Activity of Tethered Human Immunodeficiency Virus 1 Protease Containing Mutations in the Flap Region of One Subunit

Abstract

USA 87, 9660-96641 and its mutants containing amino acid substitutions or deletions or both in only one flap region were expressed in Esclzerichia coli. These mutant enzymes showed various degrees of self-processing and significantly reduced catalytic activity toward oligopeptide substrates compared with the wild type. Kinetic parameters determined for one of the oligopeptide substrates showed a dramatic increase in K,,, and decrease in k,,, values. Unexpectedly, the substrate cleavage was more efficient in low salt concentration for a mutant containing a shortened hydrophilic flap. Assays with oligopeptides representing naturally occurring cleavage sites or oligopeptides containing single amino acid substitutions at the P, and P; substrate positions showed only moderate changes in the substrate specificity of the mutant proteases. Predicted structures for the mutants were constructed by molecular modeling and used to interpret the results of kinetic measurements. In general, the data suggest that the mutated part of the flaps does not have a major role in determining substrate specificity; rather, it provides the hydrophobic environment and hydrogen-bond interactions with the conserved water that are necessary for efficient substrate binding and catalysis.Keywords: human immunodeficiency virus protease; flap mutants ; oligopeptide substrate; substrate specificity; enzyme kinetics.The protease (PR) of human retroviruses is an attractive target for chemotherapy of virus infection and associated diseases, including AIDS [I]. Knowledge of the specificity and mechanism of action of the retroviral PR have been useful in the design of potent inhibitors. The retroviral PR are aspartyl enzymes and are active as dimers of two identical subunits. Comparison of the crystal structures of human immunodeficiency virus 1 (HIV-1) PR with those of HIV-1 PR-inhibitor complexes (for review see [2]) suggested that during binding of the ligand, the two flaps close down on the ligand, providing a hydrophobic environment. The two flaps have an interchain hydrogen-bond connecting their tips. It is not clear whether the flaps contribute to the specificity and catalytic efficiency of the PR. In contrast to the flaps of retroviral PR, the cellular bilobal aspartic proteinases have only one flap, which cannot completely close down on the substrate, and there is no coordinated water molecule as observed in HIV PR-inhibitor structures [3-41.We have tested the effect of mutations in the flap region on the activity and specificity of HIV-1 PR. We used a single-chain tethered-dimer of HIV-1 PR [5]. The crystal structure of this tethered-dimer was recently determined [6] and is nearly identical to the structure of the wild-type two-chain protease. To explore the role of the hydrophobic nature of the amino acid residue at the tip of the flap (Ile50), we introduced a negatively charged (Asp) and a positively charged (His) residue into this position. We introduced mutations only into the flap region of one subunit. The mutation of only one flap was...

Structural Insights into the Mechanisms of Drug Resistance in HIV-1 Protease NL4-3

“…The packing of the flaps has been previously demonstrated to be crucial in substrate binding and drug resistance. 10,[24][25][26] The loss of packing contacts and increased mobility of the flaps would reduce the enthalpic component of TL-3 binding, consistent with the observed increase in the IC 50 for TL-3 by an additional factor of w3 (IC 1X 50 Z 22 nM; IC 3X 50 Z 64 nM). 8 In addition to increasing the IC 50 for TL-3, the flap mutations in the 3X mutant resulted in the recovery of some catalytic activity of the enzyme by increasing k cat .…”

Structural and kinetic analysis of drug resistant mutants of HIV‐1 protease

“…The three mutations in the flaps, K45I, M46L and G48V, had catalytic efficiencies ranging from 50 to 500% of the wild‐type activity on different substrates. The protease activity is sensitive to mutations in the flap region [30,31]. The G48V mutant had lower catalytic activity and the lowest measured structural stability, consistent with a deleterious effect.…”

Anisotropic Dynamics of the JE-2147−HIV Protease Complex: Drug Resistance and Thermodynamic Binding Mode Examined in a 1.09 Å Structure,

“…Previous molecular dynamics studies on the flaps and chimeric constructs of the flaps characterize them as crucial determinants of substrate binding and resistance (38)(39)(40). In the case of macrocyclic peptidomimetics, tighter packing of the active site loops of HIV Pr has been correlated with increased binding affinity (41).…”