Recognition between a short unstructured peptide and a partially folded 
fragment leads to the thioredoxin fold sharing native-like dynamics.

Binolfi, A.; Fernandez, C. O.; Sica, M. P.; Delfino, J. M.; Santos, J.

doi:10.1002/prot.24043

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Recognition between a short unstructured peptide and a partially folded fragment leads to the thioredoxin fold sharing native-like dynamics.

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Fernandez, C. O.
Department of NMR Based Structural Biology, MPI for biophysical chemistry, Max Planck Society;

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Binolfi, A., Fernandez, C. O., Sica, M. P., Delfino, J. M., & Santos, J. (2012). Recognition between a short unstructured peptide and a partially folded fragment leads to the thioredoxin fold sharing native-like dynamics. Proteins: Structure, Function, and Bioinformatics, 80(5), 1448-1464. doi:10.1002/prot.24043.

Zitierlink: https://hdl.handle.net/11858/00-001M-0000-000F-9FB6-6

Zusammenfassung

Thioredoxins (TRXs) constitute attractive α/β scaffolds for investigating molecular recognition. The interaction between the recombinant fragment spanning the sequence 1–93 of full-length TRX (TRX1-93) and the synthetic peptide comprising residues 94–108 (TRX94-108), plus a C-terminal tyrosine tag (the numbering scheme used in entry pdb 2TRX is used throughout the article, two complementary moieties of E. coli TRX, brings about the consolidation of a native-like complex. Despite its reduced thermodynamic stability, this complex is able to acquire fine structural features remarkably similar to those characteristic of full-length TRX, namely, hydrodynamic behavior, assessed by diffusion-ordered spectroscopy (DOSY)-NMR; the pattern of secondary structure, as revealed by three-bond HNHα coupling constants and secondary shifts for Hα/CO/Cα/Cβ; native-like tertiary structural signatures revealed by near-UV circular dichroism (CD) spectroscopy. The complex exhibits a relaxation behavior compatible with that expected for a native-like structure. However, heteronuclear nuclear Overhauser effect (NOE)s reveal an enhanced dynamics for the complex by comparison with full-length TRX. Furthermore, higher R2 values for residues 43–50 and 74–89 would likely result from an exchange process modulated by the peptide at the interface region. The slow kinetics of the consolidation reaction was followed by CD and real-time NMR. Equilibrium titration experiments by NMR yield a KD value of 1.4 ± 1.0 μM and a second low-affinity (>150 μM) binding event in the vicinity of the active site. Molecular dynamics simulations of both the isolated fragment TRX1-93 and the complex suggest the destabilization of α2 and α3 helical elements and the persistence of β-structure in the absence of TRX94-108. Altogether, structural and dynamic evidence presented herein points to the key role played by the C-terminal helix in establishing the overall fold. This critical switch module endows reduced TRX with the ability to act as a cooperative folding unit.