The Structure of a Type 3 Secretion System (T3SS) Ruler Protein Suggests a Molecular Mechanism for Needle Length Sensing

Abstract

The type 3 secretion system (T3SS) and the bacterial flagellum are related pathogenicity-associated appendages found at the surface of many disease-causing bacteria. These appendages consist of long tubular structures that protrude away from the bacterial surface to interact with the host cell and/or promote motility. A proposed "ruler" protein tightly regulates the length of both the T3SS and the flagellum, but the molecular basis for this length control has remained poorly characterized and controversial. Using the Pseudomonas aeruginosa T3SS as a model system, we report the first structure of a T3SS ruler protein, revealing a "ball-and-chain" architecture, with a globular C-terminal domain (the ball) preceded by a long intrinsically disordered N-terminal polypeptide chain. The dimensions and stability of the globular domain do not support its potential passage through the inner lumen of the T3SS needle. We further demonstrate that a conserved motif at the N terminus of the ruler protein interacts with the T3SS autoprotease in the cytosolic side. Collectively, these data suggest a potential mechanism for needle length sensing by ruler proteins, whereby upon T3SS needle assembly, the ruler protein's N-terminal end is anchored on the cytosolic side, with the globular domain located on the extracellular end of the growing needle. Sequence analysis of T3SS and flagellar ruler proteins shows that this mechanism is probably conserved across systems.

Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity Switch Protein in the Yersinia Type III Secretion System

“…Here we characterize the interaction between the cytoplasmic domain of YscU (YscU C ) and YscP using NMR spectroscopy. A direct interaction between YscP and YscU C homologues in P. aeruginosa (denoted PscP and PscU C , respectively) has been observed previously by NMR (22); it was shown that a conserved N-terminal segment of PscP was responsible for its interaction with PscU C . The domain architecture of PscP has been described as a "ball-and-chain" topology where the "ball", i.e.…”

“…is disordered and flexible in solution (22). A similar architecture has been determined for the YscP homologue FliK from S. enterica serovar Typhimurium (21).…”

“…It was subsequently shown that these suppressor mutants are less stable and exhibit faster dissociation kinetics than the wild-type protein, which enabled Yop secretion to occur in the absence of YscP (10, 28). Direct interactions have been observed between FlhB and FliK (Salmonella enterica serovar Typhimurium) and between the homologous proteins in Pseudomonas aeruginosa, PscU and PscP (residues 31-36), supporting the proposed functional link between YscU and YscP homologues (10,21,22).…”

“…A systematic deletion analysis of YscP in Yersinia enterocolitica led to the identification of domains with specific functions (11,19), including two distinct secretion signals (residues 1-35 and residues 97-137) and a substrate specificity switch domain located between residues 385 and 500 (11). Homologues of YscP with similar functions are found in the flagella and T3SSs of diverse bacterial species (21,22).…”

Comparison of the Structure, Regulation and Functions between Type Three and Type Six Secretion System in Gram-Negative Bacteria

“…Yet until this day, researchers are debating on the exact mechanism controlling needle length dimensions, and substrate switching. Bergeron et al and his group suggested a mechanism for sensing of needle length in T3SS through the ruler protein [12]. Ruler proteins are associated with the secretion of effector proteins in T3SS at early stages.…”