The crystal structure of the conserved GTPase of SRP54 from the archaeon Acidianus ambivalens and its comparison with related structures suggests a model for the SRP–SRP receptor complex

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101

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

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

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101

“…A minimal set of SRP components is found in bacteria, which consist of shorter RNAs (4.5S RNA) and only Ffh, the protein homologous to SRP54 (6,7). SRP54, the only protein component present in all SRPs, comprises an N-terminal domain (N, a four-helix bundle), a central GTPase domain [G, a ras-like GTPase fold, with an additional unique ␣-␤-␣ insertion box domain (IBD)], and a methionine-rich C-terminal domain (M, an all-␣ structure) (8,9). The N and G domains are structurally and functionally coupled; together, they build the NG domain that is connected to the M domain through a flexible linker (10).…”

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confidence: 99%

Interaction of signal-recognition particle 54 GTPase domain and signal-recognition particle RNA in the free signal-recognition particle

Hainzl

Huang

Sauer‐Eriksson

2007

The signal-recognition particle (SRP) is a ubiquitous protein-RNA complex that targets proteins to cellular membranes for insertion or secretion. A key player in SRP-mediated protein targeting is the evolutionarily conserved core consisting of the SRP RNA and the multidomain protein SRP54. Communication between the SRP54 domains is critical for SRP function, where signal sequence binding at the M domain directs receptor binding at the GTPase domain (NG domain). These SRP activities are linked to domain rearrangements, for which the role of SRP RNA is not clear. In free SRP, a direct interaction of the GTPase domain with SRP RNA has been proposed but has never been structurally verified. In this study, we present the crystal structure at 2.5-Å resolution of the SRP54 -SRP19 -SRP RNA complex of Methanococcus jannaschii SRP. The structure reveals an RNA-bound conformation of the SRP54 GTPase domain, in which the domain is spatially well separated from the signal peptide binding site. The association of both the N and G domains with SRP RNA in free SRP provides further structural evidence for the pivotal role of SRP RNA in the regulation of the SRP54 activity.T he signal-recognition particle (SRP) targets proteins destined for membrane insertion and secretion to or across the plasma membrane in bacteria and the endoplasmic reticulum in eukaryotes. SRP binds to the ribosome as well as to the hydrophobic signal sequences of nascent polypeptide chains as they emerge from the ribosome. The ribosome-nascent chain-SRP complex is then targeted to the membrane by an interaction between SRP and its receptor (SR in eukaryotes and FtsY in bacteria). In the presence of the translocon, the signal peptide is released into the translocon channel. SRP then dissociates from SR and the ribosome-nascent chain and is ready for another cycle of cotranslational protein targeting. The targeting cycle is coordinated by GTP binding and hydrolysis by both SRP and SR (for reviews, see refs. 1-3).SRP composition varies across the three domains of life. Mammalian SRP consists of a single Ϸ300-nt RNA (SRP RNA or 7S RNA) and six proteins, which can be divided into two major functional domains: the Alu domain (comprising the proteins SRP9 and -14) and the S domain (SRP19, -54, -68, and -72). The S domain functions in signal sequence recognition and SR interaction, whereas the Alu domain is required for translational arrest on signal sequence recognition (4). Archaeal SRPs consist of a 7S RNA that is highly similar to mammalian 7S RNAs, but only two homologues of the mammalian SRP proteins, namely SRP19 and SRP54 (5). A minimal set of SRP components is found in bacteria, which consist of shorter RNAs (4.5S RNA) and only Ffh, the protein homologous to SRP54 (6, 7). SRP54, the only protein component present in all SRPs, comprises an N-terminal domain (N, a four-helix bundle), a central GTPase domain [G, a ras-like GTPase fold, with an additional unique ␣-␤-␣ insertion box domain (IBD)], and a methionine-rich C-terminal domain (M, an all-␣ structure) ...

“…SRP54 interacts with the ribosomal proteins L23a and L35 (24) (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).A number of structures of the NG domain of Ffh from eubacteria and SRP54 from archaea (22,31,32,34) and the NG domain of FtsY from E. coli (21) have been reported. The structure of a major part of the M domain with and without RNA has also been determined for E. coli Ffh, human SRP54, and Ffh from Thermus aquaticus (35)(36)(37)(38).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Rosendal

Wild

Montoya

et al. 2003

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117

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...