Structural basis for the molecular evolution of SRP-GTPase activation by protein

Abstract

SIMIBI-class (named after the signal recognition particle, MinD, BioD) nucleotide-binding proteins appeared early in evolution 1 and contain GTPases, as well as ATPases, involved in the correct localization of cellular constituents. The MinD ATPase, as the central part of the Min system, regulates the determination of the cell division site in all bacterial species 2 . SRP-GTPases form a subfamily of the SIMIBI class, with only three members: the signal sequence-binding protein Ffh (SRP54 in Eukarya and Archaea), the SRP receptor FtsY (SR in Eukarya) and FlhF, which is involved in flagella biosynthesis [3][4][5] . They share the conserved NG domain, which contains two major additions to the conserved fold of small G proteins. First, an --element (I-box) is inserted in the effector region; second, the N domain, comprising four -helices, is attached to the N terminus of the G domain. SRP (Ffh together with the SRP RNA) and FtsY constitute the universally conserved co-translational protein-targeting machinery 6,7 . When bound to GTP, Ffh and FtsY form, through interactions between their NG domains 8,9 , a heterodimeric complex that regulates the transfer of a ribosome-nascent chain complex to a vacant translocon in the membrane with a series of conformational rearrangements 10,11 . The two GTPases share a composite active site between their G domains in which GTP hydrolysis is reciprocally activated 12 . The SRP RNA [13][14][15] and membrane lipids 16,17 play fundamental roles in activating the Ffh-FtsY GTPases. The recent structure of the SRP-FtsY complex, together with biochemical implications, suggest that the distal end of the hairpin-like SRP RNA may be involved in this activation 18 . The third SRP-GTPase FlhF, together with the MinD-type protein YlxH (also known as FlhG, FleN, motR or MinD2), is essential for the placement and assembly of flagella 19 in many polar and peritrichous flagellated bacteria [20][21][22][23][24] . FlhF is required for the targeting of the first flagellar protein, FliF, to the cell pole 25 by a mechanism that is so far poorly understood. FlhF is associated with the membrane 25,26 and localizes at the cell pole 20 . The FlhF protein (Fig. 1a) contains an N-terminal B domain that seems to be involved in FliF targeting 25 ; it shares the NG domain fold with the other two members of the SRP-GTPase subfamily. FlhF forms a stable homodimer with GTP and a composite active site that is basically identical to the active site of the Ffh-FtsY heterodimer 5 . In both the homo-and heterodimer, the two nucleotides are bound in a head-to-tail manner, with the -phosphate of one nucleotide interacting with the 3 -OH of the ribose moiety of the other. However, for the homo-and heterodimers formed by the three SRP-GTPases, the molecular mechanism of activation is still unknown. We set out to understand the activation of SRPGTPases by studying FlhF. RESULTS The SRP-GTPase FlhF is activated by YlxHAs FlhF (Fig. 1) forms a stable homodimer, and reciprocal activation has not been observed 5 , we reasoned ...

The GTPase Activity of FlhF Is Dispensable for Flagellar Localization, but Not Motility, in Pseudomonas aeruginosa

“…In this study, we showed that purified full-length P. aeruginosa FlhF is a GTPase with a high K m (ϳ30 M), a low turnover rate (k cat ϭ 0.06/s), and a low V max (25 mol/h). These kinetic parameters are similar to those reported for another SRP-GTPase, B. subtilis FlhF (20 mol/h) (13).…”

“…The FlhF proteins of Bacillus subtilis (BsFlhF) and C. jejuni (CjFlhF) were recently shown to have GTPase activity (13,14). However, the phenotypes associated with flhF deletion or mutation differ for these organisms and P. aeruginosa, making it difficult to generalize a role for FlhF in P. aeruginosa based on these studies.…”

YlxM Is a Newly Identified Accessory Protein That Influences the Function of Signal Recognition Particle Pathway Components in Streptococcus mutans

“…Proteins such as SRP68/72, SRP9/14, and SRP19 all contribute to the regulation of the pathway in ways that are not completely understood (reviewed in reference 3). It has been predicted that as-yet-unknown proteins may work in concert with the scRNA to regulate bacterial Ffh/FtsY activity (46,47). It appears that YlxM represents such an accessory factor.…”

“…YlxM may have a function analogous to that of YlxH, an accessory protein recently found to regulate the activity of FlhF, a protein involved in flagellar biosynthesis in B. subtilis (46). Like Ffh and FtsY, FlhF is a member of the SIMIBI family of GTPases.…”

HubP, a Polar Landmark Protein, Regulates Flagellar Number by Assisting in the Proper Polar Localization of FlhG in Vibrio alginolyticus

“…FlhF is a GTPase protein and is similar to the signal recognition particle receptor FtsY (8,9). The GTPase motif of FlhF influences the intracellular location of this protein (10,11), and GTPase activity is stimulated through interactions with FlhG (12)(13)(14). FlhG is an ATPase protein and is similar to MinD, a cell division regulator in prokaryotes (15)(16)(17).…”