The signal recognition particle binds to protein L23 at the peptide exit of the Escherichia coli ribosome

The signal recognition particle (SRP) from Escherichia coli, composed of Ffh protein and 4.5S RNA, mediates membrane targeting of translating ribosomes displaying a signal or signal-anchor sequence. SRP binds at the peptide exit of the large ribosomal subunit. Structural details of the interaction are not known. Here, the position of Ffh or SRP on the ribosome was probed by using site-specific UV-induced crosslinking by p-azidophenacyl bromide (AzP) attached to a number of cysteine residues engineered into surface positions of Ffh. Efficient crosslinking to vacant ribosomes took place from two positions (AzP17 and AzP25) in the N domain of Ffh, both with Ffh and SRP. Both AzP17 and AzP25 were predominantly crosslinked to ribosomal protein L23 that is located at the peptide exit of the 50S subunit. The SRP receptor, FtsY, did not change the crosslink pattern, whereas the presence of a nascent signal peptide on the ribosome resulted in a second crosslink between Ffh(AzP17) and protein L23, indicating that binding to the nascent signal peptide induced a slightly different arrangement of SRP on the ribosome. These results indicate a model of the topographical arrangement of SRP at the peptide exit of the 50S ribosomal subunit.     

Conformation of the signal recognition particle in ribosomal targeting complexes

The bacterial signal recognition particle (SRP) binds to ribosomes synthesizing inner membrane proteins and, by interaction with the SRP receptor, FtsY, targets them to the translocon at the membrane. Here we probe the conformation of SRP and SRP protein, Ffh, at different stages of targeting by measuring fluorescence resonance energy transfer (FRET) between fluorophores placed at various positions within SRP. Distances derived from FRET indicate that SRP binding to nontranslating ribosomes triggers a global conformational change of SRP that facilitates binding of the SRP receptor, FtsY. Binding of SRP to a signal-anchor sequence exposed on a ribosome-nascent chain complex (RNC) causes a further change of the SRP conformation, involving the flexible part of the Ffh(M) domain, which increases the affinity for FtsY of ribosome-bound SRP up to the affinity exhibited by the isolated NG domain of Ffh. This indicates that in the RNC–SRP complex the Ffh(NG) domain is fully exposed for binding FtsY to form the targeting complex. Binding of FtsY to the RNC–SRP complex results in a limited conformational change of SRP, which may initiate subsequent targeting steps.     

Effects of signal peptide and adenylate on the oligomerization and membrane binding of soluble SecA

SecA protein, a cytoplasmic ATPase, plays a central role in the secretion of signal peptide-containing proteins. Here, we examined effects of signal peptide and ATP on the oligomerization, conformational change, and membrane binding of SecA. The wild-type (WT) signal peptide from the ribose-binding protein inhibited ATP binding to soluble SecA and stimulated release of ATP already bound to the protein. The signal peptide enhanced the oligomerization of soluble SecA, while ATP induced dissociation of SecA oligomer. Analysis of SecA unfolding with urea or heat revealed that the WT signal peptide induces an open conformation of soluble SecA, while ATP increased the compactness of SecA. We further obtained evidences that the signal peptide-induced oligomerization and the formation of open structure enhance the membrane binding of SecA, whereas ATP inhibits the interaction of soluble SecA with membranes. On the other hand, the complex of membrane-bound SecA and signal peptide was shown to resume nucleotide-binding activity. From these results, we propose that the translocation components affect the degree of oligomerization of soluble SecA, thereby modulating the membrane binding of SecA in early translocation pathway. A possible sequential interaction of SecA with signal peptide, ATP, and cytoplasmic membrane is discussed.     

The signal recognition particle (SRP) RNA links conformational changes in the SRP to protein targeting

The RNA component of the signal recognition particle (SRP) is universally required for cotranslational protein targeting. Biochemical studies have shown that SRP RNA participates in the central step of protein targeting by catalyzing the interaction of the SRP with the SRP receptor (SR). SRP RNA also accelerates GTP hydrolysis in the SRP.SR complex once formed. Using a reverse-genetic and biochemical analysis, we identified mutations in the E. coli SRP protein, Ffh, that abrogate the activity of the SRP RNA and cause corresponding targeting defects in vivo. The mutations in Ffh that disrupt SRP RNA activity map to regions that undergo dramatic conformational changes during the targeting reaction, suggesting that the activity of the SRP RNA is linked to the major conformational changes in the signal sequence-binding subunit of the SRP. In this way, the SRP RNA may coordinate the interaction of the SRP and the SR with ribosome recruitment and transfer to the translocon, explaining why the SRP RNA is an indispensable component of the protein targeting machinery.     

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.     

Ligand crowding at a nascent signal sequence

We have systematically analyzed the molecular environment of the signal sequence of a growing secretory protein from Escherichia coli using a stage- and site-specific cross-linking approach. Immediately after emerging from the ribosome, the signal sequence of pOmpA is accessible to Ffh, the protein component of the bacterial signal recognition particle, and to SecA, but it remains attached to the surface of the ribosome via protein L23. These contacts are lost upon further growth of the nascent chain, which brings the signal sequence into sole proximity to the chaperone Trigger factor (TF). In its absence, nascent pOmpA shows extended contacts with L23, and even long chains interact in these conditions proficiently with Ffh. Our results suggest that upon emergence from the ribosome, the signal sequence of an E. coli secretory protein gradually becomes sequestered by TF. Although TF thereby might control the accessibility of pOmpA's signal sequence to Ffh and SecA, it does not influence interaction of pOmpA with SecB.     

Network models reveal stability and structural rearrangement of signal recognition particle

The signal recognition particle (SRP) and its receptors (SR) mediate the cotranslational targeting of the membrane and secretory proteins in all cells. In Escherichia coli, SRP is composed of the Ffh protein and the 4.5S SRP RNA. Ffh is a multidomain protein comprising a methionine-rich (M) domain, a helical N domain, and a Ras-like guanine triphosphatase (GTPase) (G) domain. The N and G domains are commonly referred to as one structural unit, the NG domain. In this article, the complex structure of SRP and SR is investigated with the Gaussian network model (GNM) and anisotropic network model (ANM). GNM provides the information of structure stability. It is found that the intermolecular interactions between SRP and SR can obviously decrease the fluctuation of NG domains. Nevertheless, the large structural rearrangement will take place during the cotranslational protein targeting cycle. Hence, the moving directions of fluctuation regions are further ascertained by using cross-correlation analysis and the ANM. The NG domain of Ffh undergoes a clockwise rotation around the GM linker and the M domain of Ffh shows an opposite direction to the NG domain. These functional movements will facilitate the SRP structure to transform into the free form and the sequence-bound form. These simple coarse-grained analyses can be used as a general and quick method for the mechanism studies of protein assembly and supramolecular systems.     

The cyanobacterial genome contains a single copy of the ffh gene encoding a homologue of the 54 kDa subunit of signal recognition particle

Cyanobacteria possess thylakoid membranes that differ in their protein composition from the cytoplasmic membrane. To study possible pathways of protein targeting to these membranes, we have investigated whether or not cyanobacteria have a homologue or homologues of the signal recognition particle-like chaperone Ffh. We have amplified a fragment of ffh by polymerase chain reaction and established that ffh is present as a single copy in the genomes of three cyanobacterial species. We have cloned and sequenced ffh from Synechococcus sp. PCC7942 and predict that Ffh functions as a ribonucleoprotein in cyanobacteria and chloroplasts.     

Versatile signal peptide of Flavobacterium‐originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli

Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane‐associated homodimeric metalloenzyme and has its own signal peptide in its N‐terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide‐containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin‐arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec‐avoidance sequence in the c‐region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848–854, 2016     

Characterization of the cleavage of signal peptide at the C-terminus of hepatitis C virus core protein by signal peptide peptidase

Production of hepatitis C virus (HCV) core protein requires the cleavages of polyprotein by signal peptidase and signal peptide peptidase (SPP). Cleavage of signal peptide at the C-terminus of HCV core protein by SPP was characterized in this study. The spko mutant (mutate a.a. 189–193 from ASAYQ to PPFPF) is more efficient than the A/F mutant (mutate a.a 189 and 191 from A to F) in blocking the cleavage of signal peptide by signal peptidase. The cleavage efficiency of SPP is inversely proportional to the length of C-terminal extension of the signal peptide: the longer the extension, the less efficiency the cleavage is. Thus, reducing the length of C-terminal extension of signal peptide by signal peptidase cleavage could facilitate further cleavage by SPP. The recombinant core protein fused with signal peptide from the C-terminus of p7 protein, but not those from the C-termini of E1 and E2, could be cleaved by SPP. Therefore, the sequence of the signal peptide is important but not the sole determinant for its cleavage by SPP. Replacement of the HCV core protein E.R.-associated domain (a.a. 120–150) with the E.R.-associated domain (a.a.1–50) of SARS-CoV membrane protein results in the failure of cleavage of this recombinant protein by SPP, though this protein still is E.R.-associated. This result suggests that not only E.R.-association but also specific protein sequence is important for the HCV core protein signal peptide cleavage by SPP. Thus, our results suggest that both sequences of the signal peptide and the E.R.-associated domain are important for the signal peptide cleavage of HCV core protein by SPP. Electronic Supplementary MaterialThe online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Fluorescence spectroscopy of soluble E. coli SPase I Δ2‐75 reveals conformational changes in response to ligand binding

The bacterial Sec pathway is responsible for the translocation of secretory preproteins. During the later stages of transport, the membrane‐embedded signal peptidase I (SPase I) cleaves the signal peptide from a preprotein. We used tryptophan fluorescence spectroscopy of a soluble, catalytically active E. coli SPase I Δ2‐75 enzyme to study its dynamic conformational changes while in solution and when interacting with lipids and signal peptides. We generated four single Trp SPase I Δ2‐75 mutants, W261, W284, W300, and W310. Based on fluorescence quenching experiments, W300 and W310 were found to be more solvent accessible than W261 and W284 in the absence of ligands. W300 and W310 inserted into lipids, consistent with their location at the enzyme's proposed membrane‐interface region, while the solvent accessibilities of W261, W284, and W300 were modified in the presence of signal peptide, suggesting propagation of structural changes beyond the active site in response to peptide binding. The signal peptide binding affinity for the enzyme was measured via FRET experiments and the K_d determined to be 4.4 μM. The location of the peptide with respect to the enzyme was also established; this positioning is crucial for the peptide to gain access to the enzyme active site as it emerges from the translocon into the membrane bilayer. These studies reveal enzymatic structural changes required for preprotein proteolysis as it interacts with its two key partners, the signal peptide and membrane phospholipids. Proteins 2014; 82:596–606. © 2013 Wiley Periodicals, Inc.     

Vestiges of the Bacterial Signal Recognition Particle-Based Protein Targeting in Mitochondria

The main bacterial pathway for inserting proteins into the plasma membrane relies on the signal recognition particle (SRP), composed of the Ffh protein and an associated RNA component, and the SRP-docking protein FtsY. Eukaryotes use an equivalent system of archaeal origin to deliver proteins into the endoplasmic reticulum, whereas a bacteria-derived SRP and FtsY function in the plastid. Here we report on the presence of homologs of the bacterial Ffh and FtsY proteins in various unrelated plastid-lacking unicellular eukaryotes, namely Heterolobosea, Alveida, Goniomonas, and Hemimastigophora. The monophyly of novel eukaryotic Ffh and FtsY groups, predicted mitochondrial localization experimentally confirmed for Naegleria gruberi, and a strong alphaproteobacterial affinity of the Ffh group, collectively suggest that they constitute parts of an ancestral mitochondrial signal peptide-based protein-targeting system inherited from the last eukaryotic common ancestor, but lost from the majority of extant eukaryotes. The ability of putative signal peptides, predicted in a subset of mitochondrial-encoded N. gruberi proteins, to target a reporter fluorescent protein into the endoplasmic reticulum of Trypanosoma brucei, likely through their interaction with the cytosolic SRP, provided further support for this notion. We also illustrate that known mitochondrial ribosome-interacting proteins implicated in membrane protein targeting in opisthokonts (Mba1, Mdm38, and Mrx15) are broadly conserved in eukaryotes and nonredundant with the mitochondrial SRP system. Finally, we identified a novel mitochondrial protein (MAP67) present in diverse eukaryotes and related to the signal peptide-binding domain of Ffh, which may well be a hitherto unrecognized component of the mitochondrial membrane protein-targeting machinery.     

Sensory rhodopsin I: Receptor activation and signal relay

Recent progress is summarized on the mechanism of phototransduction by sensory rhodopsin I (SR-I), a phototaxis receptor inHalobacterium halobium. Two aspects are emphasized: (i)The coupling of retinal isomerization to protein conformational changes. Retinal analogs have been used to probe chromophore-apoprotein interactions during the receptor activation process. One of the most important results is the finding of a steric trigger deriving from the interaction of residues on the protein with a methyl group near the isomerizing bond of the retinal (at carbon 13). Recent work on molecular genetic methods to further probe structure/function includes the synthesis and expression of an SR-I apoprotein gene designed for residue replacements by cassette mutagenesis, and transformation of anH. halobium mutant lacking all retinylidene proteins known in this species to SR-I⁺ and bacteriorhodopsin (BR)⁺. (ii)The relay of the SR-I signal to a post-receptor component. A carboxylmethylated protein (MPP-I) associated with SR-I and found in theH. halobium membrane exhibits homology with the signaling domain of eubacterial chemotaxis transducers (e.g.,Escherichia coli Tar, Tsr, and Trg proteins), suggesting a model based on SR-I MPP-I signal relay.     

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.     

Mammalian and Escherichia coli signal recognition particles

Recent evidence from both biochemical and genetic studies indicates that protein targeting to the pro-karyotic cytoplasmic membrane and the eukaryotic endoplasmic reticulum membrane may have more in common than previously thought. A ribonucleo-protein particle was identified in Escherichia coli that consists of at least one protein (P48 or Ffh) and one RNA molecule (4.5S RNA), both of which exhibit strong sequence similarity with constituents of the mammalian signal recognition particle (SRP). Like the mammalian SRP, the E. coli SRP binds specifically to the signal sequence of presecretory proteins. Depletion of either P48 or 4.5S RNA affects translation and results in the accumulation of precursors of several secreted proteins. This review discusses these recent studies and speculates on the position of the SRP in the complex network of protein interactions involved in translation and membrane targeting in E. coli.     

YidC-dependent translocation of green fluorescence protein fused to the FliP cleavable signal peptide

Escherichia coli FliP is a rare bacterial polytopic membrane protein synthesized with a cleavable, highly hydrophobic signal peptide. More hydrophilic Tat-dependent or Sec-dependent signal peptide is functionally capable of substituting for the FliP signal peptide, but a signal anchor of inner membrane protein fails to do so. To assess the intrinsic characteristics of the FliP signal peptide in mediating protein translocation, we fused it to green fluorescence protein and observed that the translocation of the chimera (FliPss-GFP) was dependent of Ffh, SecA, SecY and SecD. In addition, we showed for the first time the involvement of YidC in protein translocation across the inner membrane.     

The inhibitory performance of flavonoid cyanidin-3-sambubiocide against H274Y mutation in H1N1 influenza virus

Oseltamivir (Tamiflu) is the most accepted antiviral drug that targets the neuraminidase (NA) protein to inhibit the viral release from the host cell. Few H1N1 influenza strains with the H274Y mutation creates drug resistance to oseltamivir. In this study, we report that flavonoid cyanidin-3-sambubiocide (C3S) compound acts as a potential inhibitor against H274Y mutation. The drug resistance mechanism and inhibitory activity of C3S and oseltamivir against wild-type (WT) and H274Y mutant-type (MT) have been studied and compared based on the results of molecular docking, molecular dynamics, and quantum chemical methods. Oseltamivir has been found less binding affinity with MT. C3S has more binding affinity with WT and MT proteins. From the dynamical study, the 150th loop of the MT protein has found more deformation than WT. A single H274Y mutation induces the conformational changes in the 150th loop which leads to produce more resistance to oseltamivir. The 150th cavity is more attractive target for C3S to stop the conformational changes in the MT, than 430th cavity of NA protein. The C3S is stabilized with MT by more number of hydrogen bonds than oseltamivir. The electrostatic interaction energy shows a stronger C3S binding with MT and this compound may be more effective against oseltamivir-resistant virus strains.     

Motions of the SecA protein motor bound to signal peptide: Insights from molecular dynamics simulations

SecA is an essential part of the Sec pathway for protein secretion in bacteria. In this pathway, SecA interacts with the N-terminal fragment of the secretory protein – the signal peptide, and couples binding and hydrolysis of adenosine triphosphate with movement of the secretory protein across the SecY protein translocon. How interactions with the signal peptide alter the conformational dynamics and long-distance conformational couplings of SecA is a key open question that we address here with molecular dynamics techniques. Analyses of protein motions indicate that the signal peptide alters SecA dynamics not only at the site where this peptide binds, but also at a nucleotide-binding domain. Hydrogen bond clusters contribute to the long-distance propagation of changes in SecA dynamics.