Characterisation of the interaction of colicin A with its co‐receptor TolA

Abstract

Edited by Stuart Ferguson Keywords:Intrinsically disordered protein Colicin A TolA NMR Protein-protein interaction a b s t r a c t Colicin A enters Escherichia coli cells through interaction with endogenous TolA and TolB proteins. In vitro, binding of the colicin A translocation domain to TolA leads to unfolding of TolA. Through NMR studies of the colicin A translocation domain and polypeptides representing the individual TolA and TolB binding epitopes of colicin A we question if the unfolding of TolA induced by colicin A is likely to be physiologically relevant. The NMR data further reveals that the colicin A binding site on TolA is different from that for colicin N which explains why there is a difference in colicin toxicity for E. coli carrying a TolA-III homologue from Yersina enterocolitica in place of its own TolA-III.

Structural Evidence That Colicin A Protein Binds to a Novel Binding Site of TolA Protein in Escherichia coli Periplasm

“…S6) and the fact that different patterns of methylation were observed in both free and complexed TolAIII. Although differences do exist between the structures of TolAIII determined by x-ray (herein) and NMR methods (25), they are small, and the residues predicted to be important in the interaction of TolAIII with TA 53–107 by NMR (27) are identical to those residues shown to be involved in the x-ray structure of the TA 53–107 -TolAIII complex (Fig. 2).…”

“…However, we have preliminary NMR data showing that the chemical shifts of a mixture of the TA 1–52 and TA 53–107 peptides were slightly different for some residues of TA 53–107 when compared with those of TA 53–107 on its own 7 . NMR data have shown that a truncated polypeptide expressing both the TolA binding region and the TolB box of ColA causes an unfolding of TolAIII in vitro , which is not apparent when the TolA binding region and TolB boxes are physically separated and added to TolAIII concurrently (27). If TolA binds TolB and ColA at different binding interfaces in the TA 1–107 -TolA-TolB trimeric complex, then it is possible that an unoccupied TolB box of ColA could interfere and cause localized unfolding of TolAIII in the TA 1–107 -TolAIII complex as unfolding of TolAIII is not observed on binding of the translocation domain of ColN, which does not possess a TolB box (27).…”

“…E. coli B834(DE3) was used as an expression host for plasmids pYZ48 (residues 302–421 of TolA (TolAIII) cloned into pET21a via NdeI/XhoI restriction sites with an in-frame N-terminal His 6 tag) or pYZ69 (27) to purify TolAIII and TA 1–107 6 containing a thrombin recognition site after residue 52, respectively. An overnight culture of 10 ml of Luria Bertani medium (LB) was used to inoculate 0.5 liter of 2×YT broth supplemented with ampicillin (Melford Laboratories) to a final concentration of 100 μg/ml.…”

“…The protein concentration was adjusted to 1 mg/ml in 20 m m Tris, pH 8.0, 100 m m NaCl containing 5 units/mg of thrombin (Haematologic Technologies Inc., Essex Junction, VT), and the mixture was left on a circulating platform rotor at room temperature overnight. The thrombin was then removed using ρ-aminobenzamidine-agarose beads (Sigma) according to the manufacturer's instructions, and TA 53–107 was purified using metal chelate chromatography and nickel-agarose beads in His tag lysis buffer as described previously (27). TA 53–107 was finally loaded to a Superdex 75 gel filtration column (GE Healthcare) to remove any undigested TA 1–107 .…”

“…Isotopically labeled TolAIII comprising amino acids 302–421 of the E. coli TolA protein (Swiss-Prot accession number P19934) and TA 1–52 and TA 53–107 containing the TolB box and TolA binding region of ColA, respectively, were prepared as described previously (27). The B 1–13 peptide representing the N-terminal sequence of TolB lacking its signal peptide and comprising residues 21–33 of TolB (AEVRIVIDSGVDS) was purchased from United Peptide Corp. at a purity of 95%.…”