The crystal structure of the third signal-recognition particle GTPase FlhF reveals a homodimer with bound GTP

Abstract

Flagella are well characterized as the organelles of locomotion and allow bacteria to react to environmental changes. The assembly of flagella is a multistep process and relies on a complex type III export machinery located in the cytoplasmic membrane. The FlhF protein is essential for the placement and assembly of polar flagella and has been classified as a signal-recognition particle (SRP)-type GTPase. SRP GTPases appeared early in evolution and form a unique subfamily within the guanine nucleotide binding proteins with only three members: the signal sequence-binding protein SRP54, the SRP receptor FtsY, and FlhF. We report the crystal structures of FlhF from Bacillus subtilis in complex with GTP and GMPPNP. FlhF shares SRP GTPase-specific features such as the presence of an N-terminal ␣-helical domain and the I-box insertion. It forms a symmetric homodimer sequestering a composite active site that contains two head-to-tail arranged nucleotides similar to the heterodimeric SRP-targeting complex. However, significant differences to the GTPases of SRP and the SRP receptor include the formation of a stable homodimer with GTP as well as severe modifications and even the absence of motifs involved in regulation of the other two SRP GTPases. Our results provide insights into SRP GTPases and their roles in two fundamentally different protein-targeting routes that both rely on efficient protein delivery to a secretion channel.flagellum ͉ protein translocation ͉ x-ray structure analysis ͉ protein targeting

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

“…2c). The Gln8 interaction leads to a relocation of the G2 element, together with helix 1a of the I-box and the G3 element, into the composite active site, when compared to the NG-FlhF-GMP-PNP structure 5 ( Fig. 2d).…”

“…In both cases, interaction with the activator would induce the conformational stabilization of the G2 element required for efficient catalysis. Lowering entropy and enthalpy Clostrodium botulinum (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) Trepodema pallidum (1-27) Legionella pneumophila Borrelia burgdorferi (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) DQAXXLR motif *** ** is a fundamental and ancient principle in enzyme catalysis 32 , and it is also applicable to SRP-GTPases. Therefore, the activation of SRPGTPase FlhF is different from activation of the well-studied canonical small GTPases 33,34 , where a GAP provides a catalytic residue in trans and thereby completes the catalytic center (Fig.…”

“…SRP-GTPase homo-and heterodimers contain the complete catalytic machinery in a composite active site 5,8,9,30 . Our structural and biochemical studies show that YlxH binding to FlhF leads to movement and stabilization of the G2 element and allows the correct placement of the catalytic arginine required for elevated GTP hydrolysis.…”

“…FlhF and NG-FlhF were expressed and purified as previously described 5 . Briefly, proteins were expressed in Escherichia coli BL21(DE3) and purified by nickel-ion affinity and size exclusion chromatography (SEC).…”

“…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.…”