Abstract
Tarp (translocated actin recruiting phosphoprotein) is an effector protein common to all chlamydial species that functions to remodel the host-actin cytoskeleton during the initial stage of infection. In C. trachomatis, direct binding to actin monomers has been broadly mapped to a 100-residue region (726–825) which is predicted to be predominantly disordered, with the exception of a ~10-residue α-helical patch homologous to other WH2 actin-binding motifs. Biophysical investigations demonstrate that a Tarp726–825 construct behaves as a typical intrinsically disordered protein; within it, NMR relaxation measurements and chemical shift analysis identify the ten residue WH2-homologous region to exhibit partial α-helix formation. Isothermal titration calorimetry experiments on the same construct in the presence of monomeric G-actin show a well defined binding event with a 1:1 stoichiometry and Kd of 102 nM, whilst synchrotron radiation circular dichroism spectroscopy suggests the binding is concomitant with an increase in helical secondary structure. Furthermore, NMR experiments in the presence of G-actin indicate this interaction affects the proposed WH2-like α-helical region, supporting results from in silico docking calculations which suggest that, when folded, this α-helix binds within the actin hydrophobic cleft as seen for other actin-associated proteins.
Highlights
Chlamydiae are obligate intracellular pathogens which cause a significant disease burden in humans
The proline-rich domain enables Tarp to form homo-multimers that can facilitate actin filament nucleation at the site of chlamydial invasion by colocalising multiple G-actin binding domains; this is important in the case of C. pneumoniae and the C. trachomatis L2 serovar whose Tarp proteins only contain one actin binding domain
The distribution of Tarp residues perturbed upon actin binding suggests that a linear, sequential region, homologous to other WH2 domains, is alone sufficient to interact with actin
Summary
Chlamydiae are obligate intracellular pathogens which cause a significant disease burden in humans. It has become clear that the Tarp protein is involved in a multitude of protein:protein interactions, to achieve the principal objective of polymerising and bundling actin filaments en masse to facilitate host-cell internalization by parasite-specified phagocytosis Perturbation of these binding interactions, and those of the phosphorylation domain, have been linked to a reduction in chlamydial pathogenicity in a laboratory environment[7,14,16,17], and Tarp antibody recognition has been shown to confer protective immunity[18]. This work aimed to elucidate the structural and dynamic behavior of the native state of the Tarp G-actin-binding region, and its interaction with G-actin, by the application of solution state nuclear magnetic resonance (NMR) and synchrotron radiation circular dichroism (SRCD) spectroscopies and isothermal calorimetry (ITC) Based on these results, we propose a definition of the Tarp:G-actin interaction through the construction of a guided computational model
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