The effect of inhibitor binding on the structural stability and cooperativity of the HIV-1 protease.
The effects of the peptide inhibitor acetyl pepstatin on the structural stability of the HIV-1 protease have been measured by high sensitivity calorimetric techniques. At 25 degrees C and pH 3.6, acetyl pepstatin binds to HIV-1 protease with an affinity of 1.6 x 10(7 )M-1 and an enthalpy of 7.3 +/- 0.5 kcal/mol, indicating that binding is not favored enthalpically and that the favorable Gibbs energy originates from a large positive entropy. Since the binding of acetyl pepstatin is associated with a negative change in heat capacity (-450 cal/K*mol) the association reaction becomes enthalpically favored at temperatures higher than 40 degrees C. The presence of the inhibitor stabilizes the dimeric structure of the protease in a fashion that can be quantitatively described by a set of thermodynamic linkage equations. The combination of titration and differential scanning calorimetry provides an accurate way of determining binding constants for high affinity inhibitors that cannot be determined by titration calorimetry alone. A structure-based thermodynamic analysis of the binding process indicates that the stabilization effect is not distributed uniformly throughout the protease molecule. The binding of the inhibitor selectively stabilizes those conformational states in which the binding site is formed, triggering a redistribution of the state probabilities in the ensemble of conformations populated under native conditions. As a result, the stability constants for individual residues do not exhibit the same change in magnitude upon inhibitor binding. Residues in certain areas of the protein are affected significantly whereas residues in other areas are not affected at all. In particular, inhibitor binding has a significant effect on those regions that define the binding site, especially the flap region which becomes structurally stable as a result of the additional binding free energy. The induced stabilization propagates to regions not in direct contact with the inhibitor, particularly to the strand between residues Pro9 and Ala22 and the helix between Arg87 and Gly94. On the other hand, the stability of the strand between Asp60 and Leu76 is not significantly affected by inhibitor binding. The structural distribution of binding effects define cooperative pathways within the protease molecule. Proteins 1999;36:147-156.
Department of Biology and Biocalorimetry Center, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
M J
MJ
Todd
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Freire
D000465:Algorithms; D001665:Binding Sites; D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D003198:Computer Simulation; D004795:Enzyme Stability / Q000187:drug effects; D016333:HIV Protease / Q000737:chemistry* / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D015497:HIV-1 / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008958:Models, Molecular; D010436:Pepstatins / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013696:Temperature; D013816:Thermodynamics; D017186:Titrimetry
D017320:HIV Protease Inhibitors; D008024:Ligands; D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D016333:HIV Protease; C031375:pepstatin