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) regu lator in neurons. The activity of the enzyme is inhibited by a protein inhi bitor called PIN. We were able to purify large quantities of PIN overexpres sed in bacterial cells. Analytical ultracentrifugation and chemical cross-l inking studies showed that PIN exists as a monomer at low concentrations. T he 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 N-15-labeled PINE with unlabeled PIN, the PIN-bindin g region of nNOS was precisely mapped to a 17-residue peptide fragment from Met-228 to His-244 of nNOS. MMR 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 interact ion between PIN and nNOS in detail. The functional implications of the resu lts obtained from this study are discussed.