Summary Novel inhibitors are needed to counteract the rapid emergence of drug-resistant HIV variants. HIV-1 reverse transcriptase (RT) has both DNA polymerase and RNase H (RNH) enzymatic activities, but approved drugs that inhibit RT target the polymerase. Inhibitors that act against new targets, like RNH, would be effective against all of the current drug-resistant variants. Here, we present 2.80 Å and 2.04 Å resolution crystal structures of an RNH inhibitor, β-thujaplicinol, bound at the RNH active site of both HIV-1 RT and an isolated RNH domain. β-thujaplicinol chelates two divalent metal ions at the RNH active site. We provide biochemical evidence that β-thujaplicinol is a slow-binding RNH inhibitor with non-competitive kinetics and suggest that it forms a tropylium ion that interacts favorably with RT and the RNA:DNA substrate.

We define the target, mechanism, and structural basis of inhibition of bacterial RNA polymerase (RNAP) by the tetramic acid antibiotic streptolydigin (Stl). Stl binds to a site adjacent to but not overlapping the RNAP active center and stabilizes an RNAP-active-center conformational state with a straight-bridge helix. The results provide direct support for the proposals that alternative straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations exist and that cycling between straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations is required for RNAP function. The results set bounds on models for RNAP function and suggest strategies for design of novel antibacterial agents.

HIV-1 reverse transcriptase (RT) undergoes a series of conformational changes during viral replication and is a central target for antiretroviral therapy. The intrinsic flexibility of RT can provide novel allosteric sites for inhibition. Crystals of RT that diffract X-rays to better than 2 Å resolution facilitated the probing of RT for new druggable sites using fragment screening by X-ray crystallography. A total of 775 fragments were grouped into 143 cocktails, which were soaked into crystals of RT in complex with the non-nucleoside drug rilpivirine (TMC278). Seven new sites were discovered, including the Incoming Nucleotide Binding, Knuckles, NNRTI Adjacent, and 399 sites, located in the polymerase region of RT, and the 428, RNase H Primer Grip Adjacent, and 507 sites, located in the RNase H region. Three of these sites—Knuckles, NNRTI Adjacent, and Incoming Nucleotide Binding—are inhibitory and provide opportunities for discovery of new anti-AIDS drugs.

MacroH2A is a novel nucleosomal core histone that contains a large nonhistone region and a region that closely resembles a full length histone H2A. We have cloned a cDNA that contains the entire coding region of macroH2A1.2, one of the two identified subtypes of macroH2A1. MacroH2A1.2 was found to differ from the other known subtype, macroH2A1.1, in a single segment of the nonhistone region. MacroH2A1 specific antibodies revealed relatively high levels of both subtypes in adult liver and kidney. MacroH2A1.1 was much lower in fetal liver and kidney in comparison to their adult counterparts, and was not detected in adult thymus and testis, tissues with active cell division and differentiation. Both subtypes were present at very low levels or absent from mouse embryonic stem cells maintained in an undifferentiated state by growth in the presence of leukemia inhibitory factor. MacroH2A1.2 increased when the embryonic stem cells were induced to differentiate in vitro, while macroH2A1.1 remained undetectable. These results support the idea that macroH2A1.1 and macroH2A1.2 are functionally distinct, and suggest that changes in their expression may play a role in developmentally regulated changes in chromatin structure and function.

B lymphocyte stimulator (BLyS), a member of the tumor necrosis factor (TNF) superfamily, is a cytokine that induces B-cell proliferation and immunoglobulin secretion. We have determined the three-dimensional structure of BLyS to 2.0 A resolution and identified receptor recognition segments using limited proteolysis coupled with mass spectrometry. Similar to other structurally determined TNF-like ligands, the BLyS monomer is a beta-sandwich and oligomerizes to form a homotrimer. The receptor-binding region in BLyS is a deeper, more pronounced groove than in other cytokines. The conserved elements on the 'floor' of this groove allow for cytokine recognition of several structurally related receptors, whereas variations on the 'walls' and outer rims of the groove confer receptor specificity.