Molecular dynamics simulation reveals preorganization of the chloroplast FtsY towards complex formation induced by GTP binding

Two GTPases in the signal recognition particle (SRP) and SRP receptor (SR) interact with one another to mediate the cotranslational protein targeting pathway. Previous studies have shown that a universally conserved SRP RNA facilitates an efficient SRP–SR interaction in the presence of a signal sequence bound to SRP. However, a remarkable exception has been found in chloroplast SRP (cpSRP) pathway, in which the SRP RNA is missing. Based on biochemical and structural analyses, it is proposed that free cpSRP receptor (cpFtsY) has already been preorganized into a closed state for efficient cpSRP–cpFtsY association. However, no direct evidence has been reported to support this postulation thus far. In this study, we characterized the structural dynamics of cpFtsY and its conformational rearrangements induced by GTP binding using molecular dynamics (MD) simulations. Our results showed that the GTP-binding event triggered substantial conformational changes in free cpFtsY, including the relative orientation of N–G domain and several conserved motifs that are critical in complex formation. These rearrangements enabled the cpFtsY to relax into a preorganized ‘closed’ state that favored the formation of a stable complex with cpSRP54. Interestingly, the intrinsic flexibility of αN1 helix facilitated these rearrangements. In addition, GTP binding in cpFtsY was mediated by conserved residues that have been shown in other SRP GTPases. These findings suggested that GTP-bound cpFtsY could fluctuate into conformations that are favorable to form the stable complex, providing explanation of why SRP–SR interaction bypasses the requirement of the SRP RNA at a molecular level.     

Assembly of the human signal recognition particle (SRP): overlap of regions required for binding of protein SRP54 and assembly control.

Assembly of the human signal recognition particle (SRP) entails the incorporation of protein SRP54, mediated by a protein SRP1 9-induced conformational change in SRP RNA. To localize the region that controls this crucial step in the assembly of human SRP RNA, four chimeras, Ch-1 to Ch-4, composed of portions of human and Methanococcus jannashii SRP RNAs, were generated by PCR site-directed mutagenesis from a larger precursor. Protein-binding activities of the hybrid RNAs were determined using purified human SRP19 and a polypeptide (SRP54M) that corresponded to the methionine-rich domain of human SRP54. Mutant Ch-1 containing the large domain of M. jannashii SRP RNA, as well as mutant Ch-2 RNA in which helices 6 and 8 were replaced, bound SRP54M independently of SRP19. Mutant Ch-3 RNA, which contained M. jannashii helix 6, required SRP19 for binding of SRP54M, but mutant Ch-4 RNA, which possessed M. jannashii helix 8, bound SRP54M without SRP19. We concluded that the formation of a stable ternary complex did not rely on extensive conformational changes that might take place throughout the large domain of SRP, but was controlled by a smaller region encompassing certain RNA residues at positions 177 to 221. Five chimeric RNAs altered within helix 8 were used to investigate the potential role of a significant AA-to-U change and to determine the boundaries of the assembly control region. Reduced protein-binding activities of these chimeras demonstrated a considerable overlap of regions required for SRP54 binding and assembly control.     

Binding site of the M-domain of human protein SRP54 determined by systematic site-directed mutagenesis of signal recognition particle RNA.

The interaction of protein SRP54M from the human signal recognition particle with SRP RNA was studied by systematic site-directed mutagenesis of the RNA molecule. Protein binding sites were identified by the analysis of mutations that removed individual SRP RNA helices or disrupted helical sections in the large SRP domain. The strongest effects on the binding activity of a purified polypeptide that corresponds to the methionine-rich domain of SRP54 (SRP54M) were caused by changes in helix 8 of the SRP RNA. Binding of protein SRP19 was diminished significantly by mutations in helix 6 and was stringently required for SRP54M to associate. Unexpectedly, mutant RNA molecules that resembled bacterial SRP RNAs were incapable of interaction with SRP54M, showing that protein SRP19 has an essential and direct role in the formation of the ternary complex with SRP54 and SRP RNA. Our findings provide an example for how, in eukaryotes, an RNA function has become protein dependent.     

Role of SRP19 in assembly of the Archaeoglobus fulgidus signal recognition particle

Previous studies have shown that SRP19 promotes association of the highly conserved signal peptide-binding protein, SRP54, with the signal recognition particle (SRP) RNA in both archaeal and eukaryotic model systems. In vitro characterization of this process is now reported using recombinantly expressed components of SRP from the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidis. A combination of native gel mobility shift, filter binding, and Ni-NTA agarose bead binding assays were used to determine the binding constants for binary and ternary complexes of SRP proteins and SRP RNA. Archaeal SRP54, unlike eukaryotic homologues, has significant intrinsic affinity for 7S RNA (K(D) approximately 15 nM), making it possible to directly compare particles formed in the presence and absence of SRP19 and thereby assess the precise role of SRP19 in the assembly process. Chemical modification studies using hydroxyl radicals and DEPC identify nonoverlapping primary binding sites for SRP19 and SRP54 corresponding to the tips of helix 6 and helix 8 (SRP19) and the distal loop and asymmetric bulge of helix 8 (SRP54). SRP19 additionally induces conformational changes concentrated in the proximal asymmetric bulge of helix 8. Selected nucleotides in this bulge become modified as a result of SRP19 binding but are subsequently protected from modification by formation of the complete complex with SRP54. Together these results suggest a model for assembly in which bridging the ends of helix 6 and helix 8 by SRP19 induces a long-range structural change to present the proximal bulge in a conformation compatible with high-affinity SRP54 binding.     

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.     

Domain rearrangement of SRP protein Ffh upon binding 4.5S RNA and the SRP receptor FtsY

The signal recognition particle (SRP) mediates membrane targeting of translating ribosomes displaying a signal-anchor sequence. In Escherichia coli, SRP consists of 4.5S RNA and a protein, Ffh, that recognizes the signal peptide emerging from the ribosome and the SRP receptor at the membrane, FtsY. In the present work, we studied the interactions between the NG and M domains in Ffh and their rearrangements upon complex formation with 4.5S RNA and/or FtsY. In free Ffh, the NG and M domains are facing one another in an orientation that allows cross-linking between positions 231 in the G domain and 377 in the M domain. There are binding interactions between the two domains, as the isolated domains form a strong complex. The interdomain contacts are disrupted upon binding of Ffh to 4.5S RNA, consuming a part of the total binding energy of 4.5S RNA-Ffh association that is roughly equivalent to the free energy of domain binding to each other. In the SRP particle, the NG domain binds to 4.5S RNA in a region adjacent to the binding site of the M domain. Ffh binding to FtsY also requires a reorientation of NG and M domains. These results suggest that in free Ffh, the binding sites for 4.5S RNA and FtsY are occluded by strong domain-domain interactions which must be disrupted for the formation of SRP or the Ffh-FtsY complex.     

Visualizing induced fit in early assembly of the human signal recognition particle

Assembly of almost all ribonucleoprotein complexes involves induced fit in the RNA and, thus, formation of one or more intermediate states. In assembly of the human signal recognition particle (SRP), we show that SRP19 binding to SRP RNA involves obligatory intermediates. An apparent discrepancy exists between the ratio of dissociation and association rate constants, determined in a partitioning experiment, and the equilibrium binding constant; this kinetic signature reflects formation of a stable intermediate in assembly of the ribonucleoprotein complex. Assembly intermediates were observed directly by time-resolved footprinting. SRP19 binds rapidly to SRP RNA to form an initial labile, but structurally specific, encounter complex involving both helices III and IV. Two subsequent steps of structural consolidation yield the native RNA-protein interface. SRP19 binding stabilizes helix IV in the region recognized by SRP54, consistent with protein-protein cooperativity mediated in part by mutual recognition of similar RNA structures. This mechanism illustrates principles general to ribonucleoprotein assembly reactions that rely on recruitment of architectural RNA binding proteins.     

Inflammation in the avian spleen: timing is everything

Background

Human cells depend critically on the signal recognition particle (SRP) for the sorting and delivery of their proteins. The SRP is a ribonucleoprotein complex which binds to signal sequences of secretory polypeptides as they emerge from the ribosome. Among the six proteins of the eukaryotic SRP, the largest protein, SRP72, is essential for protein targeting and possesses a poorly characterized RNA binding domain.

Results

We delineated the minimal region of SRP72 capable of forming a stable complex with an SRP RNA fragment. The region encompassed residues 545 to 585 of the full-length human SRP72 and contained a lysine-rich cluster (KKKKKKKKGK) at postions 552 to 561 as well as a conserved Pfam motif with the sequence PDPXRWLPXXER at positions 572 to 583. We demonstrated by site-directed mutagenesis that both regions participated in the formation of a complex with the RNA. In agreement with biochemical data and results from chymotryptic digestion experiments, molecular modeling of SRP72 implied that the invariant W577 was located inside the predicted structure of an RNA binding domain. The 11-nucleotide 5e motif contained within the SRP RNA fragment was shown by comparative electrophoresis on native polyacrylamide gels to conform to an RNA kink-turn. The model of the complex suggested that the conserved A240 of the K-turn, previously identified as being essential for the binding to SRP72, could protrude into a groove of the SRP72 RNA binding domain, similar but not identical to how other K-turn recognizing proteins interact with RNA.

Conclusions

The results from the presented experiments provided insights into the molecular details of a functionally important and structurally interesting RNA-protein interaction. A model for how a ligand binding pocket of SRP72 can accommodate a new RNA K-turn in the 5e region of the eukaryotic SRP RNA is proposed.     

Basic amino acids in a distinct subset of signal peptides promote interaction with the signal recognition particle

Previous studies have demonstrated that signal peptides bind to the signal recognition particle (SRP) primarily via hydrophobic interactions with the 54-kDa protein subunit. The crystal structure of the conserved SRP ribonucleoprotein core, however, raised the surprising possibility that electrostatic interactions between basic amino acids in signal peptides and the phosphate backbone of SRP RNA may also play a role in signal sequence recognition. To test this possibility we examined the degree to which basic amino acids in a signal peptide influence the targeting of two Escherichia coli proteins, maltose binding protein and OmpA. Whereas both proteins are normally targeted to the inner membrane by SecB, we found that replacement of their native signal peptides with another moderately hydrophobic but unusually basic signal peptide (DeltaEspP) rerouted them into the SRP pathway. Reduction in either the net positive charge or the hydrophobicity of the DeltaEspP signal peptide decreased the effectiveness of SRP recognition. A high degree of hydrophobicity, however, compensated for the loss of basic residues and restored SRP binding. Taken together, the data suggest that the formation of salt bridges between SRP RNA and basic amino acids facilitates the binding of a distinct subset of signal peptides whose hydrophobicity falls slightly below a threshold level.     

Interaction of rice and human SRP19 polypeptides with signal recognition particle RNA

The signal recognition particle (SRP) controls the transport of secretory proteins into and across lipid bilayers. SRP-like ribonucleoprotein complexes exist in all organisms, including plants. We characterized the rice SRP RNA and its primary RNA binding protein, SRP19. The secondary structure of the rice SRP RNA was similar to that found in other eukaryotes; however, as in other plant SRP RNAs, a GUUUCA hexamer sequence replaced the highly conserved GNRA-tetranucleotide loop motif at the apex of helix 8. The small domain of the rice SRP RNA was reduced considerably. Structurally, rice SRP19 lacked two small regions that can be present in other SRP19 homologues. Conservative structure prediction and site-directed mutagenesis of rice and human SRP19 polypeptides indicated that binding to the SRP RNAs occurred via a loop that is present in the N-domain of both proteins. Rice SRP19 protein was able to form a stable complex with the rice SRP RNA in vitro. Furthermore, heterologous ribonucleoprotein complexes with components of the human SRP were assembled, thus confirming a high degree of structural and functional conservation between plant and mammalian SRP components.     

RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle

The signal recognition particle (SRP) targets nascent proteins to cellular membranes for insertion or secretion by recognizing polypeptides containing an N-terminal signal sequence as they emerge from the ribosome. GTP-dependent binding of SRP to its receptor protein leads to controlled release of the nascent chain into a membrane-spanning translocon pore. Here we show that the association of the SRP with its receptor triggers a marked conformational change in the complex, localizing the SRP RNA and the adjacent signal peptide-binding site at the SRP-receptor heterodimer interface. The orientation of the RNA suggests how peptide binding and GTP hydrolysis can be coupled through direct structural contact during cycles of SRP-directed protein translocation.     

A nomenclature for all signal recognition particle RNAs

The signal recognition particle (SRP) is a cytosolic ribonucleoprotein complex that guides secretory proteins to biological membranes in all organisms. The SRP RNA is at the center of the structure and function of the SRP. The comparison of the growing number of SRP RNA sequences provides a rich source for gaining valuable insight into the composition, assembly, and phylogeny of the SRP. In order to assist in the continuation of these studies, we propose an SRP RNA nomenclature applicable to the three divisions of life.     

Conserved but nonessential interaction of SRP RNA with translation factor EF-G

4.5S RNA is essential for viability of Escherichia coli, and forms a key component of the signal recognition particle (SRP), a ubiquitous ribonucleoprotein complex responsible for cotranslational targeting of secretory proteins. 4.5S RNA also binds independently to elongation factor G (EF-G), a five-domain GTPase that catalyzes the translocation step during protein biosynthesis on the ribosome. Point mutations in EF-G suppress deleterious effects of 4.5S RNA depletion, as do mutations in the EF-G binding site within ribosomal RNA, suggesting that 4.5S RNA might play a critical role in ribosome function in addition to its role in SRP. Here we show that 4.5S RNA and EF-G form a phylogenetically conserved, low-affinity but highly specific complex involving sequence elements required for 4.5S binding to its cognate SRP protein, Ffh. Mutational analysis indicates that the same molecular structure of 4.5S RNA is recognized in each case. Surprisingly, however, the suppressor mutant forms of EF-G bind very weakly or undetectably to 4.5S RNA, implying that cells can survive 4.5S RNA depletion by decreasing the affinity between 4.5S RNA and the translational machinery. These data suggest that SRP function is the essential role of 4.5S RNA in bacteria.     

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.     

Saccharomyces SRP RNA secondary structures: a conserved S-domain and extended Alu-domain

The contribution made by the RNA component of signal recognition particle (SRP) to its function in protein targeting is poorly understood. We have generated a complete secondary structure for Saccharomyces cerevisiae SRP RNA, scR1. The structure conforms to that of other eukaryotic SRP RNAs. It is rod-shaped with, at opposite ends, binding sites for proteins required for the SRP functions of signal sequence recognition (S-domain) and translational elongation arrest (Alu-domain). Micrococcal nuclease digestion of purified S. cerevisiae SRP separated the S-domain of the RNA from the Alu-domain as a discrete fragment. The Alu-domain resolved into several stable fragments indicating a compact structure. Comparison of scR1 with SRP RNAs of five yeast species related to S. cerevisiae revealed the S-domain to be the most conserved region of the RNA. Extending data from nuclease digestion with phylogenetic comparison, we built the secondary structure model for scR1. The Alu-domain contains large extensions, including a sequence with hallmarks of an expansion segment. Evolutionarily conserved bases are placed in the Alu- and S-domains as in other SRP RNAs, the exception being an unusual GU(4)A loop closing the helix onto which the signal sequence binding Srp54p assembles (domain IV). Surprisingly, several mutations within the predicted Srp54p binding site failed to disrupt SRP function in vivo. However, the strength of the Srp54p-scR1 and, to a lesser extent, Sec65p-scR1 interaction was decreased in these mutant particles. The availability of a secondary structure for scR1 will facilitate interpretation of data from genetic analysis of the RNA.     

An archaeal protein homologous to mammalian SRP54 and bacterial Ffh recognizes a highly conserved region of SRP RNA.

The gene encoding the 54 kDa protein of signal recognition particle (SRP54) in the hyperthermophilic archaeon Pyrococcus furiosus has been cloned and sequenced. Recombinant P. furiosus SRP54 (pf-SRP54) and the N-terminal G-domain and C-terminal M-domain (pf-SRP54M) of pf-SRP54 with an amino-terminal addition of six histidine residues were expressed in Escherichia coli and subjected to binding experiments for SRP RNA, non-conserved 213-nucleotide RNA (helices 1, 2, 3, 4 and 5) and conserved 107-nucleotide RNA (helices 6 and 8) from SRP RNA. The RNA binding properties of the purified protein were determined by filter binding assays. The histidine-tagged pf-SRP54M bound specifically to the conserved 107-nucleotide RNA in the absence of pf-SRP19, unlike the eukaryotic homologue, with an apparent binding constant (K) of 18 nM.     

A truncation in the 14 kDa protein of the signal recognition particle leads to tertiary structure changes in the RNA and abolishes the elongation arrest activity of the particle.

The signal recognition particle (SRP) provides the molecular link between synthesis of polypeptides and their concomitant translocation into the endoplasmic reticulum. During targeting, SRP arrests or delays elongation of the nascent chain, thereby presumably ensuring a high translocation efficiency. Components of the Alu domain, SRP9/14 and the Alu sequences of SRP RNA, have been suggested to play a role in the elongation arrest function of SRP. We generated a truncated SRP14 protein, SRP14-20C, which forms, together with SRP9, a stable complex with SRP RNA. However, particles reconstituted with SRP9/14-20C, RC(9/14-20C), completely lack elongation arrest activity. RC(9/14-20C) particles have intact signal recognition, targeting and ribosome binding activities. SRP9/14-20C therefore only impairs interactions with the ribosome that are required to effect elongation arrest. This result provides evidence that direct interactions between the Alu domain components and the ribosome are required for this function. Furthermore, SRP9/14-20C binding to SRP RNA results in tertiary structure changes in the RNA. Our results strongly indicate that these changes account for the negative effect of SRP14 truncation on elongation arrest, thus revealing a critical role of the RNA in this function.     

Chloroplast SRP54 Was Recruited for Posttranslational Protein Transport via Complex Formation with Chloroplast SRP43 during Land Plant Evolution

In bacteria, membrane proteins are targeted cotranslationally via a signal recognition particle (SRP). During the evolution of higher plant chloroplasts from cyanobacteria, the SRP pathway underwent striking adaptations that enable the posttranslational transport of the abundant light-harvesting chlorophyll-a/b-binding proteins (LHCPs). The conserved 54-kDa SRP subunit in higher plant chloroplasts (cpSRP54) is not bound to an SRP RNA, an essential SRP component in bacteria, but forms a stable heterodimer with the chloroplast-specific cpSRP43. This heterodimeric cpSRP recognizes LHCP and delivers it to the thylakoid membrane whereby cpSRP43 plays a central role. This study shows that the cpSRP system in the green alga Chlamydomonas reinhardtii differs significantly from that of higher plants as cpSRP43 is not complexed to cpSRP54 in Chlamydomonas and cpSRP54 is not involved in LHCP recognition. This divergence is attributed to altered residues within the cpSRP54 tail and the second chromodomain of cpSRP43 that are crucial for the formation of the binding interface in Arabidopsis. These changes are highly conserved among chlorophytes, whereas all land plants contain cpSRP proteins with typical interaction motifs. These data demonstrate that the coevolution of LHCPs and cpSRP43 occurred independently of complex formation with cpSRP54 and that the interaction between cpSRP54 and cpSRP43 evolved later during the transition from chlorophytes to land plants. Furthermore, our data show that in higher plants a heterodimeric form of cpSRP is required for the formation of a low molecular weight transit complex with LHCP.