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X-ray structure analysis of HIV-1 and SIV proteinase co-crystallized with isovaleryl pepstatin: Conformational variability among inhibitor complexes

James, Colin Hugh; (1999) X-ray structure analysis of HIV-1 and SIV proteinase co-crystallized with isovaleryl pepstatin: Conformational variability among inhibitor complexes. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Structural determination by means of X-ray crystallography using molecular replacement methodology has been made of two immunodeficiency virus aspartyl proteinases, one of Human (HIV-1Bru isolate) and one of Simian (SIVmac251-32H isolate) origin. Each proteinase (PR) was co-crystallized with the archetypal aspartyl proteinase inhibitor, isovaleryl pepstatin. This inhibitor occupied the normal substrate binding site in each case. This is the first published structure of SIV PR with a statine derivative and of an HIV-1 PR structure with isovaleryl pepstatin (acetyl pepstatin was co-crystallized with HIV-1 PR, NY5 isolate Fitzgerald et al.). Two structural determinations with the same inhibitor, both refined to high resolution (1.8Å for HIV-1 PR and 1.6Å for SIV PR), and subjected to known processing, enabled detailed comparisons to be made. The HIV-1 PR crystallized in spacegroup P2₁2₁2 with one biologically active dimer in the crystallographic asymmetric unit. The SIV PR crystallized in spacegroup C222₁, one monomer being in the asymmetric unit. The electron density map for the HIV-1 PR in the region of isovaleryl pepstatin suggested that only one orientation of the inhibitor may reside there. In the case of the SIV PR, two orientations of the inhibitor are implied by the symmetry. However, there is evidence from the data analysis, that in order for this to be the case, each inhibitor orientation is accompanied by small conformational adjustments to the side chains of amino acids which define the subsites of the binding cavity. The implication is that the symmetry has been compromised with the structure being averaged in certain regions. It has been illustrated that the presumed flexibility in the surface loop regions of both proteinases has a correspondence with the assignment of individual temperature factors used in the refinement procedure. In addition, for the asymmetric dimer, discrepancies in temperature factors between symmetry associated regions can be attributed to differences in protein contacts in the crystal packing. For published structures of HIV-1, HIV-2 and SIV proteinase types, a comparison of the positional deviation of all backbone atoms compared to those only associated with amino acids involved in ligand contacts has been made, the cluster averages of the three protein species showing little variation in the backbone conformation in the region of the binding site despite there being up to 50% overall sequence variation between types. There is a clear correspondence between the RMS deviations of the atomic positions for related atoms in a cluster and the associated temperature factors for a representative structure. Emphasis is placed on the need for positional accuracy of coordinates to be estimated when the differences between two structural determinations are being correlated to a change in conditions. Two deep pockets 'below' the proteinase binding site normally occupied by solvent water have been identified. Being in a relatively non-mobile region not directly affected by mutations found to date, these are worthy of further investigation in relation to the design of new inhibitors.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: X-ray structure analysis of HIV-1 and SIV proteinase co-crystallized with isovaleryl pepstatin: Conformational variability among inhibitor complexes
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Aspartyl proteinases
URI: https://discovery.ucl.ac.uk/id/eprint/10106562
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