The Intrinsically Disordered C. Trachomatis Tarp Binds Actin with a Partially Preformed Helix

Abstract

Tarp (Translocated actin recruiting protein) is an intrinsically disordered protein (IDP) from Chlamydia trachomatis, which is translocated into the host cell via type III secretion to initiate the infection process. Once exposed to the cytosol, Tarp recruits host-cell actin, forming actin pedestal-like structures that contribute to host cell invasion (Clifton et al., PNAS101,10166). Tarp is capable of directly nucleating the formation of actin filaments, and its actin-binding region was mapped to the region between residues 726 and 825 (Jewett et al., PNAS103, 15599). We have acquired and assigned nuclear magnetic resonance (NMR) spectra of Tarp726-825, which show the narrow spectral distribution and chemical shifts characteristic of an IDP. Tarp shows some homology to the actin binding WH2 motif from WAVE2, and therefore it is expected to bind in a similar manner, forming a helix upon binding. Indeed, NMR relaxation experiments of Tarp726-825 show slightly lower flexibility in the 10-residue region homologous to the WH2 motif. However, chemical shift index calculations show that this helix is only partially formed before binding. In the presence of globular actin, the majority of peaks in the Tarp NMR spectra are affected, indicating that the binding affects most of the amino acid residues in this Tarp fragment. Isothermal titration calorimetry experiments determined the dissociation constant for the binding to be 1.02 (± 0.32) × 10−7 M, and showed a significant decrease in entropy upon binding, indicating increased order upon binding. Based on this information, the binding of Tarp to actin was modelled in silico. Two possible low energy sites of interaction have been identified, both of which could account for these observations. It is therefore possible that Tarp binds actin through a “fuzzy” interface, with contribution from both conformations.