Histone deacetylases (HDACs)-an enzyme family that deacetylates histones and non-histone proteins-are implicated in human diseases such as cancer, and the first-generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 Å resolution crystal structure of a catalytically inactive HDAC8 active-site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active-site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active-site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis-conformation. A similar interaction is observed in a new hydroxamate inhibitor-HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild-type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/ Tyr306Phe mutants.

Mutations within PCSK9 (proprotein convertase subtilisin/ kexin type 9) are associated with dominant forms of familial hyper-and hypocholesterolemia. Although PCSK9 controls low density lipoprotein (LDL) receptor (LDLR) levels post-transcriptionally, several questions concerning its mode of action remain unanswered. We show that purified PCSK9 protein added to the medium of human endothelial kidney 293, HepG2, and Chinese hamster ovary cell lines decreases cellular LDL uptake in a dose-dependent manner. Using this cell-based assay of PCSK9 activity, we found that the relative potencies of several PCSK9 missense mutants (S127R and D374Y, associated with hypercholesterolemia, and R46L, associated with hypocholesterolemia) correlate with LDL cholesterol levels in humans carrying such mutations. Notably, we found that in vitro wild-type PCSK9 binds LDLR with an ϳ150-fold higher affinity at an acidic endosomal pH (K D ‫؍‬ 4.19 nM) compared with a neutral pH (K D ‫؍‬ 628 nM). We also demonstrate that wild-type PCSK9 and mutants S127R and R46L are internalized by cells to similar levels, whereas D374Y is more efficiently internalized, consistent with their affinities for LDLR at neutral pH. Finally, we show that LDL diminishes PCSK9 binding to LDLR in vitro and partially inhibits the effects of secreted PCSK9 on LDLR degradation in cell culture. Together, the results of our biochemical and cell-based experiments suggest a model in which secreted PCSK9 binds to LDLR and directs the trafficking of LDLR to the lysosomes for degradation.PCSK9 (proprotein convertase subtilisin/kexin type 9) encodes the ninth member of the mammalian proprotein convertase family of serine endoproteases. PCSK9 is translated as a 692-amino acid proprotein that includes several domains found in other proprotein convertases, including an N-terminal signal sequence, a prodomain, a catalytic domain, and a cysteine-rich C-terminal domain (1-3). The PCSK9 catalytic domain shares high sequence similarity with the proteinase K family of subtilases and contains a catalytic triad (Asp 186 , His 226 , and Ser 386 ) responsible for autoprocessing (1, 4). PCSK9 processing occurs in the secretory pathway, presumably in the endoplasmic reticulum, and results in proteolytic cleavage occurring after Gln 152 (FAQ2SIP). This cleavage generates a stable PCSK9 heterodimer composed of a 14-kDa prodomain fragment and a mature 57-kDa fragment containing the catalytic and C-terminal domains (4, 5). Following processing, the PCSK9 heterodimer exits the ER and is eventually secreted (1). The prodomain of PCSK9 remains strongly bound to the mature protein after secretion, presumably inhibiting the catalytic activity of PCSK9 (1, 5, 6). To date, there is no conclusive evidence that the processed secreted form of PCSK9 can cleave any substrates in a catalytic serine-dependent manner.The first evidence that PCSK9 plays a significant role in regulating plasma low density lipoprotein (LDL) 3 cholesterol (LDL-C) levels was the identification of several missense mutations in PCS...

Elicitation of potent and broadly neutralizing antibodies is an important goal in designing an effective human immunodeficiency virus-1 (HIV-1) vaccine. The HIV-1 gp41 inner-core trimer represents a functionally and structurally conserved target for therapeutics. Here we report the 2.0-A-resolution crystal structure of the complex between the antigen-binding fragment of D5, an HIV-1 cross-neutralizing antibody, and 5-helix, a gp41 inner-core mimetic. Both binding and neutralization depend on residues in the D5 CDR H2 loop protruding into the conserved gp41 hydrophobic pocket, as well as a large pocket in D5 surrounding core gp41 residues. Kinetic analysis of D5 mutants with perturbed D5-gp41 interactions suggests that D5 persistence at the fusion intermediate is crucial for neutralization. Thus, our data validate the gp41 N-peptide trimer fusion intermediate as a target for neutralizing antibodies and provide a template for identification of more potent and broadly neutralizing molecules.

HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H͞I1-BMV-D5 (D5), that binds to HR1 and inhibits the assembly of fusion intermediates in vitro. D5 inhibits the replication of diverse HIV-1 clinical isolates and therefore represents a previously unknown example of a crossneutralizing IgG selected by binding to designed antigens. NMR studies and functional analyses map the D5-binding site to a previously identified hydrophobic pocket situated in the HR1 groove. This hydrophobic pocket was proposed as a drug target and subsequently identified as a common binding site for peptide and peptidomimetic fusion inhibitors. The finding that the D5 fusioninhibitory antibody shares the same binding site suggests that the hydrophobic pocket is a ''hot spot'' for fusion inhibition and an ideal target on which to focus a vaccine-elicited antibody response. Our data provide a structural framework for the design of new immunogens and therapeutic antibodies with crossneutralizing potential.envelope ͉ fusion ͉ prehairpin ͉ vaccine

Haem binding to human serum albumin (HSA) endows the protein with peculiar spectroscopic properties. Here, the effect of ibuprofen and warfarin on the spectroscopic properties of ferric haem–human serum albumin (ferric HSA–haem) and of ferrous nitrosylated haem–human serum albumin (ferrous HSA–haem‐NO) is reported. Ferric HSA–haem is hexa‐coordinated, the haem‐iron atom being bonded to His105 and Tyr148. Upon drug binding to the warfarin primary site, the displacement of water molecules − buried in close proximity to the haem binding pocket − induces perturbation of the electronic absorbance properties of the chromophore without affecting the coordination number or the spin state of the haem‐iron, and the quenching of the 1H‐NMR relaxivity. Values of Kd for ibuprofen and warfarin binding to the warfarin primary site of ferric HSA–haem, corresponding to the ibuprofen secondary cleft, are 5.4 ± 1.1 × 10−4 m and 2.1 ± 0.4 × 10−5 m, respectively. The affinity of ibuprofen and warfarin for the warfarin primary cleft of ferric HSA–haem is lower than that reported for drug binding to haem‐free HSA. Accordingly, the Kd value for haem binding to HSA increases from 1.3 ± 0.2 × 10−8 m in the absence of drugs to 1.5 ± 0.2 × 10−7 m in the presence of ibuprofen and warfarin. Ferrous HSA–haem‐NO is a five‐coordinated haem‐iron system. Drug binding to the warfarin primary site of ferrous HSA–haem‐NO induces the transition towards the six‐coordinated haem‐iron species, the haem‐iron atom being bonded to His105. Remarkably, the ibuprofen primary cleft appears to be functionally and spectroscopically uncoupled from the haem site of HSA. Present results represent a clear‐cut evidence for the drug‐induced shift of allosteric equilibrium(a) of HSA.