Connecting sequence features within the disordered C-terminal linker of Bacillus subtilis FtsZ to functions and bacterial cell division

Intrinsically disordered regions (IDRs) can function as autoregulators of folded enzymes to which they are tethered. One example is the bacterial cell division protein FtsZ. This includes a folded core and a C-terminal tail (CTT) that encompasses a poorly conserved, disordered C-terminal linker (CTL) and a well-conserved 17-residue C-terminal peptide (CT17). Sites for GTPase activity of FtsZs are formed at the interface between GTP binding sites and T7 loops on cores of adjacent subunits within dimers. Here, we explore the basis of autoregulatory functions of the CTT in Bacillus subtilis FtsZ (Bs-FtsZ). Molecular simulations show that the CT17 of Bs-FtsZ makes statistically significant CTL-mediated contacts with the T7 loop. Statistical coupling analysis of more than 1,000 sequences from FtsZ orthologs reveals clear covariation of the T7 loop and the CT17 with most of the core domain, whereas the CTL is under independent selection. Despite this, we discover the conservation of nonrandom sequence patterns within CTLs across orthologs. To test how the nonrandom patterns of CTLs mediate CTT–core interactions and modulate FtsZ functionalities, we designed Bs-FtsZ variants by altering the patterning of oppositely charged residues within the CTL. Such alterations disrupt the core–CTT interactions, lead to anomalous assembly and inefficient GTP hydrolysis in vitro and protein degradation, aberrant assembly, and disruption of cell division in vivo. Our findings suggest that viable CTLs in FtsZs are likely to be IDRs that encompass nonrandom, functionally relevant sequence patterns that also preserve three-way covariation of the CT17, the T7 loop, and core domain.

Intrinsically disordered regions (IDRs) contribute to a multitude of protein functions (1–4). IDRs often have autoregulatory roles when tethered to folded domains either as tails or as linkers between folded domains (5–12). Of particular interest are IDRs tethered to folded domains that are enzymes (7, 13, 14). Several studies demonstrate that IDRs tethered to folded domains can function as autoregulators (12), specifically as autoinhibitors of enzymatic activities (13, 15, 16). One such example is the C-terminal tail (CTT) of the essential GTPase that controls and regulates bacterial cell division (17). The CTT encompasses a disordered C-terminal linker (CTL) and an alpha-helix-forming C-terminal peptide.Cell division in bacteria is initiated by assembly of the cytokinetic ring at the nascent division site (18–26). Polymers formed by the essential GTPase filamenting temperature-sensitive mutant Z (FtsZ) are the foundation of this ring, which is also known as the Z-ring (27–32). FtsZ is a prokaryotic homolog of tubulin. It forms single-stranded protofilaments upon binding GTP in vitro (33). Linear polymers of FtsZ, which also undergo bundling via lateral associations, serve as a platform for the cell division machinery composed of at least 30 different proteins (19, 32, 34–39). FtsZ polymers also undergo treadmilling in vivo, driven by the turnover of subunits that occurs on the order of seconds (40).Previous in vitro experiments showed that FtsZ polymerization belongs to a class of phase transitions known as reversible polymerization (41). A defining hallmark of reversible phase transitions, with subunit concentration as the conserved order parameter, is the presence of at least one threshold concentration for the occurrence of a specific phase transition. Cohan et al. recently identified two distinct threshold concentrations for Bacillus subtilis FtsZ (Bs-FtsZ) phase transitions occurring in the presence of GTP (17). In agreement with previous work on Escherichia coli FtsZ, Bs-FtsZ forms single-stranded protofilaments when the first threshold concentration, denoted as c_A, is crossed (42–47). The second threshold concentration, denoted as c_B where c_B > c_A, characterizes the threshold for bundling of protofilaments.Bs-FtsZ encompasses two domains: a folded N-terminal core and a CTT (Fig. 1A). The core domain forms a complete GTPase upon dimerization whereby the T7 loop of one protomer is inserted into the nucleotide binding site of the complementary protomer. The interface between the T7 loop and the nucleotide binding site is the active site for GTP hydrolysis (48). The CTT is further composed of an intrinsically disordered CTL and a 17-residue C-terminal peptide (CT17). The CT17 was previously termed CTP (17), and it includes a conserved “constant region” and a “variable region” (CTV) (30, 49, 50). The CT17 can form alpha-helical conformations (51–53) and is thus an alpha-molecular recognition element (54) that enables a precise network of homotypic and heterotypic protein–protein interactions. Whereas the CT17 includes a conserved region (33), the CTL is hypervariable across orthologs, varying in length, amino acid composition, and sequence (33, 49, 55, 56). Mutations in the CTL and the CTV of Bs-FtsZ disrupt lateral interactions between protofilaments (49, 56).Open in a separate windowFig. 1.Modular architecture of B. subtilis FtsZ includes a disordered CTT. (A) The electrostatic potential (103) is mapped onto the core domain in red and blue for regions of negative and positive potential, respectively. The T7 loop is highlighted in green. The CTT includes a CTL that connects the 17-residue C-terminal peptide (CT17) to the core domain. (B) The CTT is predicted to be disordered using IUPRED (60). The CTT sequence is shown with negatively and positively charged residues of the CTL in red and blue, respectively. The CT17 sequence is shown in gray. (C) Ensemble-averaged secondary structure contents of the CTT obtained from atomistic simulations. (D) UV-CD spectra of the four Bs-FtsZ constructs. See Materials and Methods for details on scaling of the [θ*].Consistent with previous work (57–59), Cohan et al. showed, through systematic deletions of each module, that the core domain of Bs-FtsZ is the main driver of GTP binding-induced polymerization (17). Deletion of the CT17 (ΔCT17), previously referred to as ΔCTP, increases c_A while also shifting c_B upward by at least threefold. Internal deletion of the CTL (ΔCTL) decreases c_A and this construct forms mini rings stabilized by cohesive interactions of the CT17. Overall, the CTL weakens the driving forces for linear polymerization and bundling, whereas the CT17 appears to be the primary driver of lateral associations. Deletion of the CTT (ΔCTT) lowers c_A by over an order of magnitude and forms long, single-stranded polymers. Cohan et al. also showed that ΔCTT is the most efficient GTPase, whereas the wild-type Bs-FtsZ is the least efficient enzyme of the four constructs studied (17).The picture that emerges is of the CTT as an autoregulator of Bs-FtsZ assembly and an autoinhibitor of enzymatic activity (17). Here, we uncover a molecular-level, mechanistic understanding of how the distinctive functions of CTTs are achieved.