Domain folding and flexibility of Escherichia coli FtsZ determined by tryptophan site‐directed mutagenesis

Abstract

FtsZ has two domains, the amino GTPase domain with a Rossmann fold, and the carboxyl domain that resembles the chorismate mutase fold. Bioinformatics analyses suggest that the interdomain interaction is stronger than the interaction of the protofilament longitudinal interfaces. Crystal B factor analysis of FtsZ and detected conformational changes suggest a connection between these domains. The unfolding/ folding characteristics of each domain of FtsZ were tested by introducing tryptophans into the flexible region of the amino (F135W) and the carboxyl (F275W and I294W) domains. As a control, the mutation F40W was introduced in a more rigid part of the amino domain. These mutants showed a native-like structure with denaturation and renaturation curves similar to wild type. However, the I294W mutant showed a strong loss of functionality, both in vivo and in vitro when compared to the other mutants. The functionality was recovered with the double mutant I294W/F275A, which showed full in vivo complementation with a slight increment of in vitro GTPase activity with respect to the single mutant. The formation of a stabilizing aromatic interaction involving a stacking between the tryptophan introduced at position 294 and phenylalanine 275 could account for these results. Folding/unfolding of these mutants induced by guanidinium chloride was compatible with a mechanism in which both domains within the protein show the same stability during FtsZ denaturation and renaturation, probably because of strong interface interactions.Keywords: FtsZ; folding; protein flexibility; GTPase FtsZ is a GTPase that forms the Z-ring, the first step in bacterial cell division. This ring is located around the middle of the cell on the cytosolic side of the inner membrane and is essential for recruiting the rest of the proteins that form the divisome (Errington et al. 2003;Vicente et al. 2006). The 3D crystal structure of FtsZ has been resolved for four microorganisms: the extremophilic archaebacteria Methanococcus jannaschii (MjFtsZ) in GDP and GTP conformations and an engineered conformation of Thermotoga maritima (TmFtsZ); and the mesophilic Pseudomonas aeruginosa (PaFtsZ) in GDP state and Mycobacterium tuberculosis

Glutamate 83 and arginine 85 of helix H3 bend are key residues for FtsZ polymerization, GTPase activity and cellular viability of Escherichia coli: lateral mutations affect FtsZ polymerization and E. coliviability

“…Thus, cells carrying FtsZ(E83Q) or FtsZ(R85Q) were affected in cell division and showed reduced survival and cell filamentation in agreement with previously described lateral-interaction-FtsZ mutants that could not complement the temperature sensitive ftsZ84 [10]. We have quantified survival rates at the restrictive temperature (42°C) as a means to evaluate the effect of FtsZ mutations on cell division, similar to studies on the effects of proteins expression from unstable plasmids [39,40]. …”

Flexibility in the Periplasmic Domain of BamA Is Important for Function

“…While the presence of interdomain dynamics has been demonstrated for many multi-domain proteins (Bertini et al, 2004; Blackledge, 2010; Diaz-Espinoza et al, 2007; Goto et al, 2001; Maciejewski et al, 2011; Ryabov and Fushman, 2006), the functional significance of these motions is often difficult to establish experimentally. Thus, here we complemented the NMR studies showing flexibility with an in vivo study of BamA mutants to probe the functional consequences of disrupting this flexibility.…”

Genetic and Biochemical Characterization of the MinC-FtsZ Interaction in Bacillus subtilis

“…Among the mutations whose GTPase was not tested, I293T is located on the bottom surface of FtsZ, very close to loop T7, which contains critical residues for GTP hydrolysis. Mutations in residues next to I293 (W319Y of Methanococcus jannaschii , I294W of E. coli ) showed 10 to 100 fold reduced GTPase [64], [65] and it is likely that I293T will have the same effect. Lastly, T232I is on a lateral surface of FtsZ, away from residues involved in GTP binding and hydrolysis.…”