The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation

Structural study of the type II 3-dehydroquinate dehydratase from Actinobacillus pleuropneumoniae

9 pages, 7 figures, 2 tables.The structure of the type II dehydroquinate dehydratase (DHQase) from Actinobacillus pleuropneumoniae, the third enzyme of the shikimate pathway, has been determined. Crystals diffracting to 1.7 Å were obtained in space and on earth using the counter-diffusion technique. The structure was solved using molecular replacement and refined to high resolution. The overall structure of the dodecameric enzyme is described and compared with structures of DHQases from other bacteria. DHQases contain a flexible loop that presumably closes over the active site upon substrate binding. The enzyme can exist in an open or closed conformation. The present structure displays the open conformation, with a sulfate anion bound in the active site. The availability of this structure opens a route to structure-based antibiotics targetting this pathogenic bacterium.We thank Professor Kabsch for providing XDS free of charge. We acknowledge the support of the European Space Agency and the European Community Action to Research Infrastructure Action of the Improving Human Potential Programme to the EMBL Hamburg Outstation, contract No. HPRI-CT-1999-00017. We thank Olivier Minster (ESA) for his support of space science. The authors acknowledge the excellent work of Dr Eva ManÄ as in managing the logistics concerning the space mission. We thank Viscount Dirk Frimout for his support for space crystallization experiments.Peer reviewe

Structural study of the type II 3-dehydroquinate dehydratase from Actinobacillus pleuropneumoniae

9 pages, 7 figures, 2 tables.The structure of the type II dehydroquinate dehydratase (DHQase) from Actinobacillus pleuropneumoniae, the third enzyme of the shikimate pathway, has been determined. Crystals diffracting to 1.7 Å were obtained in space and on earth using the counter-diffusion technique. The structure was solved using molecular replacement and refined to high resolution. The overall structure of the dodecameric enzyme is described and compared with structures of DHQases from other bacteria. DHQases contain a flexible loop that presumably closes over the active site upon substrate binding. The enzyme can exist in an open or closed conformation. The present structure displays the open conformation, with a sulfate anion bound in the active site. The availability of this structure opens a route to structure-based antibiotics targetting this pathogenic bacterium.We thank Professor Kabsch for providing XDS free of charge. We acknowledge the support of the European Space Agency and the European Community Action to Research Infrastructure Action of the Improving Human Potential Programme to the EMBL Hamburg Outstation, contract No. HPRI-CT-1999-00017. We thank Olivier Minster (ESA) for his support of space science. The authors acknowledge the excellent work of Dr Eva ManÄ as in managing the logistics concerning the space mission. We thank Viscount Dirk Frimout for his support for space crystallization experiments.Peer reviewe