Prediction of pK_a and redox properties in the thioredoxin superfamily

Abstract

Electrostatic interactions play important roles in diverse biological phenomena controlling the function of many proteins. Polar molecules can be studied with the FDPB method solving the Poisson-Boltzmann equation on a finite difference grid. A method for the prediction of pK a s and redox potentials in the thioredoxin superfamily is introduced. The results are compared with experimental pK a data where available, and predictions are made for members lacking such data. Studying CxxC motif variation in the context of different background structures permits analysis of contributions to cysteine ⌬pK a s. The motif itself and the overall framework regulate pK a variation. The reported method includes generation of multiple sidechain rotamers for the CxxC motif and is an effective predictive tool for functional pK a variation across the superfamily. Redox potential follows the trend in cysteine pK a variation, but some residual discrepancy indicates that a pH-independent factor plays a role in determining redox potentials for at least some members of the superfamily. A possible molecular basis for this feature is discussed.Keywords: electrostatics calculations; thioredoxin superfamily; ionizable groups; redox potential Proteins in the thioredoxin superfamily characteristically include a thioredoxin fold, consisting of a mixed ␤-sheet of five strands surrounded by four ␣-helices, and a CxxC sequence motif in which x denotes an unspecified amino acid, in their active site (Martin 1995;Åslund and Beckwith 1999). This sequence is CPHC starting at residue C30 in Escherichia coli DsbA, CGPC starting at C32 in human thioredoxin, CGYC starting at C98 in E. coli DsbC, and CVYC starting at C14 in T4 bacteriophage glutaredoxin. Different members of the superfamily can have opposite functions depending on the redox potential and the environment, either reducing disulfide bonds or oxidizing cysteine thiols (Debarbieux and Beckwith 2000). The CxxC motif has been extensively studied in terms of redox potential and function (Grauschopf et al. 1995; Huber-Wunderlich and Glockshuber 1998) and has been characterized as a rheostat in the active site (Chivers et al. 1997). The function of each member of the thioredoxin superfamily is determined by their redox potential and by the direction of the electron transport pathway within which each participates. Thioredoxin 1 of E. coli, with a redox potential of −270 mV, is a major reductant in the cytoplasm, whereas DsbA, a periplasmic protein with a redox potential of −122 mV, is highly oxidizing and required for the disulfide bond formation in the cell envelope (Åslund and Beckwith 1999). The active sites of members of the thioredoxin superfamily have remarkably similar conformation but exhibit extensive variation in redox equilibria.Electrostatic energies play a significant role in the control of protein function (Honig and Nicholls 1995), and accurate determination of these energies is therefore required to aid functional prediction. The magnitudes of electrostatic interactions of ...

Stabilization of the Catalytic Thiolate in a Mammalian Glutaredoxin: Structure, Dynamics and Electrostatics of Reduced Pig Glutaredoxin and its Mutants

“…However, contrary to a commonly held hypothesis, these cationic side-chains provide little stabilization for the thiolate, implying that they affect the enzymatic activity via other mechanisms. The pK a value of nucleophilic cysteine 22 (pK a 22 ) is dominated by local hydrogen-bonds, formed only in a welldefined active-site conformation, supported by a comparison between the calculated and experimental values of pK a 22 . The edge of the aromatic ring of Phe24 is polar enough to contribute to stabilize the thiolate, consistent with the conserved aromatic side-chain at this position in the glutaredoxin motif.…”

Evidence for Proton Shuffling in a Thioredoxin-Like Protein during Catalysis

“…Finite-difference Poisson-Boltzmannbased pK a calculation schemes [25][26][27] as well as ab initio calculations were employed in order to reproduce the experimentally determined pK a values of the active-site cysteines and to elucidate the molecular origins of the two opposing roles of the Cys-X-X-Cys motif in Trx-like proteins. [28][29][30][31][32] In the case of Escherichia coli C-terminal DsbD, 23 which catalyzes electron transport from cytoplasmic Trx to periplasmic target proteins, a good agreement between theory and experiment was obtained (pK a exp = 9.3-10.5, pK a th = 10.0). For the dithiol oxidase DsbA, however, the extremely low pK a of the solvent-exposed Cys30 (pK a exp = 3.5) could not be reproduced (pK a th = 8.3-9.2 33 ) with standard methods that exclusively consider the change in the electrostatic environment to determine the standard reaction free energy G 0 for protonating a titratable group.…”

“…50 The hydration entropy contribution 29,33,34 (not included in the standard pK a calculations 51 ) was estimated and taken into account in the pK a calculations performed on the simulated MD structures. This entropic contribution was estimated by counting the water molecules in the first hydration shell of all titratable amino acids in DsbL and of the respective amino acids in the tripeptide Gly-X-Gly (model for solvent-exposed amino acid, X = Asp, Glu, Lys, Cys, Arg).…”

“…However, the pK a value of Cys30 could be predicted fairly accurately if changes in hydration entropy were included in the calculation of the free energy for the protonation of titratable groups (pK a th = 4.0-4.3). 29,33,34 The empirically derived hydration entropy contribution accounts for the loss of water molecules in the hydration shell of a titratable group when it is moved from the solvent to its position in the protein.…”

Electrostatic Stabilization and General Base Catalysis in the Active Site of the Human Protein Disulfide Isomerase a Domain Monitored by Hydrogen Exchange

“…The redox potentials among the members of this family vary by more than 150 mV, which corresponds to a difference in equilibrium constants of more than 10 5 for the two-electron transfer reaction at 25 8C. This variation in redox potentials largely arises from differences in the thiol pK value of the N-terminal cysteine, [1,2] which serves as the primary A C H T U N G T R E N N U N G nucleophile that attacks the disulfide linkages of the protein substrate.…”