Protein-protein interactions at an enzyme-substrate interface: Characterization of transient reaction intermediates throughout a full catalytic cycle of Escherichia coli thioredoxin reductase

A large collection of structural snapshots along a full catalytic cycle of Escherichia coli thioredoxin reductase (TrxR) has been generated and characterized using a combination of theoretical methods. Molecular models were built starting from the available X‐ray crystallographic structures of dimer...

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Published in:Proteins, structure, function, and bioinformatics structure, function, and bioinformatics, 2010-01, Vol.78 (1), p.36-51
Main Authors: Negri, Ana, Rodríguez-Larrea, David, Marco, Esther, Jiménez-Ruiz, Antonio, Sánchez-Ruiz, José M., Gago, Federico
Format: Article
Language:English
Subjects:
Binding Sites
Calorimetry, Differential Scanning
Crystallography, X-Ray
differential scanning calorimetry
Escherichia coli - enzymology
molecular dynamics
Molecular Dynamics Simulation
NADP - metabolism
Point Mutation
Protein Binding
Protein Conformation
protein-protein interactions
Substrate Specificity
thioredoxin
thioredoxin reductase
Thioredoxin-Disulfide Reductase - chemistry
Thioredoxin-Disulfide Reductase - genetics
Thioredoxin-Disulfide Reductase - metabolism
Thioredoxins - chemistry
Thioredoxins - genetics
Thioredoxins - metabolism
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Summary:A large collection of structural snapshots along a full catalytic cycle of Escherichia coli thioredoxin reductase (TrxR) has been generated and characterized using a combination of theoretical methods. Molecular models were built starting from the available X‐ray crystallographic structures of dimeric wild‐type TrxR in the flavin‐oxidizing conformation and a C135S TrxR mutant enzyme in a flavin‐reducing conformation “trapped” by a cross‐link between Cys138 of TrxR and Cys32 of C35S mutant thioredoxin (Trx). The transition between these two extreme states, which is shown to be reproduced in a normal mode analysis, as well as natural cofactor binding and dissociation, were simulated for the wild‐type species using unrestrained and targeted molecular dynamics following docking of oxidized Trx to reduced TrxR. The whole set of simulations provides a comprehensive structural framework for understanding the mechanism of disulfide reduction in atomic detail and identifying the most likely intermediates that facilitate entry of NADPH and exit of NADP+. The crucial role assigned to Arg73 and Lys36 of Trx in substrate binding and complex stabilization was ascertained when R73G, R73D, and K36A site‐directed mutants of Trx were shown to be impaired to different extents in their ability to be reduced by TrxR. On the basis of previous findings and the results reported herein, E. coli TrxR appears as a beautifully engineered molecular machine that is capable of synchronizing cofactor capture and ejection with substrate binding and redox activity through an interdomain twisting motion. Proteins 2010. © 2009 Wiley‐Liss, Inc.
ISSN:0887-3585
1097-0134
DOI:10.1002/prot.22490