Dissecting the microscopic steps of the cyclophilin A enzymatic cycle on the biological HIV-1 capsid substrate by NMR.
Peptidyl-prolyl isomerases (PPIases) are emerging as key regulators of many diverse biological processes. Elucidating the role of PPIase activity in vivo has been challenging because mutagenesis of active-site residues not only reduces the catalytic activity of these enzymes but also dramatically affects substrate binding. Employing the cyclophilin A PPIase together with its biologically relevant and natively folded substrate, the N-terminal domain of the human immunodeficiency virus type 1 capsid (CA(N)) protein, we demonstrate here how to dissect residue-specific contributions to PPIase catalysis versus substrate binding utilizing NMR spectroscopy. Surprisingly, a number of cyclophilin A active-site mutants previously assumed to be strongly diminished in activity toward biological substrates based only on a peptide assay catalyze the human immunodeficiency virus capsid with wild-type activity but with a change in the rate-limiting step of the enzymatic cycle. The results illustrate that a quantitative analysis of catalysis using the biological substrates is critical when interpreting the effects of PPIase mutations in biological assays.
Bosco
Daryl A
DA
Department of Biochemistry and Howard Hughes Medical Institute, MS 009, Brandeis University, Waltham, MA 02454, USA.
Eisenmesser
Elan Zohar
EZ
Clarkson
Michael W
MW
Wolf-Watz
Magnus
M
Labeikovsky
Wladimir
W
Millet
Oscar
O
Kern
Dorothee
D
D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D021984:Cyclophilin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D036022:Capsid Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D021984:Cyclophilin A