Wayne A. Hendrickson

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody.

The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gpl20 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 Å resolution of an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding. The structure reveals a cavity-laden CD4–gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion. Our results provide a framework for understanding the complex biology of HIV entry into cells and should guide efforts to intervene.

The antigenic structure of the HIV gp120 envelope glycoprotein

The human immunodeficiency virus HIV-1 establishes persistent infections in humans which lead to acquired immunodeficiency syndrome (AIDS). The HIV-1 envelope glycoproteins, gp120 and gp41, are assembled into a trimeric complex that mediates virus entry into target cells. HIV-1 entry depends on the sequential interaction of the gp120 exterior envelope glycoprotein with the receptors on the cell, CD4 and members of the chemokine receptor family. The gp120 glycoprotein, which can be shed from the envelope complex, elicits both virus-neutralizing and non-neutralizing antibodies during natural infection. Antibodies that lack neutralizing activity are often directed against the gp120 regions that are occluded on the assembled trimer and which are exposed only upon shedding,. Neutralizing antibodies, by contrast, must access the functional envelope glycoprotein complex and typically recognize conserved or variable epitopes near the receptor-binding regions. Here we describe the spatial organization of conserved neutralization epitopes on gp120, using epitope maps in conjunction with the X-ray crystal structure of a ternary complex that includes a gp120 core, CD4 and a neutralizing antibody. A large fraction of the predicted accessible surface of gp120 in the trimer is composed of variable, heavily glycosylated core and loop structures that surround the receptor-binding regions. Understanding the structural basis for the ability of HIV-1 to evade the humoral immune response should assist in the design of a vaccine.

Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure.

An expression system has been established for the incorporation of selenomethionine into recombinant proteins produced from plasmids in Escherichia coli. Replacement of methionine by selenomethionine is demonstrated at the level of 100% for both T4 and E. coli thioredoxins. The natural recombinant proteins and the selenomethionyl variants of both thioredoxins crystallize isomorphously. Anomalous scattering factors were deduced from synchrotron X‐ray absorption measurements of crystals of the selenomethionyl proteins. Taken with reference to experience in the structural analysis of selenobiotinyl streptavidin by the method of multiwavelength anomalous diffraction (MAD), these data indicate that recombinant selenomethionyl proteins analyzed by MAD phasing offer a rather general means for the elucidation of atomic structures.

HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites

The ability of human immunodeficiency virus (HIV-1) to persist and cause AIDS is dependent on its avoidance of antibody-mediated neutralization. The virus elicits abundant, envelope-directed antibodies that have little neutralization capacity. This lack of neutralization is paradoxical, given the functional conservation and exposure of receptor-binding sites on the gp120 envelope glycoprotein, which are larger than the typical antibody footprint and should therefore be accessible for antibody binding. Because gp120–receptor interactions involve conformational reorganization, we measured the entropies of binding for 20 gp120-reactive antibodies. Here we show that recognition by receptor-binding-site antibodies induces conformational change. Correlation with neutralization potency and analysis of receptor–antibody thermodynamic cycles suggested a receptor-binding-site ‘conformational masking’ mechanism of neutralization escape. To understand how such an escape mechanism would be compatible with virus–receptor interactions, we tested a soluble dodecameric receptor molecule and found that it neutralized primary HIV-1 isolates with great potency, showing that simultaneous binding of viral envelope glycoproteins by multiple receptors creates sufficient avidity to compensate for such masking. Because this solution is available for cell-surface receptors but not for most antibodies, conformational masking enables HIV-1 to maintain receptor binding and simultaneously to resist neutralization.

Structure of the hydrophobic protein crambin determined directly from the anomalous scattering of sulphur

The highly ordered crystal structure of crambin has been solved at 1.5 Å resolution directly from the diffraction data of a native crystal at a wavelength remote from the sulphur absorption edge. The molecule has three disulphide bridges among its 46 amino acid residues, of which 46% are in helices and 17% are in a β-sheet. Crambin is shown to be an amphipathic protein, inasmuch as its six charged groups are segregated from hydrophobic surface elements. Phasing methods used here will also apply elsewhere.

Stereochemically restrained refinement of macromolecular structures

Publisher Summary The chapter focuses on the practical application of stereochemically-restrained refinement to macromolecular crystals. Details of computational procedures and minimization algorithms are treated and need not be considered in routine applications. However, it is important to understand the nature of the function being minimized. Thorough structural refinement has become an integral part of macromolecular crystallography. The chapter describes some extensions of the current export versions of the programs that have been implemented or are envisioned. Atomic motion and conformational heterogeneity (or disorder) is major impediments to successful refinement. The use of Fourier transformations to compute structure factors and gradient vectors might greatly improve speed for large problems. There are numerous other improvements that can be envisioned including a method for modeling the fluid solvent, an appropriate treatment of the correlation between occupancy and thermal parameters of discrete solvent molecules, restraints for nonbonded contacts from crystal packing, inclusion of attractive potentials for nonbonded contacts, provision for refining partial structures, and proper estimation of standard deviations. Extensions such as these are expected to be important in realizing the goal of producing refined structural models that reproduce the diffraction patterns to within the accuracy of the measured data and which are compatible with prior stereochemical knowledge of macromolecules.

A Structural Superfamily of Growth Factors Containing a Cystine Knot Motif

Neil Q. McDonald and Wayne A. Hendrickson Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute College of Physicians and Surgeons of Columbia University New York, New York 10032 The three-dimensional structures of proteins can provide important insights, sometimes unexpectedly, into the evo- lutionary relationships of proteins. Proteins may evolve from a common ancestor to the point at which they no longer share any significant overall sequence similarity (less than 20% identity), but their evolutionary relatedness may still be evident from a structural comparison. This is because those few invariant or conservatively replaced amino acid residues that are critical for the preservation of the three-dimensional structure can be identified from structural information. Locating such weaksignalsof relat- edness by sequence comparisons alone requires large numbers of sequences, whereas two related three- dimensional structures can be expected to show a good superposition of these invariant residues in topologically equivalent positions, despite great sequence divergence elsewhere. Polypeptide growth factors, a diverse group of regula- tory agents that control cell survival, proliferation, and dif- ferentiation, provide a good illustration of this phenome- non (Table 1). Nerve growth factor (NGF), transforming growth factor 82 (TGFj32), and platelet-derived growth fac- tor BB (PDGF-BB) have been found to adopt similar pro- tomeric structures that contain a motif we shall refer to as the cystine knot. These growth factors contain this motif and aconserved b-strand structure, arguing that they form a structural superfamily. The evolutionary and functional implications that arise from this observation, which is the focus of this minireview, are discussed here. The determination of the crystal structure of dimeric NGF revealed a novel three-dimensional fold. Each sub- unit consists of predominantly P-strand secondary struc- ture and an unusual clustering of three cystine bridges (McDonald et al., 1991). Subsequently, the structure of TGFj32 (Daopin et al., 1992a; Schlunegger and Griitter, 1992) showed a tertiary fold similar to NGF (Swindells, 1992; Murzin and Chothia, 1992). This observation has been confirmed by a structural comparison of the NGF and TGF82 molecules (Daopin et al., 1992b). The structure determination of PDGF-BB has provided another example of this structural fold (Oefner et al., 1992). Figure 1 shows a schematic summary of the three pro- tomer structures of NGF, TGFpP, and PDGF-BB that high- lights the common features characterizing the fold, partic- ularly the cystine knot motif (open circles) and two pairs of antiparallel 8 strands (labeled 81-84). In the NGF struc- ture, the disulfide bridges Cy~~-Cys~~ and Cys”-Cy~~~~ form a 1Cmembered ring through which the Cy~~~-Cys~ juncture passes. These three disulfide bridges have topo-

DC-SIGN-mediated internalization of HIV is required for trans-enhancement of T cell infection

Fusion of the human immunodeficiency virus (HIV) to the plasma membrane of target cells is mediated by interaction of its envelope glycoprotein, gp120, with CD4 and appropriate chemokine receptors. gp120 additionally binds to DC-SIGN, a C-type lectin expressed on immature dendritic cells. This interaction does not result in viral fusion, but instead contributes to enhanced infection in trans of target cells that express CD4 and chemokine receptors. Here we show that DC-SIGN mediates rapid internalization of intact HIV into a low pH nonlysosomal compartment. Internalized virus retains competence to infect target cells. Removal of the DC-SIGN cytoplasmic tail reduced viral uptake and abrogated the trans-enhancement of T cell infection. We propose that HIV binds to DC-SIGN to gain access to an intracellular compartment that contributes to augmentation or retention of viral infectivity.

Structure of human follicle-stimulating hormone in complex with its receptor

Follicle-stimulating hormone (FSH) is central to reproduction in mammals. It acts through a G-protein-coupled receptor on the surface of target cells to stimulate testicular and ovarian functions. We present here the 2.9-Å-resolution structure of a partially deglycosylated complex of human FSH bound to the extracellular hormone-binding domain of its receptor (FSHRHB). The hormone is bound in a hand-clasp fashion to an elongated, curved receptor. The buried interface of the complex is large (2,600 Å2) and has a high charge density. Our analysis suggests that all glycoprotein hormones bind to their receptors in this mode and that binding specificity is mediated by key interaction sites involving both the common α- and hormone-specific β-subunits. On binding, FSH undergoes a concerted conformational change that affects protruding loops implicated in receptor activation. The FSH–FSHRHB complexes form dimers in the crystal and at high concentrations in solution. Such dimers may participate in transmembrane signal transduction.

Structures of HIV-1 gp120 Envelope Glycoproteins from Laboratory-Adapted and Primary Isolates

BACKGROUND The gp120 exterior envelope glycoprotein of HIV-1 binds sequentially to CD4 and chemokine receptors on cells to initiate virus entry. During natural infection, gp120 is a primary target of the humoral immune response, and it has evolved to resist antibody-mediated neutralization. We previously reported the structure at 2.5 A of a gp120 core from the HXBc2 laboratory-adapted isolate in complex with a 2 domain fragment of CD4 and the antigen binding fragment of a human antibody. This revealed atomic details of gp120-receptor interactions and suggested multiple mechanisms of immune evasion. RESULTS We have now extended the HXBc2 structure in P222, crystals to 2.2 A. The enhanced resolution enabled a more accurate modeling of less-well-ordered regions and provided conclusive identification of the density in the central cavity at the crux of the gp120-CD4 interaction as isopropanol from the crystallization medium. We have also determined the structure of a gp120 core from the primary clinical HIV-1 isolate, YU2, in the same ternary complex but in a C2 crystal lattice. Comparisons of HXBc2 and YU2 showed that while CD4 binding was rigid, portions of the gp120 core were conformationally flexible; overall differences were minor, with sequence changes concentrated on a surface expected to be exposed on the envelope oligomer. CONCLUSIONS Despite dramatic antigenic differences between primary and laboratory-adapted HIV-1, the gp120 cores from these isolates are remarkably similar. Taken together with chimeric substitution and sequence analysis, this indicates that neutralization resistance is specified by quaternary interactions involving the major variable loops and thus affords a mechanism for viral adaptation. Conservation of the central cavity suggests the possibility of therapeutic inhibitors. The structures reported here extend in detail and generality our understanding of the biology of the gp120 envelope glycoprotein.