLIG), a pre‐CD4 binding‐intermediate conformation (pCD4), a CD4 bound‐intermediate conformation (bCD4), and a CD4/co‐receptor‐bound gp120 (LIG) from a series of partial structures. We also describe a simple pathway illustrating the transition between these four states. Guided by the positions of BMS‐626529 resistance substitutions and structure–activity relationship data for the AI series, putative binding sites for BMS‐626529 were identified, supported by biochemical and biophysical data. BMS‐626529 was docked into the UNLIG model and molecular dynamics simulations were used to demonstrate the thermodynamic stability of the different gp120 UNLIG/BMS‐626529 models. We propose that BMS‐626529 binds to the UNLIG conformation of gp120 within the structurally conserved outer domain, under the antiparallel β20–β21 sheet, and adjacent to the CD4 binding loop. Through this binding mode, BMS‐626529 can inhibit both CD4‐induced and CD4‐independent formation of the “open state” four‐stranded gp120 bridging sheet, and the subsequent formation and exposure of the chemokine co‐receptor binding site. This unique mechanism of action prevents the initial interaction of HIV‐1 with the host CD4+ T cell, and subsequent HIV‐1 binding and entry. Our findings clarify the novel mechanism of BMS‐626529, supporting its ongoing clinical development. Proteins 2015; 83:331–350. © 2014 Wiley Periodicals, Inc.     

The Highly Conserved Layer-3 Component of the HIV-1 gp120 Inner Domain Is Critical for CD4-Required Conformational Transitions

The trimeric envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) mediates virus entry into host cells. CD4 engagement with the gp120 exterior envelope glycoprotein subunit represents the first step during HIV-1 entry. CD4-induced conformational changes in the gp120 inner domain involve three potentially flexible topological layers (layers 1, 2, and 3). Structural rearrangements between layer 1 and layer 2 have been shown to facilitate the transition of the envelope glycoprotein trimer from the unliganded to the CD4-bound state and to stabilize gp120-CD4 interaction. However, our understanding of CD4-induced conformational changes in the gp120 inner domain remains incomplete. Here, we report that a highly conserved element of the gp120 inner domain, layer 3, plays a pivot-like role in these allosteric changes. In the unliganded state, layer 3 modulates the association of gp120 with the Env trimer, probably by influencing the relationship of the gp120 inner and outer domains. Importantly, layer 3 governs the efficiency of the initial gp120 interaction with CD4, a function that can also be fulfilled by filling the Phe43 cavity. This work defines the functional importance of layer 3 and completes a picture detailing the role of the gp120 inner domain in CD4-induced conformational transitions in the HIV-1 Env trimer.     

Fluctuation dynamics analysis of gp120 envelope protein reveals a topologically based communication network

Human Immunodeficiency Virus (HIV) infection is initiated by binding of the viral glycoprotein gp120, to the cellular receptor CD4. On CD4 binding, gp120 undergoes conformational change, permitting binding to the chemokine receptor. Crystal structures of gp120 ternary complex reveal the CD4 bound conformation of gp120. We report here the application of the Gaussian network model (GNM) to the crystal structures of gp120 bound to CD4 or CD4 mimic and 17b, to study the collective motions of the gp120 core and determine the communication propensities of the residue network. The GNM fluctuation profiles identify residues in the inner domain and outer domain that may facilitate conformational change or stability, respectively. Communication propensities delineate a residue network that is topologically suited for signal propagation from the Phe43 cavity throughout the gp120 outer domain. These results provide a new context for interpreting gp120 core envelope structure–function relationships. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Enhanced dynamics of HIV gp120 glycoprotein by small molecule binding

HIV cell entry and infection are driven by binding events to the CD4 and chemokine receptors with associated conformational change of the viral glycoprotein, gp120. Scyllatoxin miniprotein CD4 mimetics and a small molecule inhibitor of CD4 binding, NBD-556, also effectively induce gp120 conformational change. In this study we examine the fluctuation profile of gp120 in context of CD4, a miniprotein mimetic, and NBD-556 with the aim of understanding the effect of ligand binding on gp120 conformational dynamics. Analysis of molecular dynamics trajectories indicate that NBD-556 binding in the Phe 43 cavity enhances the overall mobility of gp120, especially in the outer domain in comparison to CD4 or miniprotein bound complex. Interactions with the more flexible bridging sheet strengthen upon NBD-556 binding and may contribute to gp120 restructuring. The enhanced mobility of D368, E370, and I371 with NBD-556 bound in the Phe 43 cavity suggests that interactions with α3-helix in the outer domain are not optimal, providing further insights into gp120--small molecule interactions that may impact small molecule designs.     

A beta strand lock exchange for signal transduction in TonB-dependent transducers on the basis of a common structural motif

Transport of molecules larger than 600 Da across the outer membrane involves TonB-dependent receptors and TonB-ExbB-ExbD of the inner membrane. The transport is energy consuming, and involves direct interactions between a short N-terminal sequence of receptor, called the TonB box, and TonB. We solved the structure of the ferric pyoverdine (Pvd-Fe) outer membrane receptor FpvA from Pseudomonas aeruginosa in its apo form. Structure analyses show that residues of the TonB box are in a beta strand which interacts through a mixed four-stranded beta sheet with the periplasmic signaling domain involved in interactions with an inner membrane sigma regulator. In this conformation, the TonB box cannot form a four-stranded beta sheet with TonB. The FhuA-TonB or BtuB-TonB structures show that the TonB-FpvA interactions require a conformational change which involves a beta strand lock-exchange mechanism. This mechanism is compatible with movements of the periplasmic domain deduced from crystallographic analyses of FpvA, FpvA-Pvd, and FpvA-Pvd-Fe.     

Mutagenic stabilization and/or disruption of a CD4-bound state reveals distinct conformations of the human immunodeficiency virus type 1 gp120 envelope glycoprotein

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein is conformationally flexible. Upon binding to the host cell receptor CD4, gp120 assumes a conformation that is recognized by the second receptor, CCR5 and/or CXCR4, and by the CD4-induced (CD4i) antibodies. Guided by the X-ray crystal structure of a gp120-CD4-CD4i antibody complex, we introduced changes into gp120 that were designed to stabilize or disrupt this conformation. One mutant, 375 S/W, in which the tryptophan indole group is predicted to occupy the Phe 43 cavity in the gp120 interior, apparently favors a gp120 conformation closer to that of the CD4-bound state. The 375 S/W mutant was recognized as well as or better than wild-type gp120 by CD4 and CD4i antibodies, and the large decrease in entropy observed when wild-type gp120 bound CD4 was reduced for the 375 S/W mutant. The recognition of the 375 S/W mutant by CD4BS antibodies, which are directed against the CD4-binding region of gp120, was markedly reduced compared with that of the wild-type gp120. Compared with the wild-type virus, viruses with the 375 S/W envelope glycoproteins were resistant to neutralization by IgG1b12, a CD4BS antibody, were slightly more sensitive to soluble CD4 neutralization and were neutralized more efficiently by the 2G12 antibody. Another mutant, 423 I/P, in which the gp120 bridging sheet was disrupted, did not bind CD4, CCR5, or CD4i antibodies, even though recognition by CD4BS antibodies was efficient. These results indicate that CD4BS antibodies recognize conformations of gp120 different from that recognized by CD4 and CD4i antibodies.     

Localized changes in the gp120 envelope glycoprotein confer resistance to human immunodeficiency virus entry inhibitors BMS-806 and #155

BMS-806 and the related compound, #155, are novel inhibitors of human immunodeficiency virus type 1 (HIV-1) entry that bind the gp120 exterior envelope glycoprotein. BMS-806 and #155 block conformational changes in the HIV-1 envelope glycoproteins that are induced by binding to the host cell receptor, CD4. We tested a panel of HIV-1 envelope glycoprotein mutants and identified several that were resistant to the antiviral effects of BMS-806 and #155. In the CD4-bound conformation of gp120, the amino acid residues implicated in BMS-806 and #155 resistance line the "phenylalanine 43 cavity" and a water-filled channel that extends from this cavity to the inner domain. Structural considerations suggest a model in which BMS-806 and #155 bind gp120 prior to receptor binding and, upon CD4 binding, are accommodated in the Phe-43 cavity and adjacent channel. The integrity of the nearby V1/V2 variable loops and N-linked carbohydrates on the V1/V2 stem indirectly influences sensitivity to the drugs. A putative binding site for BMS-806 and #155 between the gp120 receptor-binding regions and the inner domain, which is thought to interact with the gp41 transmembrane envelope glycoprotein, helps to explain the mode of action of these drugs.     

Molecular dynamics simulations of the unfolding of beta(2)-microglobulin and its variants

In this study, we examined the unfolding processes of native beta(2)-microglobulin and two related variants, one with an N-terminal hexapeptide deletion DeltaN6 and another with Lys57-Asp58 cleavage, by high-temperature molecular dynamics simulations. Three simulation models were used, molecular dynamics (MD) simulations with explicit water solvation, MD simulations with the CHARMM EEF1 force field and Langevin dynamics with the CHARMM EEF1 force field. Our simulations reproduce many of the experimentally observed structural changes. The most striking agreement is in the beta-strands to alpha-helix transition. In our simulations, strands beta(3), beta(4) and beta(5) consistently change to alpha-helix, whereas beta(8) changes to an alpha-helix only briefly. Through comparisons of the conformational behavior of the native, the DeltaN6 and the Lys-cut beta(2)-m, using the three simulation methods, we identified the consensus conformational changes that differentiate between the native beta(2)-m and its two variants. We found that the main effect of the removal of the N-terminal hexapeptide is to increase the separation between strands beta(2) and beta(6) and to facilitate the beta to alpha transition. On the other hand, the lysine cleavage only increases the flexibility of strand beta(5) and does not affect the interactions between strands beta(2) and beta(6). These conformational changes may relate to polymerization tendencies of these variants.     

Lineage-Specific Differences between Human and Simian Immunodeficiency Virus Regulation of gp120 Trimer Association and CD4 Binding

Metastable conformations of the gp120 and gp41 envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) must be maintained in the unliganded state of the envelope glycoprotein trimer. Binding of gp120 to the primary receptor, CD4, triggers the transition to an open conformation of the trimer, promoting interaction with the CCR5 chemokine receptor and ultimately leading to gp41-mediated virus-cell membrane fusion and entry. Topological layers in the gp120 inner domain contribute to gp120-trimer association in the unliganded state and to CD4 binding. Here we describe similarities and differences between HIV-1 and SIVmac gp120. In both viruses, the gp120 N/C termini and the inner domain β-sandwich and layer 2 support the noncovalent association of gp120 with the envelope glycoprotein trimer. Layer 1 of the SIVmac gp120 inner domain contributes more to trimer association than the corresponding region of HIV-1 gp120. On the other hand, layer 1 plays an important role in stabilizing the CD4-bound conformation of HIV-1 but not SIVmac gp120 and thus contributes to HIV-1 binding to CD4. In SIVmac, CD4 binding is instead enhanced by tryptophan 375, which fills the Phe 43 cavity of gp120. Activation of SIVmac by soluble CD4 is dependent on tryptophan 375 and on layer 1 residues that determine a tight association of gp120 with the trimer. Distinct biological requirements for CD4 usage have resulted in lineage-specific differences in the HIV-1 and SIV gp120 structures that modulate trimer association and CD4 binding.     

Conformational rearrangement within the soluble domains of the CD4 receptor is ligand-specific

Ligand binding induces shape changes within the four modular ectodomains (D1-D4) of the CD4 receptor, an important receptor in immune signaling. Small angle x-ray scattering (SAXS) on both a two-domain and a four-domain construct of the soluble CD4 (sCD4) is consistent with known crystal structures demonstrating a bilobal and a semi-extended tetralobal Z conformation in solution, respectively. Detection of conformational changes within sCD4 as a result of ligand binding was followed by SAXS on sCD4 bound to two different glycoprotein ligands: the tick saliva immunosuppressor Salp15 and the HIV-1 envelope protein gp120. Ab initio modeling of these data showed that both Salp15 and gp120 bind to the D1 domain of sCD4 and yet induce drastically different structural rearrangements. Upon binding, Salp15 primarily distorts the characteristic lobal architecture of the sCD4 without significantly altering the semi-extended shape of the sCD4 receptor. In sharp contrast, the interaction of gp120 with sCD4 induces a shape change within sCD4 that can be described as a Z-to-U bi-fold closure of the four domains across its flexible D2-D3 linker. Placement of known crystal structures within the boundaries of the SAXS-derived models suggests that the ligand-induced shape changes could be a result of conformational changes within this D2-D3 linker. Functionally, the observed shape changes in CD4 receptor causes dissociation of lymphocyte kinase from the cytoplasmic domain of Salp15-bound CD4 and facilitates an interaction between the exposed V3 loops of CD4-bound gp120 molecule to the extracellular loops of its co-receptor, a step essential for HIV-1 viral entry.     

Molecular mechanism of HIV-1 gp120 mutations that reduce CD4 binding affinity

The interaction of the HIV-1 fusion protein gp120 with its cellular receptor CD4 represents a crucial step of the viral infection process, thus rendering gp120 a promising target for the intervention with anti-HIV drugs. Naturally occurring mutations of gp120, however, can decrease its affinity for anti-infective ligands like therapeutic antibodies or soluble CD4. To understand this phenomenon on a structural level, we performed molecular dynamics simulations of two gp120 variants (termed gp120_3-2 and gp120_2-1), which exhibit a significantly decreased binding of soluble CD4. In both variants, the exchange of a nonpolar residue byglutamate was identified as an important determinant for reduced binding. However, those glutamates are located at different sequence positions and affect different steps of the recognition process: E471 in gp120_3-2 predominantly affects the CD4-bound conformation, whereas E372 in gp120_2-1 mainly modulates the conformational sampling of free gp120. Despite these differences, there exists an interesting similarity between the two variants: both glutamates exert their function by modulating the conformation and interactions of glycine-rich motifs (G366–G367, G471–G473) resulting in an accumulation of binding incompetent gp120 conformations or a loss of intermolecular gp120–CD4 hydrogen bonds. Thus, the present data suggests that interference with the structure and dynamics of glycine-rich stretches might represent a more widespread mechanism, by which gp120 mutations reduce binding affinity. This knowledge should be helpful to predict the resistance of novel gp120 mutations or to design gp120–ligands with improved binding properties.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:41     

Molecular dynamics simulations of a bacterial autotransporter: NalP from Neisseria meningitidis

NalP is an autotransporter secretory protein found in the outer membrane of Neisseria meningitidis. The crystal structure of the NalP translocator domain revealed a transmembrane beta-barrel containing a central alpha-helix. The role of this alpha-helix, and of the conformational dynamics of the beta-barrel pore have been studied via atomistic molecular dynamics simulations. Three simulations, each of 10 ns duration, of NalP embedded within a solvated DMPC bilayer were performed. The helix was removed from the barrel interior in one simulation. The conformational stability of the protein is similar to that of other outer membrane proteins, e.g., OmpA, in comparable simulations. The transmembrane beta-barrel is stable even in the absence of the alpha-helix. Removal of the helix results in an influx of water into the pore region, suggesting the helix acts as a 'plug'. Water molecules entering the resultant pore form hydrogen bonds with the barrel lining that compensate for the loss of helix-barrel hydrogen bonds. The dimensions of the pore fluctuate over the course of the simulation revealing it to be flexible, but only wide enough to allow transport of the passenger domain in an unfolded or extended conformation. The simulations help us to understand the role of the central helix in plugging the pore and in maintaining the width of the barrel, and show that the NalP monomer is sufficient for the transport of the passenger domain in an unfolded or extended conformation.     

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure–function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca²⁺ removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca²⁺ removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein–ligand binding, including the concept of the free energy landscape (FEL) of the protein–solvent system, how the ruggedness and variability of FEL determine protein’s dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth.     

Specific structure appears at the N terminus in the sub-millisecond folding intermediate of the alpha subunit of tryptophan synthase, a TIM barrel protein

Competing views of the products of sub-millisecond folding reactions observed in many globular proteins have been ascribed either to the formation of discrete, partially folded states or to the random collapse of the unfolded chain under native-favoring conditions. To test the validity of these alternative interpretations for the stopped-flow burst-phase reaction in the (betaalpha)8, TIM barrel motif, a series of alanine replacements were made at five different leucine or isoleucine residues in the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli. This protein has been proposed to fold, in the sub-millisecond time range, to an off-pathway intermediate with significant stability and approximately 50% of the far-UV circular dichroism (CD) signal of the native conformation. Individual alanine replacements at any of three isoleucine or leucine residues in either alpha1, beta2 or beta3 completely eliminate the off-pathway species. These variants, within 5 ms, access an intermediate whose properties closely resemble those of an on-pathway equilibrium intermediate that is highly populated at moderate urea concentrations in wild-type alphaTS. By contrast, alanine replacements for leucine residues in either beta4 or beta6 destabilize but preserve the off-pathway, burst-phase species. When considered with complementary thermodynamic and kinetic data, this mutational analysis demonstrates that the sub-millisecond appearance of CD signal for alphaTS reflects the acquisition of secondary structure in a distinct thermodynamic state, not the random collapse of an unfolded chain. The contrasting results for replacements in the contiguous alpha1/beta2/beta3 domain and the C-terminal beta4 and beta6 strands imply a heterogeneous structure for the burst-phase species. The alpha1/beta2/beta3 domain appears to be tightly packed, and the C terminus appears to behave as a molten-globule-like structure whose folding is tightly coupled to that of the alpha1/beta2/beta3 domain.     

Heavy Chain-Only IgG2b Llama Antibody Effects Near-Pan HIV-1 Neutralization by Recognizing a CD4-Induced Epitope That Includes Elements of Coreceptor- and CD4-Binding Sites

The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and β19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor- and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy.     

Beta-hairpin folding mechanism of a nine-residue peptide revealed from molecular dynamics simulations in explicit water

The beta-hairpin fold mechanism of a nine-residue peptide, which is modified from the beta-hairpin of alpha-amylase inhibitor tendamistat (residues 15-23), is studied through direct folding simulations in explicit water at native folding conditions. Three 300-nanosecond self-guided molecular dynamics (SGMD) simulations have revealed a series of beta-hairpin folding events. During these simulations, the peptide folds repeatedly into a major cluster of beta-hairpin structures, which agree well with nuclear magnetic resonance experimental observations. This major cluster is found to have the minimum conformational free energy among all sampled conformations. This peptide also folds into many other beta-hairpin structures, which represent some local free energy minimum states. In the unfolded state, the N-terminal residues of the peptide, Tyr-1, Gln-2, and Asn-3, have a confined conformational distribution. This confinement makes beta-hairpin the only energetically favored structure to fold. The unfolded state of this peptide is populated with conformations with non-native intrapeptide interactions. This peptide goes through fully hydrated conformations to eliminate non-native interactions before folding into a beta-hairpin. The folding of a beta-hairpin starts with side-chain interactions, which bring two strands together to form interstrand hydrogen bonds. The unfolding of the beta-hairpin is not simply the reverse of the folding process. Comparing unfolding simulations using MD and SGMD methods demonstrate that SGMD simulations can qualitatively reproduce the kinetics of the peptide system.