Modulation of the acetylation state of histones plays a pivotal role in the regulation of gene expression. Histone deacetylases (HDACs) catalyze the removal of acetyl groups from lysines near the N termini of histones. This reaction promotes the condensation of chromatin, leading to repression of transcription. HDAC deregulation has been linked to several types of cancer, suggesting a potential use for HDAC inhibitors in oncology. Here we describe the first crystal structures of a human HDAC: the structures of human HDAC8 complexed with four structurally diverse hydroxamate inhibitors. This work sheds light on the catalytic mechanism of the HDACs, and on differences in substrate specificity across the HDAC family. The structure also suggests how phosphorylation of Ser39 affects HDAC8 activity.

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The structural mechanism by which non-structural protein 3 (NS3) from the hepatitis C virus (HCV) translocates along RNA is currently unknown. HCV NS3 is an ATP-dependent motor protein essential for viral replication and a member of the superfamily 2 (SF2) helicases. Crystallographic analysis using a labeled RNA oligonucleotide allowed us to unambiguously track the positional changes of RNA bound to full-length HCV NS3 during two discrete steps of the ATP hydrolytic cycle. The crystal structures of HCV NS3, NS3 bound to bromine-labeled RNA, and a tertiary complex of NS3 bound to labeled RNA and a non-hydrolyzable ATP analog provide a direct view of how large domain movements resulting from ATP binding and hydrolysis allow the enzyme to translocate along the phosphodiester backbone. While directional translocation of HCV NS3 by a single base pair per ATP hydrolyzed is observed, the 3’-end of the RNA does not shift register with respect to a conserved tryptophan residue, supporting a “spring-loading” mechanism that leads to larger steps by the enzyme as it moves along a nucleic acid substrate.

The crystal structure of the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas foetus has been determined and refined against X-ray data to 1.9 A resolution. T. foetus HGXPRTase crystallizes as an asymmetric dimer, with GMP bound to only one of the two molecules that form the asymmetric unit. Each molecule of HGXPRTase is formed by two lobes joined by a short "hinge" region, and the GMP binds in a cavity between the two lobes. A comparison of the two molecules in the asymmetric unit shows that the hinge region is flexible and that ligand binding affects the relative positions of the two lobes. The binding of GMP brings the two lobes closer together, rotating one lobe by about 5 degrees relative to the other. T. foetus appears to depend on HGXPRTase for its supply of GMP, making this enzyme a target for antiparasite drug design. A comparison of the structures of T. foetus HGXPRTase and human HGPRTase reveals that, while these enzymes retain a similar polypeptide fold, there are substantial differences between the active sites of these two homologs. These differences suggest that it will be possible to find compounds that selectively inhibit the parasite enzyme.

Background: Idelalisib is a PI3Kδ inhibitor used to treat hematological malignancies.Results: Idelalisib is selective, noncovalent, reversible, and ATP-competitive.Conclusion: The crystal structure helps explain the potency and selectivity of idelalisib. The biophysical and biochemical data clarify the details of the inhibitor's interactions with PI3Kδ.Significance: Its use in humans makes it important to understand how idelalisib inhibits PI3Kδ.

The X-ray structure of GMD reveals that it is a member of the short-chain dehydrogenase/reductase (SDR) family of proteins. We have modeled the binding of NADP and GDP-mannose to the enzyme and mutated four of the active-site residues to determine their function. The combined modeling and mutagenesis data suggests that at position 133 threonine substitutes serine as part of the serine-tyrosine-lysine catalytic triad common to the SDR family and Glu 135 functions as an active-site base.

Inosine-5'-monophosphate dehydrogenase (IMPDH) is an attractive drug target for the control of parasitic infections. The enzyme catalyzes the oxidation of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), the committed step in de novo guanosine monophosphate (GMP) biosynthesis. We have determined the crystal structures of IMPDH from the protozoan parasite Tritrichomonas foetus in the apo form at 2.3 A resolution and the enzyme-XMP complex at 2.6 A resolution. Each monomer of this tetrameric enzyme is comprised of two domains, the largest of which includes an eight-stranded parallel beta/alpha-barrel that contains the enzyme active site at the C termini of the barrel beta-strands. A second domain, comprised of residues 102-220, is disordered in the crystal. IMPDH is expected to be active as a tetramer, since the active site cavity is formed by strands from adjacent subunits. An intrasubunit disulfide bond, seen in the crystal structure, may stabilize the protein in a less active form, as high concentrations of reducing agent have been shown to increase enzyme activity. Disorder at the active site suggests that a high degree of flexibility may be inherent in the catalytic function of IMPDH. Unlike IMPDH from other species, the T. foetus enzyme has a single arginine that is largely responsible for coordinating the substrate phosphate in the active site. This structural uniqueness may facilitate structure-based identification and design of compounds that specifically inhibit the parasite enzyme.

Falcipain-2 (FP2), the major cysteine protease of the human malaria parasite Plasmodium falciparum, is a hemoglobinase and promising drug target. Here we report the crystal structure of FP2 in complex with a protease inhibitor, cystatin. The FP2 structure reveals two previously undescribed cysteine protease structural motifs, designated FP2 nose and FP2arm, in addition to details of the active site that will help focus inhibitor design. Unlike most cysteine proteases, FP2 does not require a prodomain but only the short FP2 nose motif to correctly fold and gain catalytic activity. Our structure and mutagenesis data suggest a molecular basis for this unique mechanism by highlighting the functional role of two Tyr within FP2 nose and a conserved Glu outside this motif. The FP2arm motif is required for hemoglobinase activity. The structure reveals topographic features and a negative charge cluster surrounding FP2 arm that suggest it may serve as an exo-site for hemoglobin binding. Motifs similar to FP2 nose and FP2arm are found only in related plasmodial proteases, suggesting that they confer malariaspecific functions.cysteine protease ͉ falcipain 2 ͉ inhibitor ͉ malaria ͉ x-ray structure A n estimated 500 million people are infected with malaria worldwide, resulting in over a million deaths annually, mainly among children in subSaharan Africa (www.who.int͞healthtopics͞ malaria͞en). There is an urgent need for new antimalarial therapy, because widespread drug resistance has limited the utility of most available treatments (1). Plasmodium falciparum is responsible for nearly all severe illness and death from malaria. During its life cycle within host red blood cells, the protozoan parasite relies on hemoglobin hydrolysis to supply amino acids for protein synthesis and to maintain osmotic stability (2, 3). The proteases involved in hemoglobin hydrolysis have been validated as promising drug targets (2). Both aspartyl proteases (plasmepsins) and cysteine proteases (falcipains) play a role. Inhibition of either protease class is lethal to the parasite, and inhibition of both is synergistic for parasite killing (4-6). Falcipain-2 (FP2), falcipain-2Ј (FP2Ј), and falcipain-3 (FP3) are papain-family (C1) Clan CA cysteine proteases that cleave native or denatured human hemoglobin (7,8). FP2, the most-abundant and best-studied enzyme among the falcipains, is a prime target for drug development. When the FP2 gene was deleted from P. falciparum by homologous recombination, trophozoites accumulated undegraded hemoglobin (9). Moreover, synthetic inhibitors targeting C1 proteases halted P. falciparum growth in cell culture and cured malaria in animal models (6,(10)(11)(12).When compared with family C1 proteases from other organisms, mature falcipains contain two unique sequence motifs: an extension at the N terminus (17 aa for FP2) and an insertion between the catalytic His and Asn near the C terminus (14 aa for FP2). Although the N-terminal extension has been shown to mediate folding of FP2 in the absence of a prodomain (13,14), ...