Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants.
Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Instituto de Química y Fisicoquímica Biológicas, CONICET, Junín 956, C1113AAD, Buenos Aires, Argentina.
Martín E
ME
Noguera
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Instituto de Química y Fisicoquímica Biológicas, CONICET, Junín 956, C1113AAD, Buenos Aires, Argentina.
Diego S
DS
Vazquez
Laboratorio de Fisicoquímica Biológica, Instituto de Química Biológica and Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, Uruguay.
Gerardo
G
Ferrer-Sueta
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Instituto de Química y Fisicoquímica Biológicas, CONICET, Junín 956, C1113AAD, Buenos Aires, Argentina.
William A
WA
Agudelo
Department of Integrative Biology, IGBMC, CNRS, INSERM, Université de Strasbourg, Illkirch, France.
Eduardo
E
Howard
Instituto de Biología Molecular y Celular de Rosario (IBR), Rosario, Santa Fe, Argentina.
Rodolfo M
RM
Rasia
Laboratorio de Fisicoquímica Biológica, Instituto de Química Biológica and Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, Uruguay.
Bruno
B
Manta
Department of Integrative Biology, IGBMC, CNRS, INSERM, Université de Strasbourg, Illkirch, France.
Alexandra
A
Cousido-Siah
Department of Integrative Biology, IGBMC, CNRS, INSERM, Université de Strasbourg, Illkirch, France.
André
A
Mitschler
Department of Integrative Biology, IGBMC, CNRS, INSERM, Université de Strasbourg, Illkirch, France.
Alberto
A
Podjarny
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Instituto de Química y Fisicoquímica Biológicas, CONICET, Junín 956, C1113AAD, Buenos Aires, Argentina.
Javier
J
Santos
D000478:Alkylation; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation / Q000235:genetics*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics*
D050505:Mutant Proteins; D013879:Thioredoxins; D003545:Cysteine