Broad Neutralization of Viral Pathogens

Influenza, Hepatitis C, and HIV-1 continue to constitute significant threats to global health. We have structurally and functionally characterized several potent, broadly neutralizing antibodies (bnAbs) against HIV-1, influenza and hepatitis C viruses. The surface antigens of these viruses are the main target of neutralizing antibodies. However, most antibodies are strain-specific and protect only against highly related strains within the same subtype. Recently, a number of antibodies have been identified that are much broader and neutralize across multiple subtypes and types of these viruses through binding to functionally conserved sites, such as the receptor binding site or the fusion domain. For example, co-crystal structures of bnAbs with influenza hemagglutinin (HA) identified highly conserved sites in the fusion domain (stem) and in the receptor binding site (head) as target for broad neutralization[1]. HCV is also genetically diverse, but some antibodies have potent neutralizing activity across most genotypes of the virus. One family of these antibodies targets a conserved antigenic site on the HCV E2 envelope glycoprotein that overlaps with the CD81 receptor-binding site[2]. For HIV-1, structural and functional characterization of different families of bnAbs have led to identification of novel epitopes on HIV-1 Env, many of which involve glycans. These glycan-dependent Abs have unique features that enable them to penetrate the glycan shield and bind complex epitopes that consist of sugars and underlying protein segments on gp120 on HIV-1 Env. Recent x-ray[3] and EM structures of a soluble form of HIV-1 Env have revealed that the epitopes are more extensive and complex than previously appreciated. This structural information is now being used to aid in structure-assisted vaccine design for HIV-1, HCV and for a more universal flu vaccine. IAW is supported by NIH grants AI100663, AI082362, AI84817, AI099275 and GM094586 and the Crucell Vaccine Institute.

Bovine antibodies with ultra long H3 Complementarity Determining Regions

About 10% of the bovine antibody repertoire exhibit extremely long H3 complementarity determining regions (CDRs). These H3 CDRs are usually described as `loops' in the more familiar mouse and human antibody Fab structures, but the ultra long bovine H3 CDRs are actually small, cysteine-rich protein domains that vary in size from 44 to 64 amino acids. We have recently determined the structures for two bovine antibody Fab fragments, and will describe these, as well as compare them with two other previously determined bovine Fab structures (Wang et al., Cell, 2013). One new Fab has a relatively short H3 CDR region of 44 residues, with just one disulfide bond, while the other boasts one of the longest H3 CDRs, with 63 residues and four disulfide bonds. These H3 CDRs fold to form apparently rigid `stem' regions, that present the disulfide bonded `knob' domain far above the five other Fab CDR loops. Despite extreme diversity in sequence, length and disulfide bonding patterns, the CDRs share structural homology, both in their long stems and in the more variable knob regions.

JCSG - Adapting Structural Genomics to Eukaryotic Complexes

For over a decade, the Joint Center for Structural Genomics (JCSG.org) has been at the forefront of developing tools and methodologies that enable the application of high-throughput structural biology (HTBSB) approaches to a broad range of challenging biological and biomedical problems. In PSI:Biology (2010-2015) to meet the challenges and embrace the opportunities that arise from our Partnerships projects, we have leveraged our gene-to-structure pipeline to explore challenging projects focused on structural characterization of interaction networks involved in stem cell regulation, T-cell activation and nuclear receptor signaling. These highly collaborative efforts have enabled development of systematic and integrative approaches for identifying and investigating networks of key multi-domain eukaryotic proteins and higher order assemblies of multi-component eukaryotic protein/protein and protein/nucleic acid complexes. In parallel, our biomedical theme project has focused on investigating host/microbe interactions of the microbial communities that inhabit specific niches and environments of the human body, e.g. the human gut microbiome. These efforts to date have been centered on secreted proteins from commensal bacteria in the human gut. The symbiotic relationship and influence on human development, physiology, immunity, and nutrition represent an exciting new frontier for HTBSB where we can investigate how these microorganisms contribute to human health as well as to disease. The JCSG also strives to promote widespread use of PSI resources, materials, methodologies and data to the general scientific community, via Community Nominated Target (CNT) projects and development and use of new technologies and methodologies. We also continue to contribute to the original PSI mission of structural coverage of the expanding protein universe. Supported by NIH U54-GM094586.

Understanding disease mutations from the crystal structure of SOX9 with DNA

SRY(Sex determining Region Y)-box or SOX transcription factors are important in early development and maintenance of different cell pools after birth. Of the ~20 SOX proteins (SRY, SOX1-SOX15, SOX17, SOX18, SOX21 and SOX30), SOX2, SOX9 and SOX10 mutations are primarily disease-associated: SOX2 with Combined Pituitary Hormone Deficiency, Microphthalmia, Septo-optic dysplasia and anophthalmic syndrome; SOX9 with Campomelic Dysplasia (affects development of the reproductive and skeletal system); and SOX10 (~94% sequence identity to SOX9) with Waardenburg Syndrome (affects audition and pigmentation in hair, eyes and skin; and specifically with WS types 2 and 4). As part of our Protein Structure Initiative (PSI)-Biology partnership, we performed structural and mutational analyses including x-ray crystallography and surface plasmon resonance assays, on the DNA-binding HMG domain of SOX9 with duplex DNA. Crystals were obtained in C222 space group and the structure was determined by molecular replacement to 2.77 Å resolution with final Rcryst/Rfree of 24.8/27.8%. The overall structure of the SOX9-DNA complex is similar to other SOX/SRY protein complexes. The SOX9-DNA protein-DNA interactions suggested a panel of mutations to assay for biochemical activity, which allowed us to understand the molecular basis of five mutations identified in Campomelic Dysplasia. These mutated residues have direct contact with DNA as well as indirect contacts, i.e., these mutations lead to allosteric secondary structure changes in the protein, which affect residues in direct contact with DNA. Due to the very high sequence identity between SOX9 and SOX10, our crystal structure also helps to rationalize the effect of SOX10 mutations in Waardenburg Syndrome. This work is supported by NIH grants U54 GM094586 and U01 GM094614. SSRL operations are funded by DOE BES, and the SSRL SMB program by DOE BER, NIH NCRR BTP and NIH NIGMS.