Conformational changes in the GTPase modules of the signal reception particle and its receptor drive initiation of protein translocation

During cotranslational protein targeting, two guanosine triphosphatase (GTPase) in the signal recognition particle (SRP) and its receptor (SR) form a unique complex in which hydrolyses of both guanosine triphosphates (GTP) are activated in a shared active site. It was thought that GTP hydrolysis drives the recycling of SRP and SR, but is not crucial for protein targeting. Here, we examined the translocation efficiency of mutant GTPases that block the interaction between SRP and SR at specific stages. Surprisingly, mutants that allow SRP-SR complex assembly but block GTPase activation severely compromise protein translocation. These mutations map to the highly conserved insertion box domain loops that rearrange upon complex formation to form multiple catalytic interactions with the two GTPs. Thus, although GTP hydrolysis is not required, the molecular rearrangements that lead to GTPase activation are essential for protein targeting. Most importantly, our results show that an elaborate rearrangement within the SRP-SR GTPase complex is required to drive the unloading and initiate translocation of cargo proteins.     

Concerted complex assembly and GTPase activation in the chloroplast signal recognition particle

The universally conserved signal recognition particle (SRP) and SRP receptor (SR) mediate the cotranslational targeting of proteins to cellular membranes. In contrast, a unique chloroplast SRP in green plants is primarily dedicated to the post-translational targeting of light harvesting chlorophyll a/b binding (LHC) proteins. In both pathways, dimerization and activation between the SRP and SR GTPases mediate the delivery of cargo; whether and how the GTPase cycle in each system adapts to its distinct substrate proteins were unclear. Here, we show that interactions at the active site essential for GTPase activation in the chloroplast SRP and SR play key roles in the assembly of the GTPase complex. In contrast to their cytosolic homologues, GTPase activation in the chloroplast SRP-SR complex contributes marginally to the targeting of LHC proteins. These results demonstrate that complex assembly and GTPase activation are highly coupled in the chloroplast SRP and SR and suggest that the chloroplast GTPases may forego the GTPase activation step as a key regulatory point. These features may reflect adaptations of the chloroplast SRP to the delivery of their unique substrate protein.     

Dual recognition of the ribosome and the signal recognition particle by the SRP receptor during protein targeting to the endoplasmic reticulum

We have analyzed the interactions between the signal recognition particle (SRP), the SRP receptor (SR), and the ribosome using GTPase assays, biosensor experiments, and ribosome binding assays. Possible mechanisms that could contribute to an enhanced affinity between the SR and the SRP-ribosome nascent chain complex to promote protein translocation under physiological ionic strength conditions have been explored. Ribosomes or 60S large ribosomal subunits activate the GTPase cycle of SRP54 and SRalpha by providing a platform for assembly of the SRP-SR complex. Biosensor experiments revealed high-affinity, saturable binding of ribosomes or large ribosomal subunits to the SR. Remarkably, the SR has a 100-fold higher affinity for the ribosome than for SRP. Proteoliposomes that contain the SR bind nontranslating ribosomes with an affinity comparable to that shown by the Sec61 complex. An NH2-terminal 319-residue segment of SRalpha is necessary and sufficient for binding of SR to the ribosome. We propose that the ribosome-SR interaction accelerates targeting of the ribosome nascent chain complex to the RER, while the SRP-SR interaction is crucial for maintaining the fidelity of the targeting reaction.     

Targeting proteins to membranes: structure of the signal recognition particle

In all three kingdoms of life, co-translational targeting of secretory and membrane proteins to the prokaryotic plasma membrane or eukaryotic endoplasmic reticulum is mediated by a ribonucleoprotein complex, the signal recognition particle (SRP), and its membrane-associated receptor (SR). SRP binds to signal sequences of nascent proteins as they emerge from the exit tunnel of the ribosome. The resulting targeting complex, composed of the SRP and the ribosome-nascent chain complex (RNC), then docks with the SR in a GTP-dependent manner. Passing through a complex series of conformational states, SRP and SR deliver the RNC to the translocon, which in turn mediates protein translocation across or integration into the membrane. The core structural and mechanistic principles of SRP-dependent protein targeting are universally conserved. Recent structural investigations combining X-ray crystallography and cryo-electron microscopy have provided new insights into three essentials steps of the SRP-dependent protein targeting cycle: the assembly and interaction of the SRP ribonucleoprotein core, the GTP-dependent SRP-SR association, and the interaction between SRP and the ribosome.     

Co-translational protein targeting by the signal recognition particle

The signal recognition particle (SRP) mediates the co-translational targeting of nascent proteins to the eukaryotic endoplasmic reticulum membrane, or the bacterial plasma membrane. During this process, two GTPases, one in the SRP and one in the SRP receptor (SR), form a complex in which both proteins reciprocally activate the GTPase reaction of one another. The recent crystal structures of the T. aquaticus SRP.SR complex show that the two GTPases associate via an unusually extensive and highly cooperative interaction surface, and form a composite active site at the interface. GTPase activation proceeds through a unique mechanism, stimulated by both interactions between the twinned GTP molecules across the dimer interface and by conformational rearrangements that position catalytic residues in each active site with respect to the bound substrates. Distinct classes of mutations have been isolated that inhibit specific stages during SRP-SR complex formation and activation, suggesting discrete conformational stages during formation of the active SRP.SR complex. Each stage provides a potential control point in the targeting reaction at which regulation by additional components can be exerted, thus ensuring the binding and release of cargo at the appropriate time.     

Structures of SRP54 and SRP19, the two proteins that organize the ribonucleic core of the signal recognition particle from Pyrococcus furiosus

In all organisms the Signal Recognition Particle (SRP), binds to signal sequences of proteins destined for secretion or membrane insertion as they emerge from translating ribosomes. In Archaea and Eucarya, the conserved ribonucleoproteic core is composed of two proteins, the accessory protein SRP19, the essential GTPase SRP54, and an evolutionarily conserved and essential SRP RNA. Through the GTP-dependent interaction between the SRP and its cognate receptor SR, ribosomes harboring nascent polypeptidic chains destined for secretion are dynamically transferred to the protein translocation apparatus at the membrane. We present here high-resolution X-ray structures of SRP54 and SRP19, the two RNA binding components forming the core of the signal recognition particle from the hyper-thermophilic archaeon Pyrococcus furiosus (Pfu). The 2.5 A resolution structure of free Pfu-SRP54 is the first showing the complete domain organization of a GDP bound full-length SRP54 subunit. In its ras-like GTPase domain, GDP is found tightly associated with the protein. The flexible linker that separates the GTPase core from the hydrophobic signal sequence binding M domain, adopts a purely alpha-helical structure and acts as an articulated arm allowing the M domain to explore multiple regions as it scans for signal peptides as they emerge from the ribosomal tunnel. This linker is structurally coupled to the GTPase catalytic site and likely to propagate conformational changes occurring in the M domain through the SRP RNA upon signal sequence binding. Two different 1.8 A resolution crystal structures of free Pfu-SRP19 reveal a compact, rigid and well-folded protein even in absence of its obligate SRP RNA partner. Comparison with other SRP19*SRP RNA structures suggests the rearrangement of a disordered loop upon binding with the RNA through a reciprocal induced-fit mechanism and supports the idea that SRP19 acts as a molecular scaffold and a chaperone, assisting the SRP RNA in adopting the conformation required for its optimal interaction with the essential subunit SRP54, and proper assembly of a functional SRP.     

The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane

The signal recognition particle receptor (SR) is required for the cotranslational targeting of both secretory and membrane proteins to the endoplasmic reticulum (ER) membrane. During targeting, the SR interacts with the signal recognition particle (SRP) which is bound to the signal sequence of the nascent protein chain. This interaction catalyzes the GTP-dependent transfer of the nascent chain from SRP to the protein translocation apparatus in the ER membrane. The SR is a heterodimeric protein comprised of a 69-kD subunit (SR alpha) and a 30- kD subunit (SR beta) which are associated with the ER membrane in an unknown manner. SR alpha and the 54-kD subunits of SRP (SRP54) each contain related GTPase domains which are required for SR and SRP function. Molecular cloning and sequencing of a cDNA encoding SR beta revealed that SR beta is a transmembrane protein and, like SR alpha and SRP54, is a member of the GTPase superfamily. Although SR beta defines its own GTPase subfamily, it is distantly related to ARF and Sar1. Using UV cross-linking, we confirm that SR beta binds GTP specifically. Proteolytic digestion experiments show that SR alpha is required for the interaction of SRP with SR. SR alpha appears to be peripherally associated with the ER membrane, and we suggest that SR beta, as an integral membrane protein, mediates the membrane association of SR alpha. The discovery of its guanine nucleotide-binding domain, however, makes it likely that its role is more complex than that of a passive anchor for SR alpha. These findings suggest that a cascade of three directly interacting GTPases functions during protein targeting to the ER membrane.     

A structural step into the SRP cycle

Co-translational targeting of secretory and membrane proteins to the translocation machinery is mediated by the signal recognition particle (SRP) and its membrane-bound receptor (SR) in all three domains of life. Although the overall composition of the SRP system differs, the central ribonucleoprotein core and the general mechanism of GTP-dependent targeting are highly conserved. Recently, structural studies have contributed significantly to our understanding of the molecular organization of SRP. SRP appears as a structurally flexible particle modulated and regulated by its interactions with the ribosome-nascent chain complex, the translocon and the SR. The SRP core (SRP54 with its cognate RNA binding site) plays a central role in these interactions and communicates the different binding states by long-range interdomain communication. Based on recent structures of SRP54, a model for signal peptide binding stimulating the GTP affinity during the first step of the SRP cycle is presented. The model is placed in the context of the recent structures of mammalian SRP bound to a ribosome-nascent chain complex and of a subcomplex of SRP-SR.     

X-ray structures of the signal recognition particle receptor reveal targeting cycle intermediates

The signal recognition particle (SRP) and its conjugate receptor (SR) mediate cotranslational targeting of a subclass of proteins destined for secretion to the endoplasmic reticulum membrane in eukaryotes or to the plasma membrane in prokaryotes. Conserved active site residues in the GTPase domains of both SRP and SR mediate discrete conformational changes during formation and dissociation of the SRP.SR complex. Here, we describe structures of the prokaryotic SR, FtsY, as an apo protein and in two different complexes with a non-hydrolysable GTP analog (GMPPNP). These structures reveal intermediate conformations of FtsY containing GMPPNP and explain how the conserved active site residues position the nucleotide into a non-catalytic conformation. The basis for the lower specificity of binding of nucleotide in FtsY prior to heterodimerization with the SRP conjugate Ffh is also shown. We propose that these structural changes represent discrete conformational states assumed by FtsY during targeting complex formation and dissociation.     

Advances in the structure and functions of signal recognition particle in protein targeting

The signal recognition particle (SRP) is a unique moiety in living cells, which has been conserved during evolution for protein targeting and translocation across membranes in collaboration with its receptor (SR). The structural and functional features of its components, (six polypeptides and RNA) are being rapidly elucidated. We have endeavored in this review to epitomize most recent advances in this field. Its two domains (S and Alu) play important roles in signal recognition, elongation arrest and protein targeting of the polypeptide being synthesized in the cytoplasm. SRP14 and SRP9 help in the elongation arrest by interacting with signal peptide. GTPase activity of SRP54 releases SRP from SR. In addition, alpha and beta subunits of SR also possess GTPase activities and the three GTPases help in docking of nascent peptide chain-ribosome complex to the translocation site. Further strides in proteomics employing mass spectrometry and X-ray crystallography are expected to throw more light on the molecular events occurring during protein targeting and translocation.     

Molecular dynamics simulations reveal structural coordination of Ffh-FtsY heterodimer toward GTPase activation

Two homologous GTPases (guanine-triphosphatases) in the signal recognition particle (SRP) and its receptor (SR) use their cumulative energy during GTP (guanine-triphosphate) hydrolysis to control the co-translational protein targeting process. Distinct from classical GTPases, which rely on external factors to hydrolyze GTP, SRP GTPases stimulate one another's activity in a self-sufficient manner upon SRP-SR complex association. Although both ground-state and putative transition-state GTP analogs have been used to recapitulate the state of GTPase activation, the underlying mechanism of the activated state still remains elusive. In particular, several residues that were placed in pending positions have been shown to be important to GTP hydrolysis in biochemical studies. Here, we examined the stability and dynamics of three interaction networks involving these residues and discovered that they contribute to the GTPase activation via well-tuned conformational changes. The crystallographically identified pending residues Ffh:R191/FtsY:R195 undergo extensive conformational rearrangements to form persisted interactions with FtsY:E284/Ffh:E274, explaining the biochemically observed defective effect of R191 mutant to the activation of both GTPases. In addition, the side chain of FtsY:R142, one of the most important catalytic residues, rotates to an extended conformation that could more efficiently maintain the electrostatic balance for GTP hydrolysis. Finally, the invariant residues Ffh:G190 and FtsY:G194, instead of the supposed auxiliary water molecules, are proposed to stabilize the nucleophilic waters during GTPase activation. In complementary to experimental observations, these findings suggest a more favorable interaction model for SRP GTPase activation and would thus benefit to our understanding of how SRP GTPases regulate the protein targeting pathway.     

Co-translational protein targeting to the bacterial membrane

Co-translational protein targeting by the Signal Recognition Particle (SRP) is an essential cellular pathway that couples the synthesis of nascent proteins to their proper cellular localization. The bacterial SRP, which contains the minimal ribonucleoprotein core of this universally conserved targeting machine, has served as a paradigm for understanding the molecular basis of protein localization in all cells. In this review, we highlight recent biochemical and structural insights into the molecular mechanisms by which fundamental challenges faced by protein targeting machineries are met in the SRP pathway. Collectively, these studies elucidate how an essential SRP RNA and two regulatory GTPases in the SRP and SRP receptor (SR) enable this targeting machinery to recognize, sense and respond to its biological effectors, i.e. the cargo protein, the target membrane and the translocation machinery, thus driving efficient and faithful co-translational protein targeting. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

The conformation of bound GMPPNP suggests a mechanism for gating the active site of the SRP GTPase

BACKGROUND: The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein that mediates cotranslational targeting of secreted and membrane proteins to the membrane. Targeting is regulated by GTP binding and hydrolysis events that require direct interaction between structurally homologous "NG" GTPase domains of the SRP signal recognition subunit and its membrane-associated receptor, SR alpha. Structures of both the apo and GDP bound NG domains of the prokaryotic SRP54 homolog, Ffh, and the prokaryotic receptor homolog, FtsY, have been determined. The structural basis for the GTP-dependent interaction between the two proteins, however, remains unknown. RESULTS: We report here two structures of the NG GTPase of Ffh from Thermus aquaticus bound to the nonhydrolyzable GTP analog GMPPNP. Both structures reveal an unexpected binding mode in which the beta-phosphate is kinked away from the binding site and magnesium is not bound. Binding of the GTP analog in the canonical conformation found in other GTPase structures is precluded by constriction of the phosphate binding P loop. The structural difference between the Ffh complex and other GTPases suggests a specific conformational change that must accompany movement of the nucleotide from an "inactive" to an "active" binding mode. CONCLUSIONS: Conserved side chains of the GTPase sequence motifs unique to the SRP subfamily may function to gate formation of the active GTP bound conformation. Exposed hydrophobic residues provide an interaction surface that may allow regulation of the GTP binding conformation, and thus activation of the GTPase, during the association of SRP with its receptor.     

The structure of the mammalian signal recognition particle (SRP) receptor as prototype for the interaction of small GTPases with Longin domains

The eukaryotic signal recognition particle (SRP) and its receptor (SR) play a central role in co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum. The SR is a heterodimeric complex assembled by the two GTPases SRalpha and SRbeta, which is membrane-anchored. Here we present the 2.45-A structure of mammalian SRbeta in its Mg2+ GTP-bound state in complex with the minimal binding domain of SRalpha termed SRX. SRbeta is a member of the Ras-GTPase superfamily closely related to Arf and Sar1, while SRX belongs to the SNARE-like superfamily with a fold also known as longin domain. SRX binds to the P loop and the switch regions of SRbeta-GTP. The binding mode and structural similarity with other GTPase-effector complexes suggests a co-GAP (GTPase-activating protein) function for SRX. Comparison with the homologous yeast structure and other longin domains reveals a conserved adjustable hydrophobic surface within SRX which is of central importance for the SRbeta-GTP:SRX interface. A helix swap in SRX results in the formation of a dimer in the crystal structure. Based on structural conservation we present the SRbeta-GTP:SRX structure as a prototype for conserved interactions in a variety of GTPase regulated targeting events occurring at endomembranes.     

Assembly of archaeal signal recognition particle from recombinant components

Signal recognition particle (SRP) takes part in protein targeting and secretion in all organisms. Searches for components of archaeal SRP in primary databases and completed genomes indicated that archaea possess only homologs of SRP RNA, and proteins SRP19 and SRP54. A recombinant SRP was assembled from cloned, expressed and purified components of the hyperthermophilic archaeon Archaeoglobus fulgidus. Recombinant Af-SRP54 associated with the signal peptide of bovine pre-prolactin translated in vitro. As in mammalian SRP, Af-SRP54 binding to Af-SRP RNA required protein Af-SRP19, although notable amounts bound in absence of Af-SRP19. Archaeoglobus fulgidus SRP proteins also bound to full-length SRP RNA of the archaeon Methanococcus jannaschii, to eukaryotic human SRP RNA, and to truncated versions which corresponded to the large domain of SRP. Dependence on SRP19 was most pronounced with components from the same species. Reconstitutions with heterologous components revealed a significant potential of human SRP proteins to bind to archaeal SRP RNAs. Surprisingly, M.jannaschii SRP RNA bound to human SRP54M quantitatively in the absence of SRP19. This is the first report of reconstitution of an archaeal SRP from recombinantly expressed purified components. The results highlight structural and functional conservation of SRP assembly between archaea and eucarya.     

Molecular evolution of SRP cycle components: functional implications.

Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein that targets a subset of nascent presecretory proteins to the endoplasmic reticulum membrane. We have considered the SRP cycle from the perspective of molecular evolution, using recently determined sequences of genes or cDNAs encoding homologs of SRP (7SL) RNA, the Srp54 protein (Srp54p), and the alpha subunit of the SRP receptor (SR alpha) from a broad spectrum of organisms, together with the remaining five polypeptides of mammalian SRP. Our analysis provides insight into the significance of structural variation in SRP RNA and identifies novel conserved motifs in protein components of this pathway. The lack of congruence between an established phylogenetic tree and size variation in 7SL homologs implies the occurrence of several independent events that eliminated more than half the sequence content of this RNA during bacterial evolution. The apparently non-essential structures are domain I, a tRNA-like element that is constant in archaea, varies in size among eucaryotes, and is generally missing in bacteria, and domain III, a tightly base-paired hairpin that is present in all eucaryotic and archeal SRP RNAs but is invariably absent in bacteria. Based on both structural and functional considerations, we propose that the conserved core of SRP consists minimally of the 54 kDa signal sequence-binding protein complexed with the loosely base-paired domain IV helix of SRP RNA, and is also likely to contain a homolog of the Srp68 protein. Comparative sequence analysis of the methionine-rich M domains from a diverse array of Srp54p homologs reveals an extended region of amino acid identity that resembles a recently identified RNA recognition motif. Multiple sequence alignment of the G domains of Srp54p and SR alpha homologs indicates that these two polypeptides exhibit significant similarity even outside the four GTPase consensus motifs, including a block of nine contiguous amino acids in a location analogous to the binding site of the guanine nucleotide dissociation stimulator (GDS) for E. coli EF-Tu. The conservation of this sequence, in combination with the results of earlier genetic and biochemical studies of the SRP cycle, leads us to hypothesize that a component of the Srp68/72p heterodimer serves as the GDS for both Srp54p and SR alpha. Using an iterative alignment procedure, we demonstrate similarity between Srp68p and sequence motifs conserved among GDS proteins for small Ras-related GTPases. The conservation of SRP cycle components in organisms from all three major branches of the phylogenetic tree suggests that this pathway for protein export is of ancient evolutionary origin.     

Structure and DNA-binding activity of the Pyrococcus furiosus SMC protein hinge domain

Structural Maintenance of Chromosomes (SMC) proteins are essential for a wide range of processes including chromosome structure and dynamics, gene regulation, and DNA repair. While bacteria and archaea have one SMC protein that forms a homodimer, eukaryotes possess three distinct SMC complexes, consisting of heterodimeric pairs of six different SMC proteins. SMC holocomplexes additionally contain several specific regulatory subunits. The bacterial SMC complex is required for chromosome condensation and segregation. In eukaryotes, this function is carried out by the condensin (SMC2-SMC4) complex. SMC proteins consist of N-terminal and C-terminal domains that fold back onto each other to create an ATPase "head" domain, connected to a central "hinge" domain via a long coiled-coil region. The hinge domain mediates dimerization of SMC proteins and binds DNA. This activity implicates a direct involvement of the hinge domain in the action of SMC proteins on DNA. We studied the SMC hinge domain from the thermophilic archaeon Pyrococcus furiosus. Its crystal structure shows that the SMC hinge domain fold is largely conserved between archaea and bacteria as well as eukarya. Like the eukaryotic condensin hinge domain, the P. furiosus SMC hinge domain preferentially binds single-stranded DNA (ssDNA), but its affinity for DNA is weaker than that of its eukaryotic counterpart, and point mutations reveal that its DNA-binding surface is more confined. The ssDNA-binding activity of its hinge domain might play a role in the DNA-loading process of the prokaryotic SMC complex during replication.     

Molecular Mechanism of GTPase Activation at the Signal Recognition Particle (SRP) RNA Distal End

The signal recognition particle (SRP) RNA is a universally conserved and essential component of the SRP that mediates the co-translational targeting of proteins to the correct cellular membrane. During the targeting reaction, two functional ends in the SRP RNA mediate distinct functions. Whereas the RNA tetraloop facilitates initial assembly of two GTPases between the SRP and SRP receptor, this GTPase complex subsequently relocalizes ∼100 Å to the 5′,3′-distal end of the RNA, a conformation crucial for GTPase activation and cargo handover. Here we combined biochemical, single molecule, and NMR studies to investigate the molecular mechanism of this large scale conformational change. We show that two independent sites contribute to the interaction of the GTPase complex with the SRP RNA distal end. Loop E plays a crucial role in the precise positioning of the GTPase complex on these two sites by inducing a defined bend in the RNA helix and thus generating a preorganized recognition surface. GTPase docking can be uncoupled from its subsequent activation, which is mediated by conserved bases in the next internal loop. These results, combined with recent structural work, elucidate how the SRP RNA induces GTPase relocalization and activation at the end of the protein targeting reaction.     

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

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.     

Structure of the chloroplast signal recognition particle (SRP) receptor: domain arrangement modulates SRP-receptor interaction

The signal recognition particle (SRP) pathway mediates co-translational targeting of nascent proteins to membranes. Chloroplast SRP is unique in that it does not contain the otherwise universally conserved SRP RNA, which accelerates the association between the SRP guanosine-5′-triphosphate (GTP) binding protein and its receptor FtsY in classical SRP pathways. Recently, we showed that the SRP and SRP receptor (SR) GTPases from chloroplast (cpSRP54 and cpFtsY, respectively) can interact with one another 400-fold more efficiently than their bacterial homologues, thus providing an explanation as to why this novel chloroplast SRP pathway bypasses the requirement for the SRP RNA. Here we report the crystal structure of cpFtsY from Arabidopsis thaliana at 2.0 Å resolution. In this chloroplast SR, the N-terminal “N” domain is more tightly packed, and a more extensive interaction surface is formed between the GTPase “G” domain and the N domain than was previously observed in many of its bacterial homologues. As a result, the overall conformation of apo-cpFtsY is closer to that found in the bacterial SRP•FtsY complex than in free bacterial FtsY, especially with regard to the relative orientation of the N and G domains. In contrast, active-site residues in the G domain are mispositioned, explaining the low basal GTP binding and hydrolysis activity of free cpFtsY. This structure emphasizes proper N-G domain arrangement as a key factor in modulating the efficiency of SRP-receptor interaction and helps account, in part, for the faster kinetics at which the chloroplast SR interacts with its binding partner in the absence of an SRP RNA.