Zhen-Yu J. Sun

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-A 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.

The RAG1 and RAG2 proteins catalyze V(D)J recombination and are essential for generation of the diverse repertoire of antigen receptor genes and effective immune responses. RAG2 is composed of a "core" domain that is required for the recombination reaction and a C-terminal nonessential or "non-core" region. Recent evidence has emerged arguing that the non-core region plays a critical regulatory role in the recombination reaction, and mutations in this region have been identified in patients with immunodeficiencies. Here we present the first structural data for the RAG2 protein, using NMR spectroscopy to demonstrate that the C terminus of RAG2 contains a noncanonical PHD finger. All of the non-core mutations of RAG2 that are implicated in the development of immunodeficiencies are located within the PHD finger, at either zinc-coordinating residues or residues adjacent to an alpha-helix on the surface of the domain that participates in binding to the signaling molecules, phosphoinositides. Functional analysis of disease and phosphoinositide-binding mutations reveals novel intramolecular interactions within the non-core region and suggests that the PHD finger adopts two distinct states. We propose a model in which the equilibrium between these states modulates recombination activity. Together, these data identify the PHD finger as a novel and functionally important domain of RAG2.

The initial step of protein NMR resonance assignments typically identifies the sequence positions of 1H-15N HSQC cross-peaks. This is usually achieved by tediously comparing strips of multiple triple-resonance experiments. More conveniently, this could be obtained directly with hNcaNH and hNcocaNH-type experiments. However, in large proteins and at very high fields, rapid transverse relaxation severely limits the sensitivity of these experiments, and the limited spectral resolution obtainable in conventionally recorded experiments leaves many assignments ambiguous. We have developed alternative hNcaNH experiments that overcome most of these limitations. The TROSY technique was implemented for semiconstant time evolutions in both indirect dimensions, which results in remarkable sensitivity and resolution enhancements. Non-uniform sampling in both indirect dimensions combined with Maximum Entropy (MaxEnt) reconstruction enables such dramatic resolution enhancement while maintaining short measuring times. Experiments are presented that provide either bidirectional or unidirectional connectivities. The experiments do not involve carbonyl coherences and thus do not suffer from fast chemical shift anisotropy-mediated relaxation otherwise encountered at very high fields. The method was applied to a 300 microM sample of a 37 kDa fragment of the E. coli enterobactin synthetase module EntF, for which high-resolution spectra with an excellent signal-to-noise ratio were obtained within 4 days each.