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

BINOLFI, A; FERNANDEZ, CO; SICA, MP; DELFINO, JM; SANTOA, J

PROTEINS: STRUCTURE, FUNCTION AND GENETICS

WILEY-LISS, DIV JOHN WILEY & SONS INC

Lugar: New York; Año: 2012 p. 1448 - 1464

0887-3585

Thioredoxins (TRXs) constitute attractive a/b scaffolds forinvestigating molecular recognition. The interactionbetween the recombinant fragment spanning the sequence1?93 of full-length TRX (TRX1-93) and the synthetic peptidecomprising residues 94?108 (TRX94-108), plus a C-terminaltyrosine tag (the numbering scheme used in entrypdb 2TRX is used throughout the article, two complementarymoieties of E. coli TRX, brings about the consolidationof a native-like complex. Despite its reduced thermodynamicstability, this complex is able to acquire fine structuralfeatures remarkably similar to those characteristic offull-length TRX, namely, hydrodynamic behavior, assessedby diffusion-ordered spectroscopy (DOSY)-NMR; the patternof secondary structure, as revealed by three-bondHNHa coupling constants and secondary shifts for Ha/CO/Ca/Cb; native-like tertiary structural signatures revealed bynear-UV circular dichroism (CD) spectroscopy. The complexexhibits a relaxation behavior compatible with thatexpected for a native-like structure. However, heteronuclearnuclear Overhauser effect (NOE)s reveal an enhanced dynamics for the complex by comparison with full-lengthTRX. Furthermore, higher R2 values for residues 43?50 and74?89 would likely result from an exchange process modulatedby the peptide at the interface region. The slow kineticsof the consolidation reaction was followed by CD and realtimeNMR. Equilibrium titration experiments by NMR yielda KD value of 1.4 6 1.0 lM and a second low-affinity (>150lM) binding event in the vicinity of the active site. Moleculardynamics simulations of both the isolated fragment TRX1-93and the complex suggest the destabilization of a2 and a3 helicalelements and the persistence of b-structure in the absenceof TRX94-108. Altogether, structural and dynamic evidencepresented herein points to the key role played by theC-terminal helix in establishing the overall fold. This criticalswitch module endows reduced TRX with the ability to act asa cooperative folding unit.