Protein inhibitor of neuronal nitric-oxide synthase, PIN, binds to a 17-amino acid residue fragment of the enzyme.
Neuronal nitric-oxide synthase (nNOS) is the primary nitric oxide (NO) regulator in neurons. The activity of the enzyme is inhibited by a protein inhibitor called PIN. We were able to purify large quantities of PIN overexpressed in bacterial cells. Analytical ultracentrifugation and chemical cross-linking studies showed that PIN exists as a monomer at low concentrations. The protein forms a high order aggregate at elevated concentrations. We have shown, using NMR spectroscopy, that the previously identified PIN-binding domain (PINB) of nNOS (residues 161-245) adopts a random coil structure in solution. By titrating 15N-labeled PINB with unlabeled PIN, the PIN-binding region of nNOS was precisely mapped to a 17-residue peptide fragment from Met-228 to His-244 of nNOS. NMR titration experiments also showed that PIN binds to nNOS with a 1:2 stoichiometry. A synthetic peptide corresponding to the identified PIN-binding region of nNOS was used to study the interaction between PIN and nNOS in detail. The functional implications of the results obtained from this study are discussed.
Fan
J S
JS
Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, People's Republic of China.
Zhang
Q
Q
Li
M
M
Tochio
H
H
Yamazaki
T
T
Shimizu
M
M
Zhang
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D017931:DNA Primers; D029721:Drosophila Proteins; D004398:Dyneins; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
D002352:Carrier Proteins; D017931:DNA Primers; D029721:Drosophila Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D004398:Dyneins