Structure, dynamics and electrostatics of the active site of glutaredoxin 3 from Escherichia coli: Comparison with functionally related proteins

The chemistry of active-site cysteine residues is central to the activity o f thiol-disulfide oxidoreductases of the thioredoxin superfamily. In these reactions, a nucleophilic thiolate is required, but the associated pK(a), v alues differ vastly in the superfamily, from less than 4 in DsbA to greater than 7 in Trx. The factors that stabilize this thiolate are, however, not clearly established. The glutaredoxins (Grxs), which are members of this su perfamily, contain a Cys-Pro-Tyr-Cys motif in their active site. In reduced Grxs, the pK(a) of the N-terminal active-site nucleophilic cysteine residu e is lowered significantly, and the stabilization of the corresponding thio late is expected to influence the redox potential of these enzymes. Here, w e use a combination of long molecular dynamics (MD) simulations, pK(a) calc ulations, and experimental investigations to derive the structure and dynam ics of the reduced active site from Escherichia coli Grx3, and investigate the factors that stabilize the thiolate. Several different MD simulations c onverged toward a consensus conformation for the active-site cysteine resid ues (Cys11 and Cys14), after a number of local conformational changes. Key features of the model were tested experimentally by measurement of NMR scal ar coupling constants, and determination of pK(a) values of selected residu es. The pK(a) values of the Grx3 active-site residues were calculated durin g the MD simulations, and support the underlying structural model. The stru cture of Grx3, in combination with the pK(a) calculations, indicate that th e pK(a) of the N-terminal active-site cysteine residue in Grx3 is intermedi ate between that of its counterpart in DsbA and Trx. The pK(a) values in be st agreement with experiment are obtained with a low (<4) protein dielectri c constant. The calculated pK(a) values fluctuate significantly in response to protein dynamics, which underscores the importance of the details of th e underlying structures when calculating pK(a) values. The thiolate of Cys1 1 is stabilized primarily by direct hydrogen bonding with the amide protons of Tyr13 and Cys14 and the thiol proton of Cys14, rather than by long rang e interactions from charged groups or from a helix macrodipole. From the co mparison of reduced Grx3 with other members of the thioredoxin superfamily, a unifying theme for the structural basis of thiol pK(a) differences in th is superfamily begins to emerge. (C) 2001 Academic Press.