Structure of a GDP:AlF4 Complex of the SRP GTPases Ffh and FtsY, and Identification of a Peripheral Nucleotide Interaction Site

Focia, Pamela J.; Gawronski-Salerno, Joseph; Coon, John S.; Freymann, Douglas

doi:10.1016/j.jmb.2006.05.031

Cited by 32 publications

(45 citation statements)

References 62 publications

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“…In a previous crystal structure of the Ffh-FtsY NG domain heterodimer containing GDP-AlF 4 -, a peripheral nucleotide was found to bind in the same position as C83 (ref. 31), and mutation of C83 indeed affects SRPFtsY activity in vitro 18 . However, the low resolution of the SRP-FtsY structure and the absence of GDP-aluminum fluoride to mimic the transition state do not allow one to deduce the exact structural consequences of this RNA-GTPase contact for catalysis.…”

Section: Mechanism Of Srp-gtpase Activation Is Conservedmentioning

confidence: 90%

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

Bange

Kümmerer

Grudnik

et al. 2011

Nat Struct Mol Biol

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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 ...

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Section: Mechanism Of Srp-gtpase Activation Is Conservedmentioning

confidence: 90%

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

Bange

Kümmerer

Grudnik

et al. 2011

Nat Struct Mol Biol

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“…Probing of mutant RNAs with proteins that were BABE-Fe modified at residues E153 of Ffh and A359 of FtsY showed a loss of cleavage at residues 47-44 relative to wild type, suggesting that positions of these residues relative to the BABE-Fe moiety have changed relative to the wildtype tetraloop. Intriguingly, a recent crystal structure of the Ffh-FtsY heterodimer revealed a GMP bound at the heterodimer interface where the 4.5S RNA is predicted to bind, suggesting a possible site of interaction for nucleotides in or near the 4.5S RNA tetraloop (Focia et al 2006). …”

Section: Discussionmentioning

confidence: 99%

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

Siu

Spanggord²,

Doudna³

2006

RNA

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The signal recognition particle (SRP) cotranslationally targets proteins to cell membranes by coordinated binding and release of ribosome-associated nascent polypeptides and a membrane-associated SRP receptor. GTP uptake and hydrolysis by the SRPreceptor complex govern this targeting cycle. Because no GTPase-activating proteins (GAPs) are known for the SRP and SRP receptor GTPases, however, it has been unclear whether and how GTP hydrolysis is stimulated during protein trafficking in vivo. Using both biochemical and genetic experiments, we show here that SRP RNA enhances GTPase activity of the SRP-receptor complex above a critical threshold required for cell viability. Furthermore, this stimulation is a property of the SRP RNA tetraloop. SRP RNA tetraloop mutants that confer defective growth phenotypes can assemble into SRP-receptor complexes, but fail to stimulate GTP hydrolysis in these complexes in vitro. Tethered hydroxyl radical probing data reveal that specific positioning of the RNA tetraloop within the SRP-receptor complex is required to stimulate GTPase activity to a level sufficient to support cell growth. These results explain why no external GAP is needed and why the phylogenetically conserved SRP RNA tetraloop is required in vivo.

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“…The early intermediate, which lacks stabilizing interactions from the ␥-phosphate of GTP, is very unstable without cargo (5,14), hence it cannot accumulate under the nanomolar concentrations of (11,14), as described in the text, and the positions of fluorescence probes that detect the different conformational stages, as described in the text. SRP and SR used here (Fig.…”

Section: Cargo Stabilizes the Early Intermediate By Two Orders Of Magmentioning

confidence: 99%

“…These GTPases do not exhibit substantial conformational changes depending on whether GTP or GDP is bound (5)(6)(7), and further, their intrinsic nucleotide exchange rates are 10 2 -10 4 -fold faster than those of classical GTPases (8,9). Thus, no external GEFs are required to switch these GTPases from the GDP-to the GTPbound state, and the facilitation of nucleotide exchange by an external GEF cannot be the mechanism to turn these GTPases to the ''on'' state.…”

mentioning

confidence: 99%

Multiple conformational switches in a GTPase complex control co-translational protein targeting

Zhang

Schaffitzel

Ban

et al. 2009

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

223

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The ''GTPase switch'' paradigm, in which a GTPase switches between an active, GTP-bound state and an inactive, GDP-bound state through the recruitment of nucleotide exchange factors (GEFs) or GTPase activating proteins (GAPs), has been used to interpret the regulatory mechanism of many GTPases. A notable exception to this paradigm is provided by two GTPases in the signal recognition particle (SRP) and the SRP receptor (SR) that control the co-translational targeting of proteins to cellular membranes. Instead of the classical ''GTPase switch,'' both the SRP and SR undergo a series of discrete conformational rearrangements during their interaction with one another, culminating in their reciprocal GTPase activation. Here, we show that this series of rearrangements during SRP-SR binding and activation provide important control points to drive and regulate protein targeting. Using real-time fluorescence, we showed that the cargo for SRPribosomes translating nascent polypeptides with signal sequences-accelerates SRP⅐SR complex assembly over 100-fold, thereby driving rapid delivery of cargo to the membrane. A series of subsequent rearrangements in the SRP⅐SR GTPase complex provide important driving forces to unload the cargo during late stages of protein targeting. Further, the cargo delays GTPase activation in the SRP⅐SR complex by 8 -12 fold, creating an important time window that could further improve the efficiency and fidelity of protein targeting. Thus, the SRP and SR GTPases, without recruiting external regulatory factors, constitute a self-sufficient system that provides exquisite spatial and temporal control of a complex cellular process.conformational change ͉ fluorescence spectroscopy ͉ protein targeting and translocation ͉ signal recognition particle

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Structure of a GDP:AlF4 Complex of the SRP GTPases Ffh and FtsY, and Identification of a Peripheral Nucleotide Interaction Site

Cited by 32 publications

References 62 publications

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

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

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

Multiple conformational switches in a GTPase complex control co-translational protein targeting

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