proteins STRUCTURE O FUNCTION O BIOINFORMATICS A molecular dynamics study comparing a wild-type with a multiple drug resistant HIV protease: Differences in flap and aspartate 25 cavity dimensions Steve A. Seibold and Robert I. Cukier * Department of Chemistry, Michigan State University, East Lansing, Michigan 48824-1322 INTRODUCTION HIV protease is an essential component in the replication of the virus that causes AIDS. 1 It activates other HIV proteins by cleaving the nascent viral polyprotein chain at specific sites to produce active proteins. Numerous crystal structures have been obtained, leading to the discovery of the active site, and the prediction that the enzyme utilizes an aspartic protease acid-base mechanism for protein cleavage. 2–8 However, the structural/conformational changes that are required for binding of substrate, its cleavage, and release of product are still obscure. The HIV protease [Fig. 1(A)] is a 22 kDa homodimer. The scaf- folding of the active site, formed by the homodimer’s interface, contains two b-hairpin loops and two flexible b-hairpin structures or ‘‘flaps’’ that close down on the active site upon substrate bind- ing and open up for product release. Each 99-residue monomer contributes a catalytically essential aspartic acid (Asp25) as part of the conserved Asp-Thr-Gly sequence. 9 The variations in flap conformations observed in X-ray studies of the protease suggest that the flaps are flexible and can adopt multiple conformations. 10 NMR studies demonstrated that the flap tips (residues 46–54) are, indeed, mobile on the micro-second and sub-nanosecond time scales and move from one extreme (closed) to another (open). 11 As important as these observations are, they do not provide atomic details about the role of the atoms or residues in the flap movements, which are essential for under- standing their function in substrate interaction and in drug resist- ance. The flap tips, containing the hydrophobic sequence GGIGG, are highly mobile, which is consistent with the presence of flexible residues such as glycine. Scott and Schiffer 12 suggest that curling of the flap tips occurs to bury hydrophobic residues and aids in substrate entry to the catalytic site. A mechanism for increasing the conformational flexibility of the glycine rich flap tips was eluci- dated by free energy simulations. 13 Meagher and Carlson 14 simu- lated HIV protease and compared the MD flap fluctuation order *Correspondence to: Robert I. Cukier, Department of Chemistry, Michigan State University, East Lansing, MI 48824-1322. E-mail: cukier@cem.msu.edu Received 30 October 2006; Revised 21 February 2007; Accepted 22 March 2007 Published online 10 July 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.21535 ABSTRACT HIV proteases can develop resistance to therapeutic drugs by mutating specific residues, but still main- tain activity with their natural substrates. To gain insight into why mutations confer such resistance, long (70 ns) Molecular Dynamics simulations in explicit solvent were performed on a multiple drug resistant (MDR) mutant (with Asn25 in the crystal structure mutated in silico back to the catalytically active Asp25) and a wild type (WT) protease. HIV proteases are homodimers, with characteristic flap tips whose conformations and dynamics are known to be important influences of ligand binding to the aspartates that form the catalytic center. The WT protease undergoes a transition between 25 and 35 ns that is absent in the MDR protease. The origin of this distinction is investigated using principal com- ponent analysis, and is related to differences in motion mainly in the flap region of each monomer. Trajectory analysis suggests that the WT transition arises from a concerted motion of the flap tip dis- tances to their catalytic aspartate residues, and the distance between the two flap tips. These distances form a triangle that in the WT expands the active site from an initial (semi-open) form to an open form, in a correlated manner. In contrast, the MDR protease remains in a more closed configuration, with uncorrelated fluctuations in the distances defining the triangle. This contrasting behavior sug- gests that the MDR mutant achieves its resistance to drugs by making its active site less accessible to inhibitors. The migration of water to the active site aspartates is monitored. Water molecules move in and out of the active site and individual waters hydrogen bond to both aspartate carboxylate oxy- gens, with residence times in the ns time regime. Proteins 2007; 69:551–565. V V C 2007 Wiley-Liss, Inc. Key words: HIV protease; molecular dynamics; prin- cipal component analysis; multiple drug resistant. V V C 2007 WILEY-LISS, INC. PROTEINS 551