Conformational change of the N-domain on formation of the complex between the GTPase domains of Thermus aquaticus Ffh and FtsY

Shepotinovskaya, Irina V.; Freymann, Douglas

doi:10.1016/s0167-4838(02)00287-x

Cited by 36 publications

(44 citation statements)

References 47 publications

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“…Residues 79-108 are absent in the crystal structure of FlhF and were shown to be partially degraded before crystallization by mass spectrometry (data not shown). A similar observation was reported for FtsY from Thermus aquaticus (33) and in the structures of the SRP/SR heterodimer the The apparent molecular mass was determined by size exclusion chromatography, the molecular mass was determined by static light scattering, and the nucleotide load was determined by HPLC analysis. The molecular masses calculated from the amino acid sequence of FlhF and NG-FlhF are 42.0 kDa and 33.3 kDa (29.0 kDa without helix ␣N1), respectively.…”

Section: Resultssupporting

confidence: 68%

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: Resultssupporting

confidence: 68%

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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“…In E. coli, association between the SRP and SR GTPases is extremely slow, with a rate constant of 5 ϫ 10 3 M Ϫ1 s Ϫ1 (Peluso et al, 2001). This slow association rate does not seem to be caused by the extended A-domain of E. coli FtsY, because Thermus aquaticus FtsY, which lacks an extended A-domain, also interacts with its binding partner very slowly (Shepotinovskaya and Freymann, 2001). At this rate and the in vivo concentration of these GTPases (nanomolar range), the association between the two GTPases will take hours to complete.…”

Section: Discussionmentioning

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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“…SRP and SR GTPases form a stable complex in the presence of GppNHp, and the complex can be separated from the monomers by Superdex 200 ( Figure 2C; Shepotinovskaya and Freymann, 2001). With wild-type cpFtsY efficient complex formation with cpSRP54 was observed, whereas with mutant cpFtsY G288W no detectable complex formation could be found during gel filtration chromatography analysis ( Figure 2C).…”

Section: Cpftsy Is Intrinsically Faster Than E Coli Ftsy At Interactmentioning

confidence: 99%

A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)–SRP Receptor Interaction by the Chloroplast SRP Pathway

Jaru-Ampornpan

Nguyen

Shan

2009

MBoC

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Cotranslational protein targeting by the signal recognition particle (SRP) requires the SRP RNA, which accelerates the interaction between the SRP and SRP receptor 200-fold. This otherwise universally conserved SRP RNA is missing in the chloroplast SRP (cpSRP) pathway. Instead, the cpSRP and cpSRP receptor (cpFtsY) by themselves can interact 200-fold faster than their bacterial homologues. Here, cross-complementation analyses revealed the molecular origin underlying their efficient interaction. We found that cpFtsY is 5-to 10-fold more efficient than Escherichia coli FtsY at interacting with the GTPase domain of SRP from both chloroplast and bacteria, suggesting that cpFtsY is preorganized into a conformation more conducive to complex formation. Furthermore, the cargo-binding M-domain of cpSRP provides an additional 100-fold acceleration for the interaction between the chloroplast GTPases, functionally mimicking the effect of the SRP RNA in the cotranslational targeting pathway. The stimulatory effect of the SRP RNA or the M-domain of cpSRP is specific to the homologous SRP receptor in each pathway. These results strongly suggest that the M-domain of SRP actively communicates with the SRP and SR GTPases and that the cytosolic and chloroplast SRP pathways have evolved distinct molecular mechanisms (RNA vs. protein) to mediate this communication. INTRODUCTIONThe signal recognition particle (SRP) and the SRP receptor (SR) comprise the major cellular machineries that cotranslationally deliver newly synthesized proteins from the cytosol to target membranes (Walter and Johnson, 1994;Keenan et al., 2001). Cotranslational protein targeting begins with recognition of the cargo-ribosomes translating nascent polypeptides containing signal sequences-by the SRP (Walter et al., 1981). The cargo is brought to the vicinity of the target membrane via the interaction between the SRP and SRP receptor (FtsY in bacteria) (Gilmore et al., 1982a). On arrival at the membrane, SRP unloads its cargo to the protein-conducting channel, composed of the sec61p complex in eukaryotic cells or secYEG complex in bacteria and archaea (Simon and Blobel, 1991;Gorlich et al., 1992;Halic et al., 2006). The SRP and SRP receptor also reciprocally stimulate each other's GTPase activity (Powers and Walter, 1995). Thus, after cargo unloading, guanosine triphosphate (GTP) hydrolysis drives disassembly of the SRP • SR complex, returning the components into the cytosol for the next round of protein targeting (Connolly et al., 1991).The SRP pathway is conserved throughout all three kingdoms of life. Although the protein components of SRP and SR vary across species, the functional core of SRP is a highly conserved ribonucleoprotein complex, composed of a 54-kDa SRP GTPase (SRP54 in eukaryotes or Ffh in bacteria) and an SRP RNA (Keenan et al., 2001). The SRP receptor also contains a conserved GTPase domain that is highly homologous to the GTPase domain in SRP54, and together the GTPase domains of SRP and SR form a unique subgroup in the GTPase superfamily (Keen...

show abstract

Conformational change of the N-domain on formation of the complex between the GTPase domains of Thermus aquaticus Ffh and FtsY

Cited by 36 publications

References 47 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

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

A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)–SRP Receptor Interaction by the Chloroplast SRP Pathway

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