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.     

Molecular dynamics simulation of SRP GTPases: Towards an understanding of the complex formation from equilibrium fluctuations

Signal recognition particle (SRP) and its receptor (SR) play essential role in the SRP‐dependent protein targeting pathway. They interact with one another to precisely regulate the targeting reaction. The mechanism of this interaction consists of at least two discrete conformational states: complex formation and GTPase activation. Although structural studies have provided valuable insights into the understanding of the SRP‐SR interaction, it still remains unclear that how SRP and SR GTPases use their intrinsic conformational flexibilities to exert multiple allosteric regulations on this interaction process. Here, we use computational simulations to present the dynamic behavior of the SRP GTPases at an atomic level to gain further understanding of SRP‐SR interaction. We show that: (i) equilibrium conformational fluctuations contain a cooperative inter‐ and intradomain structural rearrangements that are functionally relevant to complex formation, (ii) a series of residues in different domains are identified to correlate with each other during conformational rearrangements, and (iii) α3 and α4 helices at domain interface actively rearrange their relative conformation to function as a bridge between the N domain and the core region of the G domain. These results, in addition to structural studies, would harness our understanding of the molecular mechanism for SRP and SR interaction. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Efficient interaction between two GTPases allows the chloroplast SRP pathway to bypass the requirement for an SRP RNA

Cotranslational protein targeting to membranes is regulated by two GTPases in the signal recognition particle (SRP) and the SRP receptor; association between the two GTPases is slow and is accelerated 400-fold by the SRP RNA. Intriguingly, the otherwise universally conserved SRP RNA is missing in a novel chloroplast SRP pathway. We found that even in the absence of an SRP RNA, the chloroplast SRP and receptor GTPases can interact efficiently with one another; the kinetics of interaction between the chloroplast GTPases is 400-fold faster than their bacterial homologues, and matches the rate at which the bacterial SRP and receptor interact with the help of SRP RNA. Biochemical analyses further suggest that the chloroplast SRP receptor is pre-organized in a conformation that allows optimal interaction with its binding partner, so that conformational changes during complex formation are minimized. Our results highlight intriguing differences between the classical and chloroplast SRP and SRP receptor GTPases, and help explain how the chloroplast SRP pathway can mediate efficient targeting of proteins to the thylakoid membrane in the absence of the SRP RNA, which plays an indispensable role in all the other SRP pathways.     

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.     

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.     

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.     

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.     

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.     

Protein translocation: checkpoint role for SRP GTPase activation

Co-translational protein targeting by the signal recognition particle (SRP) relies on a complex series of structural rearrangements in the SRP and its receptor (SR). In order to precisely coordinate the individual steps, the GTPases of the SRP and the SR form a unique complex in which GTP hydrolysis is activated in a composite active site. A recent study provides new insights on the link between the GTPases and protein translocation.     

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.     

Structure of a GDP:AlF4 complex of the SRP GTPases Ffh and FtsY, and identification of a peripheral nucleotide interaction site

The signal recognition particle (SRP) GTPases Ffh and FtsY play a central role in co-translational targeting of proteins, assembling in a GTP-dependent manner to generate the SRP targeting complex at the membrane. A suite of residues in FtsY have been identified that are essential for the hydrolysis of GTP that accompanies disengagement. We have argued previously on structural grounds that this region mediates interactions that serve to activate the complex for disengagement and term it the activation region. We report here the structure of a complex of the SRP GTPases formed in the presence of GDP:AlF4. This complex accommodates the putative transition-state analog without undergoing significant change from the structure of the ground-state complex formed in the presence of the GTP analog GMPPCP. However, small shifts that do occur within the shared catalytic chamber may be functionally important. Remarkably, an external nucleotide interaction site was identified at the activation region, revealed by an unexpected contaminating GMP molecule bound adjacent to the catalytic chamber. This site exhibits conserved sequence and structural features that suggest a direct interaction with RNA plays a role in regulating the activity of the SRP targeting complex.     

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.     

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.     

Role of SRP RNA in the GTPase cycles of Ffh and FtsY.

The bacterial homologues of the signal recognition particle (SRP) and its receptor, the Ffh*4.5S RNA ribonucleoprotein complex and the FtsY protein, respectively, form a unique complex in which both Ffh and FtsY act as GTPase activating proteins for one another, resulting in the mutual stimulation of GTP hydrolysis by both proteins. Previous work showed that 4.5S RNA enhances the GTPase activity in the presence of both Ffh and FtsY, but it was not clear how this was accomplished. In this work, kinetic and thermodynamic analyses of the GTPase reactions of Ffh and FtsY have provided insights into the role of 4.5S RNA in the GTPase cycles of Ffh and FtsY. We found that 4.5S RNA accelerates the association between Ffh and FtsY 400-fold in their GTP-bound form, analogous to its 200-fold catalytic effect on Ffh*FtsY association previously observed with the GppNHp-bound form [Peluso, P., et al. (2000) Science 288, 1640-1643]. Further, Ffh-FtsY association is rate-limiting for the observed GTPase reaction with subsaturating Ffh and FtsY, thereby accounting for the apparent stimulatory effect of 4.5S RNA on the GTPase activity observed previously. An additional step, GTP hydrolysis from the Ffh*FtsY complex, is also moderately facilitated by 4.5S RNA. These results suggest that 4.5S RNA modulates the conformation of the Ffh*FtsY complex and may, in turn, regulate its GTPase activity during the SRP functional cycle.     

Construction of the 'minimal' SRP that interacts with the translating ribosome but not with specific membrane receptors in Escherichia coli

Escherichia coli signal recognition particle (SRP) consists of 4.5S RNA and Ffh protein. In contrast to eukaryotes, it remains unclear whether translation arrest takes place in prokaryotic cells. To study this problem we constructed a fusion of the M domain of Ffh protein with a cleavable affinity tag. This mutant Ffh, in a complex with 4.5S RNA, can bind signal peptide at the translating ribosome but is unable to bind the membrane. This SRP-ribosome complex should accumulate in the cell if translation is arrested. To test this, the complex was purified from the cells by ultracentrifugation and affinity chromatography. The composition of the complex was analyzed and found to consist of ribosomal RNAs and proteins, the Ffh M domain and 4.5S RNA. The accumulation of this complex in the cell in significant amounts indicated that SRP-mediated translation arrest did occur in bacterial cells.     

The ribosome regulates the GTPase of the beta-subunit of the signal recognition particle receptor.

Protein targeting to the membrane of the ER is regulated by three GTPases, the 54-kD subunit of the signal recognition particle (SRP) and the alpha- and beta-subunit of the SRP receptor (SR). Here, we report on the GTPase cycle of the beta-subunits of the SR (SRbeta). We found that SRbeta binds GTP with high affinity and interacts with ribosomes in the GTP-bound state. Subsequently, the ribosome increases the GTPase activity of SRbeta and thus functions as a GTPase activating protein for SRbeta. Furthermore, the interaction between SRbeta and the ribosome leads to a reduction in the affinity of SRbeta for guanine nucleotides. We propose that SRbeta regulates the interaction of SR with the ribosome and thereby allows SRalpha to scan membrane-bound ribosomes for the presence of SRP. Interaction between SRP and SRalpha then leads to release of the signal sequence from SRP and insertion into the translocon. GTP hydrolysis then results in dissociation of SR from the ribosome, and SRP from the SR.     

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.     

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.     

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.     

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.