Binding mode analysis of a major T3SS translocator protein PopB with its chaperone PcrH from Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa, a Gram-negative pathogen uses a specialized set of Type III secretion system (T3SS) translocator proteins to establish virulence in the host cell. An understanding of the factors that govern translocation by the translocator protein-chaperone complex is thus of immense importance. In this work, experimental and computational techniques were used to probe into the structure of the major translocator protein PopB from P. aeruginosa and to identify the important regions involved in functioning of the translocator protein. This study reveals that the binding sites of the common chaperone PcrH, needed for maintenance of the translocator PopB within the bacterial cytoplasm, which are primarily localized within the N-terminal domain. However, disordered and flexible residues located both at the N- and C-terminal domains are also observed to be involved in association with the chaperone. This intrinsic disorderliness of the terminal domains is conserved for all the major T3SS translocator proteins and is functionally important to maintain the intrinsically disordered state of the translocators. Our experimental and computational analyses suggest that a "disorder-to-order" transition of PopB protein might take place upon PcrH binding. The long helical coiled-coil part of PopB protein perhaps helps in pore formation while the flexible apical region is involved in chaperone interaction. Thus, our computational model of translocator protein PopB and its binding analyses provide crucial functional insights into the T3SS translocation mechanism.

Exploring the “N-Terminal Anchor” Binding Interface of the T3SS Chaperone–Translocator Complexes from P. aeruginosa

“…Taken together, our results suggest that the N-terminal anchor may not be the most critical interface for the overall thermodynamic stability of, at least, the major translocator–chaperone complex (PopB-PcrH). This agrees with and expands upon studies that show (i) that the deletion of the N-terminus of PopB (PopB 60–390 ) does not significantly weaken its affinity for PcrH (WT apparent K D = 372 nM, PopB 60–390 apparent K D = 592 nM) 23 and (ii) the mutation of PopB’s N-terminal anchor consensus residues does not affect the ability of P. aeruginosa strains to either secret protein or their cytotoxicity toward macrophages. 25 The crystal structure of the highly similar major translocator–chaperone complex from A. hydrophila (AopB 40–264 -AcrH) shows two further interfaces the “N-terminal arm” and “convex surface” ( Figure 1 A).…”

“…This shows the major translocator (AopB 40–264 ) binding to the chaperone (AcrH) at three distinct interfaces (Figure A): (i) the N-terminal anchor: a short N-terminal sequence of AopB (AopB 46–55 ) binds in an extended form to the concave face of AcrH; (ii) the N-terminal arm: the two flexible N-terminal α-helices of AcrH bind into a hole formed by the coiled-coil helix and transmembrane hairpins of AopB; and (iii) the convex surface interface: the convex surface of AcrH makes widespread interactions with the coiled-coil helix and one transmembrane hairpin of AopB. The structure confirms biochemical interaction studies on the major translocator–chaperone complex in this and other bacterial species. ,− In comparison, there are no structures of the full binding region of a minor translocator–chaperone complex. However, structures of bacterial chaperones bound with N-terminal anchor peptides of either their major or minor translocators all display the same N-terminal anchor interface as described for AcrH/AopB above. ,,, This equates to a consensus translocator peptide sequence (in bold), “x P/V x L xx P xx,” which binds to the hydrophobic concave surface of the chaperone (Figures and A).…”

Using Integrated Bioinformatics Strategy to Identify Critical Factors for the Structural Integrity of Salmonella T3SS

“…Intrinsically disordered regions have been found in several individual T3SS proteins, such as Salmonella SipD [47] , Ecoli EspD [48] , Yersinia LcrV [49] , and Pseudomonas PopB [50] . These disordered regions are frequently involved in protein-protein interactions [47,48,[50][51][52] . Nonetheless, the overall abundance and functional importance of intrinsic disorder in the T3SS proteins still remains unclear.…”