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.

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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 ␣-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 noncore 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.During immune system development, immunoglobulin and T cell receptor genes are assembled from their component gene segments. This process, called V(D)J recombination, is initiated by the lymphoid-specific recombination activating genes 1 and 2 (RAG1 and RAG2) (1, 2). The RAG proteins recognize and bind recombination signal sequences flanking each coding segment and introduce double-strand DNA breaks, which are subsequently resolved into coding joints and signal joints. Processing and joining of the ends require the activity of the ubiquitously expressed proteins from the nonhomologous end joining pathway of DNA repair (reviewed in Ref.3). V(D)J recombination is critical for proper immune system function; accordingly, mutations in the RAG or nonhomologous end joining proteins result in immunodeficiencies, and inappropriate RAG activity can lead to genomic instability and cancer (4, 5).RAG2 can be divided into two functionally defined regions, an N-terminal "core" domain (amino acids 1-383) and a Cterminal "non-core" domain (amino acids 384 -527) (see Fig. 1A). The core domain is necessary and sufficient for carrying out V(D)J recombination in vivo on exogenous plasmid substrates (6, 7), as well as V(D)J cleavage in vitro (8). The noncore domain is dispensable for activity in both of these assays; however, its high conservation throughout evolution suggests it serves critical functions. Indeed, replacement of the endogenous RAG2 gene with only the core domain results in impaired development of B and T cells in mice (9, 10). Moreover, recent studies have implicated the C terminus of RAG2 in the restriction of RAG1/2-mediated transposition (11-13) ...

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.