Revisiting monomeric HIV-1 protease. Characterization and redesign for improved properties.
Interactions between the C-terminal interface residues (96-99) of the mature HIV-1 protease were shown to be essential for dimerization, whereas the N-terminal residues () and Arg(87) contribute to dimer stability (Ishima, R., Ghirlando, R., Tozser, J., Gronenborn, A. M., Torchia, D. A., and Louis, J. M. (2001) J. Biol. Chem. 276, 49110-49116). Here we show that the intramonomer interaction between the side chains of Asp(29) and Arg(87) influences dimerization significantly more than the intermonomer interaction between Asp(29) and Arg(8'). Several mutants, including T26A, destablize the dimer, exhibit a monomer fold, and are prone to aggregation. To alleviate this undesirable property, we designed proteins in which the N- and C-terminal regions can be linked intramolecularly by disulfide bonds. In particular, cysteine residues were introduced at positions 2 and 97 or 98. A procedure for the efficient preparation of intrachain-linked polypeptides is presented, and it is demonstrated that the Q2C/L97C variant exhibits a native-like single subunit fold. It is anticipated that monomeric proteases of this kind will aid in the discovery of novel inhibitors aimed at binding to the monomer at the dimerization interface. This extends the target area of current inhibitors, all of which bind across the active site formed by both subunits in the active dimer.
Louis
John M
JM
Laboratory of Chemical Physics, National Institute Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. jmlouis@helix.nih.gov
Ishima
Rieko
R
Nesheiwat
Issa
I
Pannell
Lewis K
LK
Lynch
Shannon M
SM
Torchia
Dennis A
DA
Gronenborn
Angela M
AM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine; D001224:Aspartic Acid; D003545:Cysteine; D019281:Dimerization; D004220:Disulfides / Q000032:analysis; D004795:Enzyme Stability; D014644:Genetic Variation; D016333:HIV Protease / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D004220:Disulfides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001120:Arginine; D016333:HIV Protease; D003545:Cysteine