The structure of the chloroplast signal recognition particle (SRP) receptor reveals mechanistic details of SRP GTPase activation and a conserved membrane targeting site

Two GTPases in the signal recognition particle and its receptor (FtsY) regulate protein targeting to the membrane by formation of a heterodimeric complex. The activation of both GTPases in the complex is essential for protein translocation. We present the crystal structure of chloroplast FtsY (cpFtsY) at 1.75 A resolution. The comparison with FtsY structures in different nucleotide bound states shows structural changes relevant for GTPase activation and provides insights in how cpFtsY is pre-organized for complex formation with cpSRP54. The structure contains an amino-terminal amphipathic helix similar to the membrane targeting sequence of Escherichia coli FtsY. In cpFtsY this motif is extended, which might be responsible for the enhanced attachment of the protein to the thylakoid membrane.     

Protein SRP68 of human signal recognition particle: identification of the RNA and SRP72 binding domains

The signal recognition particle (SRP) plays an important role in the delivery of secretory proteins to cellular membranes. Mammalian SRP is composed of six polypeptides among which SRP68 and SRP72 form a heterodimer that has been notoriously difficult to investigate. Human SRP68 was purified from overexpressing Escherichia coli cells and was found to bind to recombinant SRP72 as well as in vitro-transcribed human SRP RNA. Polypeptide fragments covering essentially the entire SRP68 molecule were generated recombinantly or by proteolytic digestion. The RNA binding domain of SRP68 included residues from positions 52 to 252. Ninety-four amino acids near the C terminus of SRP68 mediated the binding to SRP72. The SRP68-SRP72 interaction remained stable at elevated salt concentrations and engaged approximately 150 amino acids from the N-terminal region of SRP72. This portion of SRP72 was located within a predicted tandem array of four tetratricopeptide (TPR)-like motifs suggested to form a superhelical structure with a groove to accommodate the C-terminal region of SRP68.     

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 threefold RNA-protein interface in the signal recognition particle gates native complex assembly

Intermediate states play well-established roles in the folding and misfolding reactions of individual RNA and protein molecules. In contrast, the roles of transient structural intermediates in multi-component ribonucleoprotein (RNP) assembly processes and their potential for misassembly are largely unexplored. The SRP19 protein is unstructured but forms a compact core domain and two extended RNA-binding loops upon binding the signal recognition particle (SRP) RNA. The SRP54 protein subsequently binds to the fully assembled SRP19-RNA complex to form an intimate threefold interface with both SRP19 and the RNA and without significantly altering the structure of SRP19. We show, however, that the presence of SRP54 during SRP19-RNA assembly dramatically alters the folding energy landscape to create a non-native folding pathway that leads to an aberrant SRP19-RNA conformation. The misassembled complex arises from the surprising ability of SRP54 to bind rapidly to an SRP19-RNA assembly intermediate and to interfere with subsequent folding of one of the RNA binding loops at the three-way protein-RNA interface. An incorrect temporal order of assembly thus readily yields a non-native three-component ribonucleoprotein particle. We propose there may exist a general requirement to regulate the order of interaction in multi-component RNP assembly reactions by spatial or temporal compartmentalization of individual constituents in the cell.     

Identification and characterization of Streptococcus pneumoniae Ffh, a homologue of SRP54 subunit of mammalian signal recognition particle

Recent studies have demonstrated that bacteria possess an essential protein translocation system similar to mammalian signal recognition particle (SRP). Here we have identified the Ffh, a homologue of the mammalian SRP54 subunit from S. pneumoniae. Ffh is a 58-kDa protein with three distinct domains: an N-terminal hydrophilic domain (N-domain), a G-domain containing GTP/GDP binding motifs, and a C-terminal methionine-rich domain (M-domain). The full-length Ffh and a truncated protein containing N and G domains (Ffh-NG) were overexpressed in E. coli and purified to homogeneity. The full-length Ffh has an intrinsic GTPase activity with k(cat) of 0.144 min(-1), and the K(m) for GTP is 10.9 microM. It is able to bind to 4.5S RNA specifically as demonstrated by gel retardation assay. The truncated Ffh-NG has approximately the same intrinsic GTPase activity to the full-length Ffh, but is unable to bind to 4.5S RNA, indicating that the NG domain is sufficient for supporting intrinsic GTP hydrolysis, and that the M domain is required for RNA binding. The interaction of S. pneumoniae Ffh with its receptor, FtsY, resulted in a 20-fold stimulation in GTP hydrolysis. The stimulation was further demonstrated to be independent of the 4.5S RNA. In addition, a similar GTPase stimulation is also observed between Ffh-NG and FtsY, suggesting that the NG domain is sufficient and the M domain is not required for GTPase stimulation between Ffh and FtsY.     

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.     

Distant segments of Saccharomyces cerevisiae scR1 RNA promote assembly and function of the signal recognition particle

The conserved signal recognition particle targets ribosomes synthesizing presecretory proteins to the endoplasmic reticulum membrane. Key to the activity of SRP is its ability to bind the ribosome at distant locations, the signal sequence exit and elongation factor-binding sites. These contacts are made by the S and Alu domains of SRP, respectively. We tested earlier secondary structure predictions of the Saccharomyces cerevisiae SRP RNA, scR1, and provide and test a consensus structure. The structure contains four non-conserved insertions, helices 9-12, into the core SRP RNA fold, and an extended helix 7. Using a series of scR1 mutants lacking part or all of these structural elements, we find that they are important for the RNA in both function and assembly of the RNP. About 20% of the RNA, corresponding to the outer regions of these helices, is dispensable for function. Further, we examined the role of several features within the S-domain section of the core, helix 5, and find that its length and flexibility are important for proper SRP function and become essential in the absence of helix 10, 11 and/or 7 regions. Overall, the genetic data indicate that regions of scR1 distant in both primary sequence and secondary structure have interrelated roles in the function of the complex, and possibly mediate communication between Alu and S domains during targeting.     

The 5e motif of eukaryotic signal recognition particle RNA contains a conserved adenosine for the binding of SRP72

The signal recognition particle (SRP) plays a pivotal role in transporting proteins to cell membranes. In higher eukaryotes, SRP consists of an RNA molecule and six proteins. The largest of the SRP proteins, SRP72, was found previously to bind to the SRP RNA. A fragment of human SRP72 (72c') bound effectively to human SRP RNA but only weakly to the similar SRP RNA of the archaeon Methanococcus jannaschii. Chimeras between the human and M. jannaschii SRP RNAs were constructed and used as substrates for 72c'. SRP RNA helical section 5e contained the 72c' binding site. Systematic alteration within 5e revealed that the A240G and A240C changes dramatically reduced the binding of 72c'. Human SRP RNA with a single A240G change was unable to form a complex with full-length human SRP72. Two small RNA fragments, one composed of helical section 5ef, the other of section 5e, competed equally well for the binding of 72c', demonstrating that no other regions of the SRPR RNA were required. The biochemical data completely agreed with the nucleotide conservation pattern observed across the phylogenetic spectrum. Thus, most eukaryotic SRP RNAs are likely to require for function an adenosine within their 5e motifs. The human 5ef RNA was remarkably resistant to ribonucleolytic attack suggesting that the 240-AUC-242 "loop" and its surrounding nucleotides form a peculiar compact structure recognized only by SRP72.     

Complexes with truncated RNAs from the large domain of Archaeoglobus fulgidus signal recognition particle

Protein SRP19 is an important component of the signal recognition particle (SRP) as it promotes assembly of protein SRP54 with SRP RNA and recognizes a tetranucleotide loop. Structural features and RNA binding activities of SRP19 of the hyperthermophilic archaeon Archaeoglobus fulgidus were investigated. An updated alignment of SRP19 sequences predicted three conserved regions and two alpha-helices. With Af-SRP RNA the Af-SRP54 protein assembled into an A. fulgidus SRP which remained intact for many hours. Stable complexes were formed between Af-SRP19 and truncated SRP RNAs, including a 36-residue fragment representing helix 6 of A. fulgidus SRP RNA.     

Characterization of the SRP68/72 interface of human signal recognition particle by systematic site‐directed mutagenesis

The signal recognition particle (SRP) is a ribonucleoprotein complex which is crucial for the delivery of proteins to cellular membranes. Among the six proteins of the eukaryotic SRP, the two largest, SRP68 and SRP72, form a stable SRP68/72 heterodimer of unknown structure which is required for SRP function. Fragments 68e′ (residues 530 to 620) and 72b′ (residues 1 to 166) participate in the SRP68/72 interface. Both polypeptides were expressed in Escherichia coli and assembled into a complex which was stable at high ionic strength. Disruption of 68e′/72b′ and SRP68/72 was achieved by denaturation using moderate concentrations of urea. The four predicted tetratricopeptide repeats (TPR1 to TPR4) of 72b′ were required for stable binding of 68e′. Site‐directed mutagenesis suggested that they provide the structural framework for the binding of SRP68. Deleting the region between TPR3 and TPR4 (h120) also prevented the formation of a heterodimer, but this predicted alpha‐helical region appeared to engage several of its amino acid residues directly at the interface with 68e′. A 39‐residue polypeptide (68h, residues 570–605), rich in prolines and containing an invariant aspartic residue at position 585, was found to be active. Mutagenesis scanning of the central region of 68h demonstrated that D585 was solely responsible for the formation of the heterodimer. Coexpression experiments suggested that 72b′ protects 68h from proteolytic digestion consistent with the assertion that 68h is accommodated inside a groove formed by the superhelically arranged four TPRs of the N‐terminal region of SRP72.     

Hypothetical protein AF2241 from Archaeoglobus fulgidus adopts a cyclophilin-like fold

AF2241 is a hypothetical protein from Archaeoglobus fulgidus and it belongs to the PFam domain of unknown function 369 (DUF369). NMR structural determination reveals that AF2241 adopts a cyclophilin-like fold, with a β-barrel core composed of eight β-strands, one α-helix, and one 3₁₀ helix located at each end of the barrel. The protein displays a high structural similarity to TM1367, another member of DUF369 whose structure has been determined recently by X-ray crystallography. Structural similarity search shows that AF2241 also has a high similarity to human cyclophilin A, however, sequence alignment and electrostatic potential analysis reveal that the residues in the PPIase catalytic site of human cyclophilin A are not conserved in AF2241 or TM1367. Instead, a putative active site of AF2241 maps to a negatively charged pocket composed of 9 conserved residues. Our results suggest that although AF2241 adopts the same fold as the human cyclophilin A, it may have distinct biological function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Assembly of chloroplast signal recognition particle involves structural rearrangement in cpSRP43

Signal recognition particle in chloroplasts (cpSRP) exhibits the unusual ability to bind and target full-length proteins to the thylakoid membrane. Unlike cytosolic SRPs in prokaryotes and eukaryotes, cpSRP lacks an RNA moiety and functions as a heterodimer composed of a conserved 54-kDa guanosine triphosphatase (cpSRP54) and a unique 43-kDa subunit (cpSRP43). Assembly of the cpSRP heterodimer is a prerequisite for post-translational targeting activities and takes place through interactions between chromatin modifier domain 2 (CD2) of cpSRP43 and a unique 10-amino-acid region in cpSRP54 (cpSRP54_pep). We have used multidimensional NMR spectroscopy and other biophysical methods to examine the assembly and structure of the cpSRP43-cpSRP54 interface. Our data show that CD2 of cpSRP43 binds to cpSRP54_pep in a 1:1 stoichiometry with an apparent K_d of ∼ 1.06 μM. Steady-state fluorescence and far-UV circular dichroism data suggest that the CD2-cpSRP54_pep interaction causes significant conformational changes in both CD2 and the peptide. Comparison of the three-dimensional solution structures of CD2 alone and in complex with cpSRP54_pep shows that significant structural changes are induced in CD2 in order to establish a binding interface contributed mostly by residues in the N-terminal segment of CD2 (Phe5-Val10) and an arginine doublet (Arg536 and Arg537) in the cpSRP54 peptide. Taken together, our results provide new insights into the mechanism of cpSRP assembly and the structural forces that stabilize the functionally critical cpSRP43-cpSRP54 interaction.     

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.     

Structural diversity of signal recognition particle RNAs in plastids

One of the pathways for protein targeting to the plasma membrane in bacteria utilizes the co-translationally acting signal recognition particle (SRP), a universally conserved ribonucleoprotein complex consisting of a 54 kDa protein and a functional RNA. An interesting exception is the higher plant chloroplast SRP, which lacks the otherwise essential RNA component. Furthermore, green plant chloroplasts have an additional post-translational SRP-dependent transport system in which the chloroplast-specific cpSRP43 protein binds to imported substrate proteins and to the conserved 54 kDa SRP subunit (cpSRP54). While homologs to the bacterial SRP protein and RNA component previously have been identified in genome sequences of red algae and diatoms, a recent study investigated the evolution of the green plant SRP system.¹ Analysis of hundreds of plastid and nuclear genomes showed a surprising pattern of multiple losses of the plastid SRP RNA during evolution and a widespread presence in all non-spermatophyte plants and green algae. Contrary to expectations, all green organisms that have an identified cpSRP RNA also contain a cpSRP43. Notably, the structure of the plastid SRP RNAs is much more diverse than that of bacterial SRP RNAs. The apical GNRA tetraloop is only conserved in organisms of the red lineage and basal organisms of the green lineage, whereas further chloroplast SRP RNAs are characterized by atypical, mostly enlarged apical loops.     

Plasmodium falciparum signal recognition particle components and anti-parasitic effect of ivermectin in blocking nucleo-cytoplasmic shuttling of SRP

Signal recognition particle (SRP) is a ubiquitous ribonucleoprotein complex that targets proteins to endoplasmic reticulum (ER) in eukaryotes. Here we report that Plasmodium falciparum SRP is composed of six polypeptides; SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72 and a 303nt long SRP RNA. We generated four transgenic parasite lines expressing SRP-GFP chimeric proteins and co-localization studies showed the nucleo-cytoplasmic localization for these proteins. The evaluation of the effect of known SRP and nuclear import/export inhibitors on P. falciparum revealed that ivermectin, an inhibitor of importin α/β mediated nuclear import inhibited the nuclear import of PfSRP polypeptides at submicromolar concentration, thereby killing the parasites. These findings provide insights into dynamic structure of P. falciparum SRP and also raise the possibility that ivermectin could be used in combination with other antimalarial agents to control the disease.     

Structural and functional analysis of the gpsA gene product of Archaeoglobus fulgidus: a glycerol-3-phosphate dehydrogenase with an unusual NADP+ preference

NAD(+)-dependent glycerol-3-phosphate dehydrogenase (G3PDH) is generally absent in archaea, because archaea, unlike eukaryotes and eubacteria, utilize glycerol-1-phosphate instead of glycerol-3-phosphate for the biosynthesis of membrane lipids. Surprisingly, the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus comprises a G3PDH ortholog, gpsA, most likely due to horizontal gene transfer from a eubacterial organism. Biochemical characterization proved G3PDH-like activity of the recombinant gpsA gene product. However, unlike other G3PDHs, the up to 85 degrees C thermostable A. fulgidus G3PDH exerted a 15-fold preference for NADPH over NADH. The A. fulgidus G3PDH bears the hallmarks of adaptation to halotolerance and thermophilicity, because its 1.7-A crystal structure showed a high surface density for negative charges and 10 additional intramolecular salt bridges compared to a mesophilic G3PDH structure. Whereas all amino acid residues required for dihydroxyacetone phosphate binding and reductive catalysis are highly conserved, the binding site for the adenine moiety of the NAD(P) cosubstrate shows a structural variation that reflects the observed NADPH preference, for example, by a putative salt bridge between R49 and the 2'-phosphate.     

信号识别颗粒调控蛋白转运系统

蛋白质是一类重要的生物大分子。在蛋白质转运系统中,很多蛋白质在核糖体中合成,然后通过内质网或质膜的转运到达相应的细胞器发挥生物学功能。蛋白质的分泌表达途径总共分为三类,分别为Sec分泌途径、双精氨酸途径和信号识别颗粒转运系统。本文简要介绍蛋白质的基本转运途径,主要介绍由信号识别颗粒所介导的蛋白质转运。分别概述信号识别颗粒及其受体的组成与功能,并对其调控途径做简要的介绍;同时也简单介绍与其相关的Yid C膜蛋白家族;对信号识别颗粒蛋白调控系统存在的必需性提出新的见解。     

Thermal stability of isocitrate dehydrogenase from Archaeoglobus fulgidus studied by crystal structure analysis and engineering of chimers

Isocitrate dehydrogenase from Archaeoglobus fulgidus (AfIDH) has an apparent melting temperature (T(m)) of 98.5 degrees C. To identify the structural features involved in thermal stabilization of AfIDH, the structure was solved to 2.5 A resolution. AfIDH was strikingly similar to mesophilic IDH from Escherichia coli (EcIDH) and displayed almost the same number of ion pairs and ionic networks. However, two unique inter-domain networks were present in AfIDH; one three-membered ionic network between the large and the small domain and one four-membered ionic network between the clasp and the small domain. The latter ionic network was presumably reduced in size when the clasp domain of AfIDH was swapped with that of EcIDH and the T (m) decreased by 18 degrees C. Contrarily, EcIDH was only stabilized by 4 degrees C by the clasp domain of AfIDH, a result probably due to the introduction of a unique inter-subunit aromatic cluster in AfIDH that may strengthen the dimeric interface in this enzyme. A unique aromatic cluster was identified close to the N-terminus of AfIDH that could provide additional stabilization of this region. Common and unique heat adaptive traits of AfIDH with those recently observed for hyperthermophilic IDH from Aeropyrum pernix (ApIDH) and Thermotoga maritima (TmIDH) are discussed herein.