Identification of protein-protein interfaces by decreased amide proton solvent accessibility

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometr y was used to identify peptic fragments from protein complexes that retaine d deuterium under hydrogen exchange conditions due to decreased solvent acc essibility at the interface of the complex. Short deuteration times allowed preferential labeling of rapidly exchanging surface amides so that primari ly solvent accessibility changes and not conformational changes were detect ed. A single mass spectrum of the peptic digest mixture was analyzed to det ermine the deuterium content of all proteolytic fragments of the protein. T he protein-protein interface was reliably indicated by those peptides that retained more deuterons in the complex compared with control experiments in which only one protein was present. The method was used to identify the ki nase inhibitor [PKI(5-24)] and ATP-binding sites in the cyclic-AMP-dependen t protein kinase. Three overlapping peptides identified the ATP-binding sit e, three overlapping peptides identified the glycine-rich loop, and two pep tides identified the PKI(5-24)-binding site. A complex of unknown structure also was analyzed, human a-thrombin bound to an 83-aa fragment of human th rombomodulin [TMEGF(4-5)]. Five peptides from thrombin showed significantly decreased solvent accessibility in the complex. Three peptides identified the anion-binding exosite I, confirming ligand competition experiments. Two peptides identified a new region of thrombin near the active site providin g a potential mechanism of how thrombomodulin alters thrombin substrate spe cificity.