Self‐recognition by an intrinsically disordered protein

Abstract

The intrinsically disordered translocation domain (T-domain) of the protein antibiotic colicin N binds to periplasmic receptors of target Escherichia coli cells in order to penetrate their inner membranes. We report here that the specific 27 consecutive residues of the T-domain of colicin N known to bind to the helper protein TolA in target cells also interacts intramolecularly with folded regions of colicin N. We suggest that this specific self-recognition helps intrinsically disordered domains to bury their hydrophobic recognition motifs and protect them against degradation, showing that an impaired self-recognition leads to increased protease susceptibility.

A Common Interaction for the Entry of Colicin N and Filamentous Phage into Escherichia coli

“…23,25 We failed to identify all 85 expected backbone NH resonances of the TolAIII-bound form of the colicin N T-domain using triple-resonance experiments, in contrast to our earlier study of the free form of the T-domain. 16 Twenty-four NH resonances of the TolAIII-bound form of the colicin N T-domain are unassigned, all of them from residues between 40 and 73, including the TolA binding box (residues 40-66). However, nine of the residues in the TolA binding box (Ser45, Asn48, Lys52, Asp54, His57, Asp59, Tyr62, Ile64, and Phe66) could be assigned (see Materials and Methods), and these show very strong chemical shift perturbations upon TolAIII binding when compared to the residues that reside outside the TolA binding box (Fig.…”

“…8 To determine whether other regions of the colicin T-domain are involved in the interaction and also to determine the nature of the TolAIII-induced structural transition of the Tdomain, 3D NMR experiments using a doublelabeled sample of the full-length T-domain in complex with unlabeled TolAIII were carried out in this work, exploiting the assignment of backbone NH resonances of this intrinsically disordered domain that we have recently reported. 16 The colicin N T-domain binds TolAIII with a K d of 1 μM, 23 and such an interaction is expected to cause changes in the magnetic environment of the nuclei that are situated at the molecular interface of the binding partners, inducing chemical shift variations of the corresponding signals. As expected, titration of unlabeled TolAIII into 13 C/ 15 N-labeled colicin N Tdomain caused a significant alteration in the 2D 1 H-15 N heteronuclear single quantum coherence (HSQC) spectrum (Fig.…”

“…34 All of these motifs contain a central tyrosine residue, and it is known that a mutation of this residue in colicin N (Y62) completely abolishes binding to TolAIII 23 and reduces intramolecular binding (which we have termed self-recognition) and, hence, protection from proteolytic degradation. 16 If the S61-H67 region of the colicin N T-domain does adopt a β-conformation on binding TolAIII, it may be an example of the recently described β-strand addition mechanism, 35 where unstructured peptide stretches ('linear motifs') add a β-strand onto a β-sheet or a β-strand in its receptor protein. β-Strand additions have been seen in promiscuous protein-protein contacts; in the present case, this would be in line with our finding that the region between residues S61 and H67 also plays a role in the aforementioned self-recognition event for colicin N. 16 TolAIII changes its conformation upon binding to colicin N but retains its global fold TolA plays an important role in maintaining the integrity of the cell envelope, providing communication between the inner and the outer membranes via its anchor in the inner membrane and its interactions with other proteins, including the outermembrane-anchored PAL 33 and the periplasmic TolB.…”

“…8 However, this earlier study used only 1 H-15 N NMR of the T-dom peptide, which did not allow resonances to be assigned. By using 1 H-13 C-15 N NMR of the fulllength T-domain and the T-dom 40-76 peptide, we have been able to sequentially assign resonances of the free 16 and TolAIII-bound forms of these peptides (this work) to judge the extent and nature of the disorderto-order transition induced upon binding to the Cterminal domain of TolA, and we report these findings here together with the unexpected similarity of the colicin N-TolAIII interaction with the interaction that TolAIII undergoes with a structured region (N1 domain) of the filamentous phage g3p protein. [17][18][19] TolA spans the periplasm of Gram-negative bacteria, where it helps maintain membrane integrity 20 and has energy-dependent conformational changes.…”

Neutrons for biologists: a beginner's guide, or why you should consider using neutrons

“…There are very good websites such as Thomas (rkt.chem.ox.ac.uk) (Solovyova et al 2004), some features of layers may be profitably studied by dual-polarization interferometry, Brewster angle microscopy or ellipsometry (Armstrong et al 2003) instead of reflectometry and some dynamics questions are better answered by NMR (Hecht et al 2008). Having defined what you would like to do, you should approach a facility that has the ability to perform the measurements.…”