Refined structure of Cu-substituted alcohol dehydrogenase at 2.1 Å resolution

Liver alcohol dehydrogenase (LADH) is a ZnII-dependent dimeric enzyme. LADH with the active-site ZnII substituted by CuII resembles blue (type I) copper proteins by its spectroscopic characteristics. In this work we present the X-ray structure of the active site CuII-substituted LADH complex with NADH and dimethyl sulfoxide (DMSO). The structure was solved by molecular replacement. The space group is P21 with cell dimensions a = 44.4, b = 180.6, c = 50.8 Å and [beta] = 108°. There is one dimer of the enzyme in the asymmetric unit. The refinement was carried out to a crystallographic R-factor of 16.1% for 41 119 unique reflections in the resolution range 12.0 to 2.1 Å. The coordination geometry of CuII in LADH is compared with the active-site metal coordination in the Zn-LADH-NADH-DMSO complex and blue-copper proteins. The distances from the metal to the protein ligands (Cys46, His67 and Cys174) are similar for the ZnII and CuII ions. The distances of the O atom of the inhibitor DMSO to the CuII ion in the two subunits of the dimer are 3.19 and 3.45 Å. These are considerably longer than the corresponding distances for the ZnII enzyme, 2.19 and 2.15 Å. The CuII ion is positioned nearly in the plane of the three protein ligands (NS2) with a geometry similar to the trigonal arrangement of the three strongly bound ligands (N2S) in blue-copper proteins. This coordination probably accounts for the similarity of the spectral characteristics of CuII-LADH and type I copper proteins

Characterization of Two Malaria Parasite Organelle Translation Elongation Factor G Proteins: The Likely Targets of the Anti-Malarial Fusidic Acid

Malaria parasites harbour two organelles with bacteria-like metabolic processes that are the targets of many anti-bacterial drugs. One such drug is fusidic acid, which inhibits the translation component elongation factor G. The response of P. falciparum to fusidic acid was characterised using extended SYBR-Green based drug trials. This revealed that fusidic acid kills in vitro cultured P. falciparum parasites by immediately blocking parasite development. Two bacterial-type protein translation elongation factor G genes are identified as likely targets of fusidic acid. Sequence analysis suggests that these proteins function in the mitochondria and apicoplast and both should be sensitive to fusidic acid. Microscopic examination of protein-reporter fusions confirm the prediction that one elongation factor G is a component of parasite mitochondria whereas the second is a component of the relict plastid or apicoplast. The presence of two putative targets for a single inhibitory compound emphasizes the potential of elongation factor G as a drug target in malaria

Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase

Non-natural l-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, l-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing l-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to l and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of l-type prodrugs to assure their efficient metabolic processing

Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase

Non-natural l-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, l-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing l-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to l and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of l-type prodrugs to assure their efficient metabolic processing

A target-protection mechanism of antibiotic resistance at atomic resolution: insights into FusB-type fusidic acid resistance

Antibiotic resistance in clinically important bacteria can be mediated by proteins that physically associate with the drug target and act to protect it from the inhibitory effects of an antibiotic. We present here the first detailed structural characterization of such a target protection mechanism mediated through a protein-protein interaction, revealing the architecture of the complex formed between the FusB fusidic acid resistance protein and the drug target (EF-G) it acts to protect. Binding of FusB to EF G induces conformational and dynamic changes in the latter, shedding light on the molecular mechanism of fusidic acid resistance