Substrate recognition in HIV-1 protease: a computational study.

HIV-1 protease is a crucial enzyme for the life cycle of the human immunodeficiency virus, the retrovirus that triggers AIDS. It is well documented that HIV-1 protease mediates the cleavage of Gag, Gag-Pol, and Nef precursor polyproteins and is highly selective concerning the set of 12 different amino acid sequences that cleaves. However, the governing principles and physical parameters, which determine substrate recognition and specificity, remain poorly understood despite the many speculative proposals that abound in the literature. In fact, it has been difficult so far to circumvent the fact that protease's substrates share little sequence identity and lack an obvious consensus binding motif. We have used microsecond time scale MD simulations to quantitatively show that some sequences of the polyprotein Gag-Pol that are not cleaved (nonsubstrates) have in fact a higher affinity to the active site of HIV-1 protease than a substrate; i.e., recognition is not governed by affinity to the active site. On the basis of a detailed analysis of the results and experimental data, we propose that the recognition is based on the geometric specificity of PR:Gag and PR:Gag-Pol multiprotein complex, that selects which residues lie in the specific position that makes them accessible to the active site for cleavage.

[1]  S. Karlsen,et al.  Crystal Structural Studies of Changes in the Native Dinuclear Iron Center of Ribonucleotide Reductase Protein R2 from Mouse* , 2004, Journal of Biological Chemistry.

[2]  J. Freed,et al.  EPR distance measurements support a model for long-range radical initiation in E. coli ribonucleotide reductase. , 2005, Journal of the American Chemical Society.

[3]  Fahmi Himo,et al.  Quantum chemical modeling of enzyme active sites and reaction mechanisms , 2006 .

[4]  U. Uhlin,et al.  Structure and function of the radical enzyme ribonucleotide reductase. , 2001, Progress in biophysics and molecular biology.

[5]  K. Morokuma,et al.  A NEW ONIOM IMPLEMENTATION IN GAUSSIAN98. PART I. THE CALCULATION OF ENERGIES, GRADIENTS, VIBRATIONAL FREQUENCIES AND ELECTRIC FIELD DERIVATIVES , 1999 .

[6]  M. Fontecave,et al.  Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic Escherichia coli. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. G. Cory.  Ribonucleotide reductase as a chemotherapeutic target. , 1988, Advances in enzyme regulation.

[8]  A. Becke,et al.  Density-functional exchange-energy approximation with correct asymptotic behavior. , 1988, Physical review. A, General physics.

[9]  N. Russo,et al.  The role of quantum chemistry in the elucidation of the elementary mechanisms of catalytic processes: from atoms, to surfaces, to enzymes , 2007 .

[10]  John C. Slater,et al.  The self-consistent field for molecules and solids , 1974 .

[11]  J. Stubbe,et al.  Detection of a new substrate-derived radical during inactivation of ribonucleotide reductase from Escherichia coli by gemcitabine 5'-diphosphate. , 1998, Biochemistry.

[12]  E. De Clercq,et al.  Nucleic acid related compounds. 74. Synthesis and biological activity of 2'(and 3')-deoxy-2'(and 3')-methylenenucleoside analogues that function as mechanism-based inhibitors of S-adenosyl-L-homocysteine hydrolase and/or ribonucleotide reductase. , 1992, Journal of medicinal chemistry.

[13]  Pedro Alexandrino Fernandes,et al.  Dehydration of ribonucleotides catalyzed by ribonucleotide reductase: the role of the enzyme. , 2006, Biophysical journal.

[14]  Leif A. Eriksson,et al.  The reduction of ribonucleotides catalyzed by the enzyme ribonucleotide reductase , 2002 .

[15]  Axel D. Becke,et al.  Density‐functional thermochemistry. IV. A new dynamical correlation functional and implications for exact‐exchange mixing , 1996 .

[16]  J. Perdew,et al.  Electronic structure of solids , 2021, Modern Physics with Modern Computational Methods.

[17]  H. Zipse.  The influence of hydrogen bonding interactions on the C-H bond activation step in class I ribonucleotide reductases. , 2003, Organic & biomolecular chemistry.

[18]  J. Bollinger,et al.  2'-Deoxy-2'-methylenecytidine and 2'-deoxy-2',2'-difluorocytidine 5'-diphosphates: potent mechanism-based inhibitors of ribonucleotide reductase. , 1991, Journal of medicinal chemistry.

[19]  P. Kollman,et al.  A well-behaved electrostatic potential-based method using charge restraints for deriving atomic char , 1993 .

[20]  Robert J. Woods,et al.  Solvated ensemble averaging in the calculation of partial atomic charges , 2001, J. Comput. Chem..

[21]  S. Elledge,et al.  The DNA damage response: putting checkpoints in perspective , 2000, Nature.

[22]  Parr,et al.  Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. , 1988, Physical review. B, Condensed matter.

[23]  J. Stubbe,et al.  Protein Radicals in Enzyme Catalysis. , 1998, Chemical reviews.

[24]  M. Eriksson,et al.  Binding of allosteric effectors to ribonucleotide reductase protein R1: reduction of active-site cysteines promotes substrate binding. , 1997, Structure.

[25]  A. Matsuda,et al.  Design, synthesis, and antineoplastic activity of 2'-deoxy-2'-methylidenecytidine. , 1988, Journal of medicinal chemistry.

[26]  G. Nocentini,et al.  Ribonucleotide reductase inhibitors: new strategies for cancer chemotherapy. , 1996, Critical reviews in oncology/hematology.

[27]  Leif A. Eriksson,et al.  Hydrogen Atom Transfer in Ribonucleotide Reductase (RNR) , 1998 .

[28]  M. Fukuoka,et al.  Phase I trial of oral 2'-deoxy-2'-methylidenecytidine: on a daily x 14-day schedule. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[29]  Jerzy Cioslowski,et al.  A new population analysis based on atomic polar tensors , 1989 .

[30]  R. Griffin,et al.  High-field EPR detection of a disulfide radical anion in the reduction of cytidine 5'-diphosphate by the E441Q R1 mutant of Escherichia coli ribonucleotide reductase. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[31]  P. Fernandes,et al.  Understanding ribonucleotide reductase inactivation by gemcitabine. , 2007, Chemistry.

[32]  Feliu Maseras,et al.  IMOMM: A new integrated ab initio + molecular mechanics geometry optimization scheme of equilibrium structures and transition states , 1995, J. Comput. Chem..

[33]  J. Stubbe,et al.  Ribonucleotide reductases: radical enzymes with suicidal tendencies. , 1995, Biochemical Society transactions.

[34]  Nuno M F S A Cerqueira,et al.  Enzyme ribonucleotide reductase: unraveling an enigmatic paradigm of enzyme inhibition by furanone derivatives. , 2006, The journal of physical chemistry. B.

[35]  J. Stubbe.  Radicals with a controlled lifestyle. , 2003, Chemical communications.

[36]  Pedro Alexandrino Fernandes,et al.  Theoretical studies on the mechanism of inhibition of Ribonucleotide Reductase by (E)-2'-Fluoromethylene-2'-deoxycitidine-5'-diphosphate. , 2003, Journal of the American Chemical Society.

[37]  R. Griffin,et al.  Characterization of a substrate-derived radical detected during the inactivation of ribonucleotide reductase from Escherichia coli by 2'- fluoromethylene-2'-deoxycytidine 5'-diphosphate , 1998 .

[38]  R J Woods,et al.  Quantum mechanical study of the nonbonded forces in water-methanol complexes. , 2001, The journal of physical chemistry. A.

[39]  Karl N. Kirschner,et al.  Solvent interactions determine carbohydrate conformation , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[40]  Pedro Alexandrino Fernandes,et al.  General performance of density functionals. , 2007, The journal of physical chemistry. A.

[41]  B. Cooperman,et al.  A quantitative model for allosteric control of purine reduction by murine ribonucleotide reductase. , 2001, Biochemistry.