Conformation of the EPEC Tir protein in solution: investigating the impact of serine phosphorylation at positions 434/463

Abstract

The translocated intimin receptor (Tir) is a key virulence factor of enteropathogenic Escherichia coli and related bacteria. During infection, Tir is translocated via a type III secretion system into host intestinal epithelial cells, where it inserts into the target cell membrane and acts as a receptor for the bacterial adhesin intimin. The effects of phosphorylation by cAMP-dependent kinase at two serine residues (Ser-434 and Ser-463) within the C-terminal domain of Tir, which may be involved in mediating structural/electrostatic changes in the protein to promote membrane insertion or intermolecular interactions, have previously been investigated. This study has focused on defining the conformation of Tir in solution and assessing any conformational changes associated with serine phosphorylation at positions 434/463. In addition to phosphorylated protein, combinations of Ala (unphosphorylatable) and Asp (phosphate-mimic) mutations of Ser-434 and Ser-463 have been generated, and a range of techniques (sodium dodecyl sulfate polyacrylamide gel electrophoresis, circular dichroism spectroscopy, analytical ultracentrifugation) used to further dissect the structural role and functional implications of changes in residue size/charge at these positions. The results have shown that under physiological NaCl concentrations, Tir is a monomer and adopts a highly elongated state in solution, consistent with a natively unfolded conformation. Despite this, perturbations in the structure in response to buffer conditions and the nature of the residues at positions 434 and 463 are apparent, and may be functionally relevant.

FIGURE 1

12% SDS-PAGE gel of cTir WT and Ala/Asp mutant proteins. WT, wild-type protein. Ala and Asp residues are referred to by their residue number and single-letter code. Protein (8 ng) was loaded in each lane and samples were visualized by Coomassie blue staining.

FIGURE 2

SDS-PAGE gels showing the effects of phosphorylation on (A) the Ala mutants, (B) the Asp mutants, and (C) the Ala/Asp combinations in cTir and (D) FL-Tir. cTir samples were run on 12% gels, FL-Tir samples on 8% gels. WT, wild-type protein. Ala and Asp residues are referred to by their single-letter code. Phosphorylation assays were performed as described in Materials and Methods. cTir (8 ng) and FL-Tir (10 ng)were loaded in each lane as appropriate and samples were visualized by Coomassie blue staining.

FIGURE 3

Far-UV circular dichroism. (A) Wavelength scans of cTir WT and mutants. The data display two overall patterns, as discussed in the text. Traces for WT, Ser-434-Asp, Ser-463-Ala, Ser-463-Asp, Ser-434-Ala/Ser-463-Ala, and Ser-434-Ala/Ser-463-Asp are solid; Ser-434-Ala, Ser-434-Asp/Ser-463-Ala, and Ser-434-Asp/Ser-463-Asp are shaded. (B) Wavelength scans for full-length protein (solid line, WT; dashed line, Ser-434-Asp/Ser-463-Asp).

FIGURE 4

General size distribution (c(s)) analysis of the SV data converted to standard conditions for (A) cTir WT in 150 mM NaCl, c = 0.92 mg/ml (solid line), Ser-434-Asp/Ser-463-Asp mutant, c = 1 mg/ml (dashed line), and phosphorylated sample, c = 0.92 mg/ml (dotted line); (B) cTir in 50 mM NaCl, with sample concentrations ranging from 0.3 mg/ml to 1.4 mg/ml. (Inset) Linear extrapolation to zero concentrations s_w for each detected species (monomer (□), dimer (○), tetramer (▵), dashed lines) and weight-average s for all three species together (*, solid line); (C) FL-Tir, with samples and symbols as in A. The concentrations were 1.2 mg/ml for WT; 0.87 mg/ml for Ser-434-Asp/Ser-463-Asp mutant, and 0.85 mg/ml for phosphorylated protein.

FIGURE 5

PONDR prediction for intrinsic disorder from amino acid sequences (A) FL-Tir, (B) cTir WT (solid line), Ser-434-Asp/Ser-463-Asp (dashed line), and Ser-434-Ala/Ser-463-Ala (dotted line). The predicted MoRF sequence, as discussed in the text, is shown as a hatch-marked box.