FtsZ protofilaments use a hinge-opening mechanism for constrictive force generation

Abstract

The essential bacterial protein FtsZ is a guanosine triphosphatase that self-assembles into a structure at the division site termed the "Z ring". During cytokinesis, the Z ring exerts a constrictive force on the membrane by using the chemical energy of guanosine triphosphate hydrolysis. However, the structural basis of this constriction remains unresolved. Here, we present the crystal structure of a guanosine diphosphate-bound Mycobacterium tuberculosis FtsZ protofilament, which exhibits a curved conformational state. The structure reveals a longitudinal interface that is important for function. The protofilament curvature highlights a hydrolysis-dependent conformational switch at the T3 loop that leads to longitudinal bending between subunits, which could generate sufficient force to drive cytokinesis.

Figure 1. Structure of a double-stranded MtbFtsZ-GDP polymer reveals key inter-subunit contacts

(A) Ribbon representation of a double-stranded MtbFtsZ-GDP polymer containing 24 subunits. The FtsZ subunits are colored in grey and cyan to distinguish the two protofilaments, and GDP molecules are colored in orange. (B) Detailed stereo-view of the inter-subunit contacts. The interactions, especially the hydrophobic contacts, are formed by highly conserved residues (Fig. S2). Among these residues, Leu269 of the top subunit is at the center of the hydrophobic interactions, forming van der Waals contacts with several hydrophobic residues of the bottom subunit. (C, D) GTPase activities of wild-type and mutant MtbFtsZ (C) or EcFtsZ (D). Means ± SD are shown (n = 3). (E) Electron microscopy analysis of GTP-dependent polymerization of MtbFtsZ (wild-type and L269E) and EcFtsZ (wild-type and L272E). Example protofilaments (PF) are highlighted with arrows.

Figure 2. GTP hydrolysis induces a “straight” to “curved” conformational switch at the longitudinal interface

(A) Superposition of the bottom subunit of a GDP-bound MtbFtsZ dimer (molecules A and B, this study) with that of a GDP-bound SaFtsZ dimer (PDB ID: 4DXD), showing a straight-to-curved bending at the longitudinal interface. Monomers in the MtbFtsZ dimer are shown in light red and dark grey for the top and bottom subunits, whereas those in the SaFtsZ dimer are shown in light green and light grey for the top and bottom subunits, respectively. (B) Superposition of MtbFtsZ monomers in the GDP-bound polymerized state (molecule A, this study; protein shown in light red with GDP in magenta) and the GTPγS-bound monomeric state (PDB ID: 1RLU; protein shown in light green with GTP in blue), illustrating that the T3 loop of MtbFtsZ adopts two different conformations: GTP-dependent Tension (T) and Relaxed (R) states. (C) Proposed mechanism for hydrolysis-mediated conformational switch of the longitudinal interface.

Figure 3. Bending of FtsZ protofilaments produces an inward force on the membrane

(A) Schematic of membrane deformation due to the force produced by hydrolysis-induced FtsZ protofilament bending. (B) Inter-monomer contacts were maintained in two independent molecular dynamics simulations of the MtbFtsZ-GDP dimer, suggesting stability of the bent conformation. Each time course measures the closest distance between a pair of amino acids: L272(top)-F135(bottom) (blue), L272(top)-L167(bottom) (orange), and L272(top)-L176(bottom) (purple). (C) Potential of mean force measurements for MtbFtsZ-GDP (red) and MtbFtsZ-GTP (green) along the hinge-opening pathway demonstrate a preference of MtbFtsZ-GDP protofilaments for a bent conformation, and suggest several energetically feasible intermediate conformations.