The intrinsically disordered C-terminal linker of FtsZ regulates protofilament dynamics and superstructurein vitro

Abstract

The bacterial tubulin FtsZ 2 polymerizes to form a discontinuous cytokinetic ring that drives bacterial cell division by directing local cell wall synthesis. FtsZ comprises a polymerizing GTPase domain, an intrinsically disordered C-terminal linker (CTL) and a C-terminal conserved α-helix (CTC). FtsZ protofilaments align circumferentially in the cell, with the CTC mediating attachment to membrane-associated division proteins. The dynamic turnover and treadmilling of clusters of FtsZ protofilaments guides cell wall synthesis and constriction. The nature and regulation of the interactions that result in the assembly of protofilaments into dynamic clusters is unknown. Here, we describe a role for the CTL of Caulobacter crescentus FtsZ as an intrinsic regulator of lateral interactions between protofilaments in vitro. FtsZ lacking its CTL (∆CTL) shows dramatically increased propensity to form long multifilament bundles compared to wildtype (WT). ∆CTL has reduced GTP hydrolysis rate compared to WT. However, reducing protofilament turnover in WT is not sufficient to induce bundling. Surprisingly, binding of the membrane-anchoring protein FzlC disrupts ∆CTL bundling in a CTC-dependent manner. Moreover, the CTL affects the ability of FtsZ curving protein FzlA to promote formation of helical bundles. We conclude that the CTL of FtsZ influences polymer structure and dynamics both through intrinsic effects on lateral interactions and turnover and by influencing extrinsic regulation of FtsZ by binding partners. Our characterization of CTL function provides a biochemical handle for understanding the relationship between Z-ring structure and function in bacterial cytokinesis.

Species‐ and C‐terminal linker‐dependent variations in the dynamic behavior of FtsZ on membranes in vitro

“…While the appearance of large ΔCTL/ΔCTL‐MTS bundles on SLBs is consistent with previous observations from electron microscopy that the CTL regulates lateral interaction between protofilaments (Sundararajan and Goley, ), we suspected that these structures are assembled in solution (upstream of the flow cell) during pre‐incubation with GTP. Moreover, these structures are only observed during initial assembly of ΔCTL/ΔCTL‐MTS polymers and are not observed at steady state.…”

“…The dimensions of the elongated structures of ΔCTL/ΔCTL‐MTS on SLB are similar to the largest multi‐filament bundles previously observed for ΔCTL polymers by electron microscopy (Sundararajan and Goley, ). Such bundles were never observed for WT FtsZ or CTL variants, namely L14 (FtsZ with a 14 amino acid CTL) and Hn CTL (FtsZ with CTL sequence from Hyphomonas neptunium ) (Sundararajan and Goley, ). When we tested the assembly of L14/L14‐MTS or Hn CTL/ Hn CTL‐MTS copolymers on SLBs, we did not observe any elongated structures.…”

“…Using our approach to understand the effects of the CTL on regulating lateral interaction between C. crescentus FtsZ protofilaments, we observed that C. crescentus ΔCTL forms networks of straight filamentous structures (Fig. A and B) similar in scale to the multi‐filament bundles observed by electron microscopy (Sundararajan and Goley, ). Moreover, we observed significantly slower dynamics for the higher order assembly of ΔCTL protofilaments compared to FtsZ protofilaments (Fig.…”

“…The CTL also appears to affect turnover of FtsZ polymers in C. crescentus and B. subtilis . Similar to Cc ΔCTL (Sundararajan and Goley, ), Bs ΔCTL has reduced GTP hydrolysis rates and forms far fewer protofilaments (by electron microscopy) compared to Bs FtsZ (wt) at 2.5 mM MgCl 2 and 50 mM KCl concentrations (Buske and Levin, ). Since neither Cc or Bs FtsZ form bundles under these conditions, it is possible that the contributions of the CTL to FtsZ turnover might be independent of its regulation of lateral interaction between protofilaments.…”

“…A). While the spatial resolution of the imaging system used here does not yield information on the organization of individual polymers within these nucleotide‐dependent clusters, these clusters likely correspond to short individual protofilaments of FtsZ or bundles of a small number of short filaments, as observed by electron microscopy (Sundararajan and Goley, ). The assembly of Ec FtsZ and Cc FtsZ polymers into distinct dynamic superstructures at steady state despite the apparent similarity in their protofilament precursors is intriguing.…”