Abstract
A pre-existing, allosteric equilibrium between closed (E*) and open (E) conformations of the active site influences the level of activity in the trypsin fold and defines ligand binding according to the mechanism of conformational selection. Using the clotting protease thrombin as a model system, we investigate the molecular determinants of the E*-E equilibrium through rapid kinetics and X-ray structural biology. The equilibrium is controlled by three residues positioned around the active site. W215 on the 215–217 segment defining the west wall of the active site controls the rate of transition from E to E* through hydrophobic interaction with F227. E192 on the opposite 190–193 segment defining the east wall of the active site controls the rate of transition from E* to E through electrostatic repulsion of E217. The side chain of E217 acts as a lever that moves the entire 215–217 segment in the E*-E equilibrium. Removal of this side chain converts binding to the active site to a simple lock-and-key mechanism and freezes the conformation in a state intermediate between E* and E. These findings reveal a simple framework to understand the molecular basis of a key allosteric property of the trypsin fold.
Highlights
Trypsin-like proteases utilize a catalytic triad for activity, composed of the highly conserved residues H57, D102 and S195
In this study we investigate the role of the side chains of W215, E217 and E192 through Ala substitutions and rapid kinetics of binding of the irreversible inhibitor H-D-Phe-Pro-Arg-CH2Cl (PPACK)
FPR binding to S195A thrombin reveals an equilibrium between two conformations, E* and E, that exchange over a longer time scale τ = (k12 + k21)−1 = 56 ms in a 1:4 ratio
Summary
Trypsin-like proteases utilize a catalytic triad for activity, composed of the highly conserved residues H57, D102 and S195. Binding of a ligand to the active site requires the 215–217 segment to assume an “open” configuration and is precluded in the “closed” one when side chains and backbone shift to occlude access to the primary specificity pocket Consistent with this scenario, recent rapid kinetics studies of proteases like chymotrypsin, thrombin, factor Xa and activated protein C8,11–16 have shown that ligand binding to the active site does not obey “induced fit”[17], where a conformational rearrangement of the complex follows the initial binding step, but rather obeys the alternative mechanism of “conformational selection”[18], where the ligand selects optimal conformations from a pre-existing equilibrium of closed (E*) and open (E) forms that precedes the binding step. The results reveal a simple mechanism for the E*-E equilibrium and provide the starting point for additional analysis
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