Evidence for a novel GTPase priming step in the SRP protein targeting pathway

Lu, Yun; Qi, Haiyan; Hyndman, Janine B.; Ulbrandt, Nancy D.; Teplyakov, A.; Tomašević, Nenad; Bernstein, Harris

doi:10.1093/emboj/20.23.6724

Cited by 40 publications

(40 citation statements)

References 43 publications

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“…Together with the ''GQ'' motif in the loop connecting the strand ␤4 and helix ␣G3, and the ''DARGG'' motif at the beginning of helix ␣G4, the ''ALLEADV'' motif plays an important role in the regulation of SRP GTPases (19,(39)(40)(41). Mutations in the ''ALLEADV'' motif of SRP54 affect signal sequence binding (39) and mutations in the ''DARGG'' motif affect the formation of the heterodimer (41). In FlhF, these three motifs are absent and the corresponding regions show only low conservation (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Bange

Petzold

Wild

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

101

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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

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Section: Resultsmentioning

confidence: 99%

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

Bange

Petzold

Wild

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

101

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“…Helix ␣G4 in the interface between the N and G domain can sense this rotation when the LN23 loop moves toward the GTP binding site. In E. coli Ffh point mutations in the highly conserved ALLEADVN motif in helix ␣N2 and in the LN23 loop have indeed an effect on signal peptide binding (29) and the mutation of a conserved glycine in the ␣G4 helix leads to a loss of complex formation with the receptor (30). Taken together, these findings suggest that signal peptide binding to the M domain may result in a similar structural rearrangement in the NG domain interface as it is needed for, or induced by, GTP binding to the G domain.…”

Section: Resultsmentioning

confidence: 99%

“…A role of SRP RNA in the regulation of the GTPase and the interaction with the SR has been shown (27,28). Interdomain communication in SRP54 has been demonstrated by mutations in a highly conserved motif in the N domain affecting signal peptide binding in the M domain (29), and the mutation of a universally conserved glycine (Gly-254 in Sulfolobus solfataricus) in the G domain, giving rise to a lethal phenotype and reduced interaction with the SR (30). Both sites are located in the interface between the N and G domains, which has been proposed to be involved in a common intramolecular signaling mechanism (29,(31)(32)(33).…”

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confidence: 99%

Crystal structure of the complete core of archaeal signal recognition particle and implications for interdomain communication

Rosendal

Wild

Montoya

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

117

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Targeting of secretory and membrane proteins by the signal recognition particle (SRP) is evolutionarily conserved, and the multidomain protein SRP54 acts as the key player in SRP-mediated protein transport. Binding of a signal peptide to SRP54 at the ribosome is coordinated with GTP binding and subsequent complex formation with the SRP receptor. Because these functions are localized to distinct domains of SRP54, communication between them is essential. We report the crystal structures of SRP54 from the Archaeon Sulfolobus solfataricus with and without its cognate SRP RNA binding site (helix 8) at 4-Å resolution. The two structures show the flexibility of the SRP core and the position of SRP54 relative to the RNA. A long linker helix connects the GTPase (G domain) with the signal peptide binding (M) domain, and a hydrophobic contact between the N and M domains relates the signal peptide binding site to the G domain. Hinge regions are identified in the linker between the G and M domains (292-LGMGD) and in the N-terminal part of the M domain, which allow for structural rearrangements within SRP54 upon signal peptide binding at the ribosome. P rotein transport to or across the plasma membrane in bacteria and the endoplasmic reticulum in eukaryotes is mediated by the signal recognition particle (SRP), a ubiquitous ribonucleoprotein particle (for reviews see refs. 1-3). SRP recognizes amino-terminal signal sequences of newly synthesized polypeptides at the ribosome, and the ribosome nascent chain SRP complex is then targeted to the membrane by an interaction between SRP and its cognate receptor (SR). In the presence of the translocon the signal peptide is released (4-6) and the translating polypeptide is translocated across or inserted into the membrane. The SRP pathway is regulated by the concerted action of GTPases in both the SRP and the SR (7-11). GTP binding is a prerequisite for SRP͞SR interaction (7), and GTP hydrolysis in SRP and SR occurs after the signal peptide is released (8) and resolves the SRP͞SR complex (12).Although the SRP pathway is evolutionarily conserved, the composition of SRP and the SR varies widely. The complex mammalian SRP consists of six protein subunits (ranging from 9 to 72 kDa) and a 7SL RNA. In eubacteria SRP consists of only one protein subunit (Ffh, fifty-four-homologue) and a 4.5S RNA. SRP in archaea represents an intermediate between these two as only two polypeptides (homologues of SRP19 and SRP54) have been identified in archaeal genomes, and the SRP RNA of Ϸ310 nts resembles the mammalian 7SL RNA (for review see ref. 13). The SRP receptor consists of only one protein (FtsY, SR␣ homologue) in eubacteria and archaea, but two proteins (SR␣ and SR␤) in eukaryotes.SRP54 is the only protein subunit that is conserved in all SRPs, and thus it is the key player in protein transport. SRP54 is essential for binding signal peptides (14-16) at the ribosome and for GTP-dependent complex formation with the SR (17, 18). SRP54 is a multidomain protein that consists of an N-terminal N domain (a f...

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“…Many mutations at this interface disrupt SRP-SR complex formation and protein targeting (Lu et al, 2001). Interestingly, unlike classical GTPases, free FtsY displays little discrimination between GTP and noncognate nucleotides.…”

Section: Introductionmentioning

confidence: 99%

Efficient Interaction between Two GTPases Allows the Chloroplast SRP Pathway to Bypass the Requirement for an SRP RNA

Jaru-Ampornpan

Chandrasekar

Shan

2007

MBoC

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Cotranslational protein targeting to membranes is regulated by two GTPases in the signal recognition particle (SRP) and the SRP receptor; association between the two GTPases is slow and is accelerated 400-fold by the SRP RNA. Intriguingly, the otherwise universally conserved SRP RNA is missing in a novel chloroplast SRP pathway. We found that even in the absence of an SRP RNA, the chloroplast SRP and receptor GTPases can interact efficiently with one another; the kinetics of interaction between the chloroplast GTPases is 400-fold faster than their bacterial homologues, and matches the rate at which the bacterial SRP and receptor interact with the help of SRP RNA. Biochemical analyses further suggest that the chloroplast SRP receptor is pre-organized in a conformation that allows optimal interaction with its binding partner, so that conformational changes during complex formation are minimized. Our results highlight intriguing differences between the classical and chloroplast SRP and SRP receptor GTPases, and help explain how the chloroplast SRP pathway can mediate efficient targeting of proteins to the thylakoid membrane in the absence of the SRP RNA, which plays an indispensable role in all the other SRP pathways.

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Evidence for a novel GTPase priming step in the SRP protein targeting pathway

Cited by 40 publications

References 43 publications

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

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

Crystal structure of the complete core of archaeal signal recognition particle and implications for interdomain communication

Efficient Interaction between Two GTPases Allows the Chloroplast SRP Pathway to Bypass the Requirement for an SRP RNA

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