Solvent accessibility of the thrombin-thrombomodulin interface.
The kinetics of solvent accessibility at the protein-protein interface between thrombin and a fragment of thrombomodulin, TMEGF45, have been monitored by amide hydrogen/deuterium (H/2H) exchange detected by MALDI-TOF mass spectrometry. The interaction is rapid and reversible, requiring development of theory and experimental methods to distinguish H/2H exchange due to solvent accessibility at the interface from H/2H exchange due to complex dissociation. Association and dissociation rate constants were measured by surface plasmon resonance and amide H/2H exchange rates were measured at different pH values and concentrations of TMEGF45. When essentially 100% of the thrombin was bound to TMEGF45, two segments of thrombin became completely solvent-inaccessible, as evidenced by the pH insensitivity of the amide H/2H exchange rates. These segments form part of anion-binding exosite I and contain the residues for which alanine substitution abolishes TM binding. Several other regions of thrombin showed slowing of amide exchange upon TMEGF45 binding, but the exchange remained pH-dependent, suggesting that these regions of thrombin were rendered only partially solvent-inaccessible by TMEGF45 binding. These partially inaccessible regions of thrombin form both surface and buried contacts into the active site of thrombin and contain residues implicated in allosteric changes in thrombin upon TM binding.
Mandell
J G
JG
Department of Chemistry and Biochemistry, University of California, San Diego, CA 92093-0359, USA.
Baerga-Ortiz
A
A
Akashi
S
S
Takio
K
K
Komives
E A
EA
D000495:Allosteric Site; D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D000838:Anions / Q000378:metabolism; D003903:Deuterium / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D023302:Protein Footprinting; D012995:Solubility; D012997:Solvents / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*; D018180:Thrombomodulin / Q000737:chemistry / Q000378:metabolism*
D000577:Amides; D000838:Anions; D010446:Peptide Fragments; D012997:Solvents; D018180:Thrombomodulin; D003903:Deuterium; D013917:Thrombin; D010434:Pepsin A