Zhen-Yu J. Sun

Structural basis for recruitment of CBP/p300 byhypoxia-inducible factor-1α

Adaptation to hypoxia is mediated by transactivation of hypoxia-responsive genes by hypoxia-inducible factor-1 (HIF-1) in complex with the CBP and p300 transcriptional coactivators. We report the solution structure of the cysteine/histidine-rich 1 (CH1) domain of p300 bound to the C-terminal transactivation domain of HIF-1α. CH1 has a triangular geometry composed of four α-helices with three intervening Zn2+-coordinating centers. CH1 serves as a scaffold for folding of the HIF-1α C-terminal transactivation domain, which forms a vise-like clamp on the CH1 domain that is stabilized by extensive hydrophobic and polar interactions. The structure reveals the mechanism of specific recognition of p300 by HIF-1α, and shows how HIF-1α transactivation is regulated by asparagine hydroxylation.

The rhesus rotavirus VP4 sialic acid binding domain has a galectin fold with a novel carbohydrate binding site

Cell attachment and membrane penetration are functions of the rotavirus outer capsid spike protein, VP4. An activating tryptic cleavage of VP4 produces the N‐terminal fragment, VP8*, which is the viral hemagglutinin and an important target of neutralizing antibodies. We have determined, by X‐ray crystallography, the atomic structure of the VP8* core bound to sialic acid and, by NMR spectroscopy, the structure of the unliganded VP8* core. The domain has the β‐sandwich fold of the galectins, a family of sugar binding proteins. The surface corresponding to the galectin carbohydrate binding site is blocked, and rotavirus VP8* instead binds sialic acid in a shallow groove between its two β‐sheets. There appears to be a small induced fit on binding. The residues that contact sialic acid are conserved in sialic acid‐dependent rotavirus strains. Neutralization escape mutations are widely distributed over the VP8* surface and cluster in four epitopes. From the fit of the VP8* core into the virion spikes, we propose that VP4 arose from the insertion of a host carbohydrate binding domain into a viral membrane interaction protein.

HIV-1 broadly neutralizing antibody extracts its epitope from a kinked gp41 ectodomain region on the viral membrane

Although rarely elicited during natural human infection, the most broadly neutralizing antibodies (BNAbs) against diverse human immunodeficiency virus (HIV)-1 strains target the membrane-proximal ectodomain region (MPER) of viral gp41. To gain insight into MPER antigenicity, immunogenicity, and viral function, we studied its structure in the lipid environment by a combination of nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and surface plasmon resonance (SPR) techniques. The analyses revealed a tilted N-terminal alpha helix (aa 664-672) connected via a short hinge to a flat C-terminal helical segment (675-683). This metastable L-shaped structure is immersed in viral membrane and, therefore, less accessible to immune attack. Nonetheless, the 4E10 BNAb extracts buried W672 and F673 after initial encounter with the surface-embedded MPER. The data suggest how BNAbs may perturb tryptophan residue-associated viral fusion involving the mobile N-terminal MPER segment and, given conservation of MPER sequences in HIV-1, HIV-2, and SIV, have important implications for structure-guided vaccine design.

Solution structure of the HIV-1 integrase-binding domain in LEDGF/p75

Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase (IN) in human cells. We have determined the NMR structure of the integrase-binding domain (IBD) in LEDGF and identified amino acid residues essential for the interaction. The IBD is a compact right-handed bundle composed of five α-helices. Based on folding topology, the IBD is structurally related to a diverse family of α-helical proteins that includes eukaryotic translation initiation factor eIF4G and karyopherin-β. LEDGF residues essential for the interaction with IN were localized to interhelical loop regions of the bundle structure. Interaction-defective IN mutants were previously shown to cripple replication although they retained catalytic function. The initial structure determination of a host cell factor that tightly binds to a retroviral enzyme lays the groundwork for understanding enzyme-host interactions important for viral replication.

Structure of a heterophilic adhesion complex between the human CD2 and CD58 (LFA-3) counterreceptors.

Interaction between CD2 and its counterreceptor, CD58 (LFA-3), on opposing cells optimizes immune recognition, facilitating contacts between helper T lymphocytes and antigen-presenting cells as well as between cytolytic effectors and target cells. Here, we report the crystal structure of the heterophilic adhesion complex between the amino-terminal domains of human CD2 and CD58. A strikingly asymmetric, orthogonal, face-to-face interaction involving the major beta sheets of the respective immunoglobulin-like domains with poor shape complementarity is revealed. In the virtual absence of hydrophobic forces, interdigitating charged amino acid side chains form hydrogen bonds and salt links at the interface (approximately 1200 A2), imparting a high degree of specificity albeit with low affinity (K(D) of approximately microM). These features explain CD2-CD58 dynamic binding, offering insights into interactions of related immunoglobulin superfamily receptors.

The αβ T Cell Receptor Is an Anisotropic Mechanosensor

Thymus-derived lymphocytes protect mammalian hosts against virus- or cancer-related cellular alterations through immune surveillance, eliminating diseased cells. In this process, T cell receptors (TCRs) mediate both recognition and T cell activation via their dimeric αβ, CD3ϵγ, CD3ϵδ, and CD3ζζ subunits using an unknown structural mechanism. Here, site-specific binding topology of anti-CD3 monoclonal antibodies (mAbs) and dynamic TCR quaternary change provide key clues. Agonist mAbs footprint to the membrane distal CD3ϵ lobe that they approach diagonally, adjacent to the lever-like Cβ FG loop that facilitates antigen (pMHC)-triggered activation. In contrast, a non-agonist mAb binds to the cleft between CD3ϵ and CD3γ in a perpendicular mode and is stimulatory only subsequent to an external tangential but not a normal force (∼50 piconewtons) applied via optical tweezers. Specific pMHC but not irrelevant pMHC activates a T cell upon application of a similar force. These findings suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into a biochemical signal upon specific pMHC ligation during immune surveillance. Activating anti-CD3 mAbs mimic this force via their intrinsic binding mode. A common TCR quaternary change rather than conformational alterations can better facilitate structural signal initiation, given the vast array of TCRs and their specific pMHC ligands.

Mechanisms Contributing to T Cell Receptor Signaling and Assembly Revealed by the Solution Structure of an Ectodomain Fragment of the CD3ϵγ Heterodimer

The T cell receptor (TCR) consists of genetically diverse disulfide-linked alpha and beta chains in noncovalent association with the invariant CD3 subunits. CD3 epsilon and CD3 gamma are integral components of both the TCR and pre-TCR. Here, we present the solution structure of a heterodimeric CD3 epsilon gamma ectodomain complex. A unique side-to-side hydrophobic interface between the two C2-set immunoglobulin-like domains and parallel pairing of their respective C-terminal beta strands are revealed. Mutational analysis confirms the importance of the distinctive linkage as well as the membrane proximal stalk motif (RxCxxCxE) for domain-domain association. These biochemical and structural analyses offer insights into the modular pairwise association of CD3 invariant chains. More importantly, the findings suggest how the rigidified CD3 elements participate in TCR-based signal transduction.

Broadly neutralizing anti-HIV-1 antibodies disrupt a hinge-related function of gp41 at the membrane interface

A vaccine capable of stimulating protective antiviral antibody responses is needed to curtail the global AIDS epidemic caused by HIV-1. Although rarely elicited during the course of natural infection or upon conventional vaccination, the membrane-proximal ectodomain region (MPER) of the HIV-1 glycoprotein of Mr 41,000 (gp41) envelope protein subunit is the target of 3 such human broadly neutralizing antibodies (BNAbs): 4E10, 2F5, and Z13e1. How these BNAbs bind to their lipid-embedded epitopes and mediate antiviral activity is unclear, but such information might offer important insight into a worldwide health imperative. Here, EPR and NMR techniques were used to define the manner in which these BNAbs differentially recognize viral membrane-encrypted residues configured within the L-shaped helix–hinge–helix MPER segment. Two distinct modes of antibody-mediated interference of viral infection were identified. 2F5, like 4E10, induces large conformational changes in the MPER relative to the membrane. However, although 4E10 straddles the hinge and extracts residues W672 and F673, 2F5 lifts up residues N-terminal to the hinge region, exposing L669 and W670. In contrast, Z13e1 effects little change in membrane orientation or conformation, but rather immobilizes the MPER hinge through extensive rigidifying surface contacts. Thus, BNAbs disrupt HIV-1 MPER fusogenic functions critical for virus entry into human CD4 T cells and macrophages either by preventing hinge motion or by perturbing MPER orientation. HIV-1 MPER features important for targeted vaccine design have been revealed, the implications of which extend to BNAb targets on other viral fusion proteins.

Antibody mechanics on a membrane-bound HIV segment essential for GP41-targeted viral neutralization

Broadly neutralizing antibodies such as 2F5 are directed against the membrane-proximal external region (MPER) of HIV-1 GP41 and recognize well-defined linear core sequences. These epitopes can be engrafted onto protein scaffolds to serve as immunogens with high structural fidelity. Although antibodies that bind to this core GP41 epitope can be elicited, they lack neutralizing activity. To understand this paradox, we used biophysical methods to investigate the binding of human 2F5 to the MPER in a membrane environment, where it resides in vivo. Recognition is stepwise, through a paratope more extensive than core binding site contacts alone, and dynamic rearrangement through an apparent scoop-like movement of heavy chain complementarity-determining region 3 (CDRH3) is essential for MPER extraction from the viral membrane. Core-epitope recognition on the virus requires the induction of conformational changes in both the MPER and the paratope. Hence, target neutralization through this lipid-embedded viral segment places stringent requirements on the plasticity of the antibody combining site.

High-Resolution Molecular and Antigen Structure of the VP8* Core of a Sialic Acid-Independent Human Rotavirus Strain

ABSTRACT The most intensively studied rotavirus strains initially attach to cells when the “heads” of their protruding spikes bind cell surface sialic acid. Rotavirus strains that cause disease in humans do not bind this ligand. The structure of the sialic acid binding head (the VP8* core) from the simian rotavirus strain RRV has been reported, and neutralization epitopes have been mapped onto its surface. We report here a 1.6-Å resolution crystal structure of the equivalent domain from the sialic acid-independent rotavirus strain DS-1, which causes gastroenteritis in humans. Although the RRV and DS-1 VP8* cores differ functionally, they share the same galectin-like fold. Differences between the RRV and DS-1 VP8* cores in the region that corresponds to the RRV sialic acid binding site make it unlikely that DS-1 VP8* binds an alternative carbohydrate ligand in this location. In the crystals, a surface cleft on each DS-1 VP8* core binds N-terminal residues from a neighboring molecule. This cleft may function as a ligand binding site during rotavirus replication. We also report an escape mutant analysis, which allows the mapping of heterotypic neutralizing epitopes recognized by human monoclonal antibodies onto the surface of the VP8* core. The distribution of escape mutations on the DS-1 VP8* core indicates that neutralizing antibodies that recognize VP8* of human rotavirus strains may bind a conformation of the spike that differs from those observed to date.