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SecA is a widely conserved ATPase that drives the secretion of proteins across the cell membrane via the SecYEG translocon, while the SRP system is a key player in the insertion of membrane proteins via SecYEG. How SecA gains access to substrate proteins in Bacillus subtilis cells and copes with an increase in substrate availability during biotechnologically desired, high-level expression of secreted proteins is poorly understood. Using single molecule tracking, we found that SecA localization closely mimics that of ribosomes, and its molecule dynamics change similarly to those of ribosomes after inhibition of transcription or translation. These data suggest that B. subtilis SecA associates with signal peptides as they are synthesized at the ribosome, similar to the SRP system. In agreement with this, SecA is a largely mobile cytosolic protein; only a subset is statically associated with the cell membrane, i.e., likely with the Sec translocon. SecA dynamics were considerably different during the late exponential, transition, and stationary growth phases, revealing that single molecule dynamics considerably alter during different genetic programs in cells. During overproduction of a secretory protein, AmyE, SecA showed the strongest changes during the transition phase, i.e., where general protein secretion is high. To investigate whether the overproduction of AmyE also has an influence on other proteins that interact with SecYEG, we analyzed the dynamics of SecDF, YidC, and FtsY with and without AmyE overproduction. SecDF and YidC did not reveal considerable differences in single molecule dynamics during overexpression, while the SRP component FtsY changed markedly in its behavior and became more statically engaged. These findings indicate that the SRP pathway becomes involved in protein secretion upon an overload of proteins carrying a signal sequence. Thus, our data reveal high plasticity of the SecA and SRP systems in dealing with different needs for protein secretion. [ABSTRACT FROM AUTHOR]

The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins via a hydrophilic groove located within the inner leaflet of the membrane. All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates.

YidC belongs to an evolutionarily conserved family of insertases, YidC/Oxa1/Alb3, in bacteria, mitochondria, and chloroplasts, respectively. Unlike Gram-negative bacteria, Gram-positives including Streptococcus mutans harbor two paralogs of YidC. The mechanism for paralog-specific phenotypes of bacterial YidC1 versus YidC2 has been partially attributed to the differences in their cytoplasmic domains. However, we previously identified a W138R gain-of-function mutation in the YidC1 transmembrane helix 2. YidC1W138R mostly phenocopied YidC2, yet the mechanism remained unknown. Primary sequence comparison of streptococcal YidCs led us to identify and mutate the YidC1W138 analog, YidC2S152 to W/A, which resulted in a loss of YidC2- and acquisition of YidC1-like phenotype. The predicted lipid-facing side chains of YidC1W138/YidC2S152 led us to propose a role for membrane phospholipids in specific-residue dependent phenotypes of S. mutans YidC paralogs. Cardiolipin (CL), a prevalent phospholipid in the S. mutans cytoplasmic membrane during acid stress, is encoded by a single gene, cls. We show a concerted mechanism for cardiolipin and YidC2 under acid stress based on similarly increased promoter activities and similar elimination phenotypes. Using coarse grain molecular dynamics simulations with the Martini2.2 Forcefield, YidC1 and YidC2 wild-type and mutant interactions with CL were assessed in silico. We observed substantially increased CL interaction in dimeric versus monomeric proteins, and variable CL occupancy in YidC1 and YidC2 mutant constructs that mimicked characteristics of the other wild-type paralog. Hence, paralog-specific amino acid- CL interactions contribute to YidC1 and YidC2-associated phenotypes that can be exchanged by point mutation at positions 138 or 152, respectively.

Protein translocases, such as the bacterial SecY complex, the Sec61 complex of the endoplasmic reticulum (ER) and the mitochondrial translocases, facilitate the transport of proteins across membranes. In addition, they catalyze the insertion of integral membrane proteins into the lipid bilayer. Several membrane insertases cooperate with these translocases, thereby promoting the topogenesis, folding and assembly of membrane proteins. Oxa1 and BamA family members serve as core components in the two major classes of membrane insertases. They facilitate the integration of proteins with a-helical transmembrane domains and of ß-barrel proteins into lipid bilayers, respectively. Members of the Oxa1 family were initially found in the internal membranes of bacteria, mitochondria and chloroplasts. Recent studies, however, also identified several Oxa1-type insertases in the ER, where they serve as catalytically active core subunits in the ER membrane protein complex (EMC), the guided entry of tail-anchored (GET) and the GET- and EMC-like (GEL) complex. The outer membrane of bacteria, mitochondria and chloroplasts contain ß-barrel proteins, which are inserted by members of the BamA family. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of these different types of membrane insertases and discuss their function. [ABSTRACT FROM AUTHOR]

SecA is a widely conserved ATPase that drives the secretion of proteins across the cell membrane via the SecYEG translocon, while the SRP system is a key player in the insertion of membrane proteins via SecYEG. How SecA gains access to substrate proteins in Bacillus subtilis cells and copes with an increase in substrate availability during biotechnologically desired, high-level expression of secreted proteins is poorly understood. Using single molecule tracking, we found that SecA localization closely mimics that of ribosomes, and its molecule dynamics change similarly to those of ribosomes after inhibition of transcription or translation. These data suggest that B. subtilis SecA associates with signal peptides as they are synthesized at the ribosome, similar to the SRP system. In agreement with this, SecA is a largely mobile cytosolic protein; only a subset is statically associated with the cell membrane, i.e., likely with the Sec translocon. SecA dynamics were considerably different during the late exponential, transition, and stationary growth phases, revealing that single molecule dynamics considerably alter during different genetic programs in cells. During overproduction of a secretory protein, AmyE, SecA showed the strongest changes during the transition phase, i.e., where general protein secretion is high. To investigate whether the overproduction of AmyE also has an influence on other proteins that interact with SecYEG, we analyzed the dynamics of SecDF, YidC, and FtsY with and without AmyE overproduction. SecDF and YidC did not reveal considerable differences in single molecule dynamics during overexpression, while the SRP component FtsY changed markedly in its behavior and became more statically engaged. These findings indicate that the SRP pathway becomes involved in protein secretion upon an overload of proteins carrying a signal sequence. Thus, our data reveal high plasticity of the SecA and SRP systems in dealing with different needs for protein secretion.

Aeromonas salmonicida is a common pathogenic bacterial species found in both freshwater and marine fish, leading to significant economic losses in the aquaculture industry. YidC is an accessory to SecYEG and is essential for the SecYEG transporter to insert into the bacterial membrane. However, the roles of the yidC gene on the host immune response remain unclear. Here, we compared the pathogenicity of yidC gene-deleted (Δ yidC) strain and wild-type (SRW-OG1) strain of mesophilic A. salmonicida to Orange-spotted grouper (Epinephelus coioides), and explored the impacts of yidC gene on the immune response of E. coioides to mesophilic A. salmonicida infection by using Red/ET recombineering. In this study, the E. coioides in the Secondary infected group had a 53.9 % higher survival rate than those in the Primary infected group. In addition, the adhesion ability of Δ yidC strain decreased by about 83.36 % compared with that of the wild-type (SRW-OG1) strain. Further comparison of the biological phenotype of SRW-OG1 and Δ yidC revealed that this yidC gene could regulate the expression of genes related to iron metabolism and have no effect on bacterial growth under the limited iron concentration. In the low concentration of Fe3+ and Fe2+ environment, SRW-OG1 can obtain iron ions by regulating yidC. Based on the above results, yidC gene contributed to the pathogenicity of mesophilic A. salmonicida to E. coioides , deletion of yidC gene promoted the inflammation and immune response of E. coioides to mesophilic A. salmonicida infection. • The adhesion ability of Δ yidC strain decreased by about 83.36 % compared with that of the SRW-OG1 strain. • The SRW-OG1 strain ceased to grow when 300 μmol/L 2,2′-bipyridine was added to the culture medium. • In the low concentration of Fe3+ and Fe2+ environment, SRW-OG1 can obtain iron ions by regulating yidC. • The Δ yidC strain shows promising immune protection against Orange-spotted grouper (Epinephelus coioides). [ABSTRACT FROM AUTHOR]

Lactococcus lactis is a crucial food-grade cell factory for secreting valuable peptides and proteins primarily via the Sec-dependent pathway. YidC, a membrane insertase, facilitates protein insertion into the lipid membrane for the translocation. However, the mechanistic details of how YidC affects protein secretion in L. lactis remain elusive. This study investigates the effects of deleting yidC1 / yidC2 on L. lactis phenotypes and protein secretion. Compared to the original strain, deleting yidC2 significantly decreased the relative biomass, electroporation efficiency, and F-ATP activity by 25%, 47%, and 33%, respectively, and weakened growth and stress resistance, whereas deleting yidC1 had a minimal impact. The absence of either yidC1 or yidC2 reduced target proteins secretion. Meanwhile, there is a considerable alteration in the transcription levels of genes involved in the secretion pathway, with secY transcription increasing over 135-fold. Our results provide a theoretical foundation for further improving target protein secretion and investigating the YidC function.

Abstract Background Protein transporters translocate hydrophilic segments of polypeptide across hydrophobic cell membranes. Two protein transporters are ubiquitous and date back to the last universal common ancestor: SecY and YidC. SecY consists of two pseudosymmetric halves, which together form a membrane-spanning protein-conducting channel. YidC is an asymmetric molecule with a protein-conducting hydrophilic groove that partially spans the membrane. Although both transporters mediate insertion of membrane proteins with short translocated domains, only SecY transports secretory proteins and membrane proteins with long translocated domains. The evolutionary origins of these ancient and essential transporters are not known. Results The features conserved by the two halves of SecY indicate that their common ancestor was an antiparallel homodimeric channel. Structural searches with SecY’s halves detect exceptional similarity with YidC homologs. The SecY halves and YidC share a fold comprising a three-helix bundle interrupted by a helical hairpin. In YidC, this hairpin is cytoplasmic and facilitates substrate delivery, whereas in SecY, it is transmembrane and forms the substrate-binding lateral gate helices. In both transporters, the three-helix bundle forms a protein-conducting hydrophilic groove delimited by a conserved hydrophobic residue. Based on these similarities, we propose that SecY originated as a YidC homolog which formed a channel by juxtaposing two hydrophilic grooves in an antiparallel homodimer. We find that archaeal YidC and its eukaryotic descendants use this same dimerisation interface to heterodimerise with a conserved partner. YidC’s sufficiency for the function of simple cells is suggested by the results of reductive evolution in mitochondria and plastids, which tend to retain SecY only if they require translocation of large hydrophilic domains. Conclusions SecY and YidC share previously unrecognised similarities in sequence, structure, mechanism, and function. Our delineation of a detailed correspondence between these two essential and ancient transporters enables a deeper mechanistic understanding of how each functions. Furthermore, key differences between them help explain how SecY performs its distinctive function in the recognition and translocation of secretory proteins. The unified theory presented here explains the evolution of these features, and thus reconstructs a key step in the origin of cells.

In 1971, Blobel proposed the first statement of the Signal Hypothesis which suggested that proteins have amino-terminal sequences that dictate their export and localization in the cell. A cytosolic binding factor was predicted, and later the protein conducting channel was discovered that was proposed in 1975 to align with the large ribosomal tunnel. The 1975 Signal Hypothesis also predicted that proteins targeted to different intracellular membranes would possess distinct signals and integral membrane proteins contained uncleaved signal sequences which initiate translocation of the polypeptide chain. This review summarizes the central role that the signal peptides play as address codes for proteins, their decisive role as targeting factors for delivery to the membrane and their function to activate the translocation machinery for export and membrane protein insertion. After shedding light on the navigation of proteins, the importance of removal of signal peptide and their degradation are addressed. Furthermore, the emerging work on signal peptidases as novel targets for antibiotic development is described. [ABSTRACT FROM AUTHOR]

The YidC/Oxa1/Alb3 family of membrane proteins function to insert proteins into membranes in bacteria, mitochondria, and chloroplasts. Recent X-ray structures of YidC from Bacillus halodurans and Escherichia coli revealed a hydrophilic groove that is accessible from the lipid bilayer and the cytoplasm. Here, we explore the water accessibility within the conserved core region of the E. coli YidC using in vivo cysteine alkylation scanning and molecular dynamics (MD) simulations of YidC in POPE/POPG membranes. As expected from the structure, YidC possesses an aqueous membrane cavity localized to the membrane inner leaflet. Both the scanning data and the MD simulations show that the lipid-exposed transmembrane helices 3, 4, and 5 are short, leading to membrane thinning around YidC. Close examination of the MD data reveals previously unrecognized structural features that are likely important for protein stability and function.

Protein integration into biomembranes is an essential biological phenomenon common to all organisms. While various factors involved in protein integration, such as SRP, SecYEG and YidC, are proteinaceous, we identified a glycolipid named MPIase (Membrane Protein Integrase), which is present in the cytoplasmic membrane of E. coli. In vitro experiments using inverted membrane vesicles prepared from MPIase-depleted strains, and liposomes containing MPIase showed that MPIase is required for insertion of a subset of membrane proteins, which has been thought to be SecYEG-independent and YidC-dependent. Also, SecYEG-dependent substrate membrane proteins require MPIase in addition. Furthermore, MPIase is also essential for insertion of proteins with multiple negative charges, which requires both YidC and the proton motive force (PMF). MPIase directly interacts with SecYEG and YidC on the membrane. MPIase not only cooperates with these factors but also has a molecular chaperone-like function specific to the substrate membrane proteins through direct interaction with the glycan chain. Thus, MPIase catalyzes membrane insertion by accepting nascent membrane proteins on the membrane through its chaperone-like function, i.e., direct interaction with the substrate proteins, and then MPIase functionally interacts with SecYEG and YidC for substrate delivery, and acts with PMF to facilitate and complete membrane insertion when necessary. In this review, we will outline the mechanisms underlying membrane insertion catalyzed by MPIase, which cooperates with proteinaceous factors and PMF.

YidC is a membrane protein that facilitates the insertion of newly synthesized proteins into lipid membranes. Through YidC, proteins are inserted into the lipid bilayer via the SecYEG-dependent complex. Additionally, YidC functions as a chaperone in protein folding processes. Several studies have provided evidence of its independent insertion mechanism. However, the mechanistic details of the YidC SecY-independent protein insertion mechanism remain elusive at the molecular level. This study elucidates the insertion mechanism of YidC at an atomic level through a combination of equilibrium and non-equilibrium molecular dynamics (MD) simulations. Different docking models of YidC-Pf3 in the lipid bilayer were built in this study to better understand the insertion mechanism. To conduct a complete investigation of the conformational difference between the two docking models developed, we used classical molecular dynamics simulations supplemented with a non-equilibrium technique. Our findings indicate that the YidC transmembrane (TM) groove is essential for this high-affinity interaction and that the hydrophilic nature of the YidC groove plays an important role in protein transport across the cytoplasmic membrane bilayer to the periplasmic side. At different stages of the insertion process, conformational changes in YidC’s TM domain and membrane core have a mechanistic effect on the Pf3 coat protein. Furthermore, during the insertion phase, the hydration and dehydration of the YidC’s hydrophilic groove are critical. These results demonstrate that Pf3 coat protein interactions with the membrane and YidC vary in different conformational states during the insertion process. Finally, this extensive study directly confirms that YidC functions as an independent insertase.

In 1971, Blobel proposed the first statement of the Signal Hypothesis which suggested that proteins have amino-terminal sequences that dictate their export and localization in the cell. A cytosolic binding factor was predicted, and later the protein conducting channel was discovered that was proposed in 1975 to align with the large ribosomal tunnel. The 1975 Signal Hypothesis also predicted that proteins targeted to different intracellular membranes would possess distinct signals and integral membrane proteins contained uncleaved signal sequences which initiate translocation of the polypeptide chain. This review summarizes the central role that the signal peptides play as address codes for proteins, their decisive role as targeting factors for delivery to the membrane and their function to activate the translocation machinery for export and membrane protein insertion. After shedding light on the navigation of proteins, the importance of removal of signal peptide and their degradation are addressed. Furthermore, the emerging work on signal peptidases as novel targets for antibiotic development is described.

The large abundance of small open reading frames (smORFs) in prokaryotic and eukaryotic genomes and the plethora of smORF‐encoded small proteins became only apparent with the constant advancements in bioinformatic, genomic, proteomic, and biochemical tools. Small proteins are typically defined as proteins of < 50 amino acids in prokaryotes and of less than 100 amino acids in eukaryotes, and their importance for cell physiology and cellular adaptation is only beginning to emerge. In contrast to antimicrobial peptides, which are secreted by prokaryotic and eukaryotic cells for combatting pathogens and competitors, small proteins act within the producing cell mainly by stabilizing protein assemblies and by modifying the activity of larger proteins. Production of small proteins is frequently linked to stress conditions or environmental changes, and therefore, cells seem to use small proteins as intracellular modifiers for adjusting cell metabolism to different intra‐ and extracellular cues. However, the size of small proteins imposes a major challenge for the cellular machinery required for protein folding and intracellular trafficking and recent data indicate that small proteins can engage distinct trafficking pathways. In the current review, we describe the diversity of small proteins in prokaryotes and eukaryotes, highlight distinct and common features, and illustrate how they are handled by the protein trafficking machineries in prokaryotic and eukaryotic cells. Finally, we also discuss future topics of research on this fascinating but largely unexplored group of proteins. [ABSTRACT FROM AUTHOR]

Background: Protein transporters translocate hydrophilic segments of polypeptide across hydrophobic cell membranes. Two protein transporters are ubiquitous and date back to the last universal common ancestor: SecY and YidC. SecY consists of two pseudosymmetric halves, which together form a membrane-spanning protein-conducting channel. YidC is an asymmetric molecule with a protein-conducting hydrophilic groove that partially spans the membrane. Although both transporters mediate insertion of membrane proteins with short translocated domains, only SecY transports secretory proteins and membrane proteins with long translocated domains. The evolutionary origins of these ancient and essential transporters are not known. Results: The features conserved by the two halves of SecY indicate that their common ancestor was an antiparallel homodimeric channel. Structural searches with SecY's halves detect exceptional similarity with YidC homologs. The SecY halves and YidC share a fold comprising a three-helix bundle interrupted by a helical hairpin. In YidC, this hairpin is cytoplasmic and facilitates substrate delivery, whereas in SecY, it is transmembrane and forms the substrate-binding lateral gate helices. In both transporters, the three-helix bundle forms a protein-conducting hydrophilic groove delimited by a conserved hydrophobic residue. Based on these similarities, we propose that SecY originated as a YidC homolog which formed a channel by juxtaposing two hydrophilic grooves in an antiparallel homodimer. We find that archaeal YidC and its eukaryotic descendants use this same dimerisation interface to heterodimerise with a conserved partner. YidC's sufficiency for the function of simple cells is suggested by the results of reductive evolution in mitochondria and plastids, which tend to retain SecY only if they require translocation of large hydrophilic domains. Conclusions: SecY and YidC share previously unrecognised similarities in sequence, structure, mechanism, and function. Our delineation of a detailed correspondence between these two essential and ancient transporters enables a deeper mechanistic understanding of how each functions. Furthermore, key differences between them help explain how SecY performs its distinctive function in the recognition and translocation of secretory proteins. The unified theory presented here explains the evolution of these features, and thus reconstructs a key step in the origin of cells. [ABSTRACT FROM AUTHOR]

Integral and membrane-anchored proteins are pivotal to survival and virulence of the dental pathogen, Streptococcus mutans. The bacterial chaperone/insertase, YidC, contributes to membrane protein translocation. Unlike Escherichia coli , most Gram-positive bacteria contain two YidC paralogs. Herein, we evaluated structural features that functionally delineate S. mutans YidC1 and YidC2. Bacterial YidCs contain five transmembrane domains (TMD), two cytoplasmic loops, and a cytoplasmic tail. Because S. mutans YidC1 (SmYidC1) and YidC2 (SmYidC2) cytoplasmic domains (CD) are less well conserved than are TMD, we engineered ectopic expression of the 14 possible YidC1-YidC2 CD domain swap combinations. Growth and stress tolerance of each was compared to control strains ectopically expressing unmodified yidC1 or yidC2. Acid and osmotic stress sensitivity are associated with yidC2 deletion. Sensitivity to excess zinc was further identified as a Δ yidC1 phenotype. Overall, YidC1 tolerated CD substitutions better than YidC2. Preferences toward particular CD combinations suggested potential intramolecular interactions. In silico analysis predicted salt-bridges between C1 and C2 loops of YidC1, and C1 loop and C-terminal tail of YidC2, respectively. Mutation of contributing residues recapitulated Δ yidC1 - and Δ yidC2 -associated phenotypes. Taken together, this work revealed the importance of cytoplasmic domains in distinct functional attributes of YidC1 and YidC2, and identified key residues involved in interdomain interactions. [ABSTRACT FROM AUTHOR]

Integral and membrane-anchored proteins are pivotal to survival and virulence of the dental pathogen, Streptococcus mutans. The bacterial chaperone/insertase, YidC, contributes to membrane protein translocation. Unlike Escherichia coli, most Gram-positive bacteria contain two YidC paralogs. Herein, we evaluated structural features that functionally delineate S. mutans YidC1 and YidC2. Bacterial YidCs contain five transmembrane domains (TMD), two cytoplasmic loops, and a cytoplasmic tail. Because S. mutans YidC1 (SmYidC1) and YidC2 (SmYidC2) cytoplasmic domains (CD) are less well conserved than are TMD, we engineered ectopic expression of the 14 possible YidC1-YidC2 CD domain swap combinations. Growth and stress tolerance of each was compared to control strains ectopically expressing unmodified yidC1 or yidC2. Acid and osmotic stress sensitivity are associated with yidC2 deletion. Sensitivity to excess zinc was further identified as a ΔyidC1 phenotype. Overall, YidC1 tolerated CD substitutions better than YidC2. Preferences toward particular CD combinations suggested potential intramolecular interactions. In silico analysis predicted salt-bridges between C1 and C2 loops of YidC1, and C1 loop and C-terminal tail of YidC2, respectively. Mutation of contributing residues recapitulated ΔyidC1- and ΔyidC2-associated phenotypes. Taken together, this work revealed the importance of cytoplasmic domains in distinct functional attributes of YidC1 and YidC2, and identified key residues involved in interdomain interactions.

Nocardia seriolae has been identified as the causative agent of fish nocardiosis, resulting in serious economic losses in aquaculture. With an aim to screen potential candidates for vaccine development against N. seriolae , the in vivo -induced genes of N. seriolae in hybrid snakehead (Channa maculate ♀ × Channa argus ♂) model were profiled via in vivo -induced antigen technology (IVIAT) in the present study, and 6 in vivo -induced genes were identified as follows: IS701 family transposase (is701), membrane protein insertase YidC (yidC), ergothioneine biosynthesis glutamate-cysteine ligase (egtA), molybdopterin respectively-dependent oxidoreductase (mol), phosphoketolase family protein (Ppl), hypothetical protein 6747 (hp6747). Additionally, the yidC was inserted into eukaryotic expression vector pcDNA3.1-myc-his-A to construct a DNA vaccine named as pcDNA-YidC to evaluate immunoprotection in hybrid snakehead after artificial challenge with N. serioale. Results showed that the transcription of yidC was detected in spleen, trunk kidney, muscle and liver in vaccinated fish, suggesting that this antigenic gene can be recombinantly expressed in fish. Meanwhile, indexes of humoral immunity were evaluated in the vaccinated fish through assessing specific-antibody IgM and serum enzyme activities, including lysozyme (LZM), superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). Quantitative real-time PCR analysis indicated that pcDNA-YidC DNA vaccine could notably enhance the expression of immune-related genes (CD4 、 CD8α 、 MHCIIα 、 TNFα 、 IL-1β and MHCIα) in 4 tissues (spleen, trunk kidney, muscle and liver) of the vaccinated fish. Finally, an immuno-protection with a relative survival rate of 65.71 % was displayed in vaccinated fish in comparison to the control groups. Taken together, these results indicate that pcDNA-YidC DNA vaccine could boost strong immune responses in hybrid snakehead and show preferably protective efficacy against N. seriolae , indicating that IVIAT is a helpful strategy to screen the highly immunogenic antigens for vaccine development against fish nocardiosis. • Six antigenic genes of Nocardia seriolae were screened via IVIAT technology. • The pcDNA-yidC was constructed and used as DNA vaccine to induce immune responses. • The trials of pcDNA-yidC DNA vaccine could provide 65.71 % RPS. • IVIAT can be used as a strategy to study the antigenic genes for vaccine development against N. seriolae. [ABSTRACT FROM AUTHOR]

The spatial and temporal coordination of protein transport is an essential cornerstone of the bacterial adaptation to different environmental conditions. By adjusting the protein composition of extra-cytosolic compartments, like the inner and outer membranes or the periplasmic space, protein transport mechanisms help shaping protein homeostasis in response to various metabolic cues. The universally conserved SecYEG translocon acts at the center of bacterial protein transport and mediates the translocation of newly synthesized proteins into and across the cytoplasmic membrane. The ability of the SecYEG translocon to transport an enormous variety of different substrates is in part determined by its ability to interact with multiple targeting factors, chaperones and accessory proteins. These interactions are crucial for the assisted passage of newly synthesized proteins from the cytosol into the different bacterial compartments. In this review, we summarize the current knowledge about SecYEG-mediated protein transport, primarily in the model organism Escherichia coli, and describe the dynamic interaction of the SecYEG translocon with its multiple partner proteins. We furthermore highlight how protein transport is regulated and explore recent developments in using the SecYEG translocon as an antimicrobial target.

During synthesis of membrane proteins, transmembrane segments (TMs) of nascent proteins emerging from the ribosome are inserted into the central pore of the translocon (SecYEG in bacteria) and access the phospholipid bilayer through the open lateral gate formed of two helices of SecY. Here we use single-molecule fluorescence resonance energy transfer to monitor lateral-gate fluctuations in SecYEG embedded in nanodiscs containing native membrane phospholipids. We find the lateral gate to be highly dynamic, sampling the whole range of conformations between open and closed even in the absence of ligands, and we suggest a statistical model-free approach to evaluate the ensemble dynamics. Lateral gate fluctuations take place on both short (submillisecond) and long (subsecond) timescales. Ribosome binding and TM insertion do not halt fluctuations but tend to increase sampling of the open state. When YidC, a constituent of the holotranslocon, is bound to SecYEG, TM insertion facilitates substantial opening of the gate, which may aid in the folding of YidC-dependent polytopic membrane proteins. Mutations in lateral gate residues showing in vivo phenotypes change the range of favored states, underscoring the biological significance of lateral gate fluctuations. The results suggest how rapid fluctuations of the lateral gate contribute to the biogenesis of inner-membrane proteins. [ABSTRACT FROM AUTHOR]

To understand how YidC and SecYEG function together in membrane protein topogenesis, insertion and folding of the lactose permease of Escherichia coli (LacY), a 12-transmembrane helix protein LacY that catalyzes symport of a galactoside and an H(+), was studied. Although both the SecYEG machinery and signal recognition particle are required for insertion of LacY into the membrane, YidC is not required for translocation of the six periplasmic loops in LacY. Rather, YidC acts as a chaperone, facilitating LacY folding. Upon YidC depletion, the conformation of LacY is perturbed, as judged by monoclonal antibody binding studies and by in vivo cross-linking between introduced Cys pairs. Disulfide cross-linking also demonstrates that YidC interacts with multiple transmembrane segments of LacY during membrane biogenesis. Moreover, YidC is strictly required for insertion of M13 procoat protein fused into the middle cytoplasmic loop of LacY. In contrast, the loops preceding and following the inserted procoat domain are dependent on SecYEG for insertion. These studies demonstrate close cooperation between the two complexes in membrane biogenesis and that YidC functions primarily as a foldase for LacY.

The c subunits of F0F1‐ATP synthase (F0c) assemble into a ring structure, following membrane insertion that is dependent on both glycolipid MPIase and protein YidC. We analyzed the insertion and assembly processes of Propionigenium modestum F0c (Pm‐F0c), of which the ring structure is resistant to SDS. Ring assembly of Pm‐F0c requires P. modestum UncI (Pm‐UncI). Ring assembly of in vitro synthesized Pm‐F0c was observed when both YidC and Pm‐UncI were reconstituted into liposomes of Escherichia coli phospholipids. Under the physiological conditions where spontaneous insertion had been blocked by diacylglycerol, MPIase was necessary for Pm‐F0c insertion allowing the subsequent YidC/Pm‐UncI‐dependent ring assembly. Thus, we have succeeded in the complete reconstitution of membrane insertion and subsequent ring assembly of Pm‐F0c. [ABSTRACT FROM AUTHOR]

Background Mycoplasma hominis , an opportunistic pathogen in human genitourinary tract, can cause chronic infection in the prostate. Intracellular survival of M. hominis leads to a prolonged presence in the host cells that can affect the cell's biological cycle. In the present study, we aimed to evaluate the frequency of M. hominis DNA in prostate tissue of Iranian patients with prostate cancer (PCa) in comparison to a control group with benign prostatic hyperplasia (BPH). Methods This research was a retrospective case-control study using 61 archived formalin-fixed paraffin-embedded (FFPE) blocks of prostate tissue from patients with PCa and 70 FFPE blocks of patients with BPH. Real-time PCR, targeting two different genes, 16S rRNA and yidC , in the M. hominis genome was performed for all specimens. Results Out of 61 blocks of prostate biopsy from patients with PCa, eight samples (13%) were positive for M. hominis , while the bacterium was not detected in any of the 70 blocks of patients with BPH (P value, 0.002). Conclusions The high frequency of M. hominis in patients with PCa likely shows a hidden role of the organism in prostate cancer during its chronic, apparently silent and asymptomatic colonization in prostate. [ABSTRACT FROM AUTHOR]

Proper membrane insertion is crucial for the structure and function of membrane proteins in all cells. The YidC insertase plays an essential role in this process, but the molecular mechanism of YidC-mediated insertion remains unknown. Here we track the stepwise movement of Pf3 coat through YidC by obtaining a series of translational arrested intermediates, and investigate them by thiol cross-linking. We show that Pf3 is inserted as a helical hairpin, i.e., the prospective transmembrane segment moves along the YidC greasy slide comprised of TM3 and TM5, whereas the N-terminal tail transiently folds back into the hydrophilic groove of YidC located in the inner leaflet of the membrane until it is translocated to the periplasm in a subsequent step involving the electrochemical membrane potential. In addition to providing virtual insights about how YidC inserts single-spanning membrane proteins, our study also demonstrates a valuable in vivo tracking method that can be applied to study more complicated substrates or other translocases. Image 1 • TM segment of Pf3 moves up the YidC greasy slide during membrane insertion. • The N-tail stably contacts the YidC hydrophilic groove after the TM reached the top of the slide. • The Pf3 N-tail can access the groove of YidC even without the membrane potential. • The tracking method can be used in the study of other substrates or translocases. • The Pf3 coat protein forms a helical hairpin during its insertion through YidC. [ABSTRACT FROM AUTHOR]

The Sec translocon provides a polypeptide-conducting channel, which is insulated from the hydrophobic lipidic environment of the membrane, for translocation of hydrophilic passenger polypeptides. Its lateral gate allows a downstream hydrophobic segment (stop-transfer sequence) to exit the channel laterally for integration into the lipid phase. We note that this channel model only partly accounts for the translocon function. The other essential role of translocon is to facilitate de novo insertion of the N-terminal topogenic segment of a substrate polypeptide into the membrane. Recent structural studies suggest that de novo insertion does not use the polypeptide-conducting channel; instead, it takes place directly at the lateral gate, which is prone to opening. We propose that the de novo insertion process, in concept, is similar to that of insertases (such as YidC in bacteria and EMC3 in eukaryotes), in which an intramembrane surface of the machinery provides the halfway point of insertion.

A comparative proteomic analysis was utilized to evaluate similarities and differences in membrane samples derived from the cariogenic bacterium Streptococcus mutans, including the wild-type strain and four mutants devoid of protein translocation machinery components, specifically ∆ffh, ∆yidC1, ∆yidC2, or ∆ffh/yidC1. The purpose of this work was to determine the extent to which the encoded proteins operate individually or in concert with one another and to identify the potential substrates of the respective pathways. Ffh is the principal protein component of the signal recognition particle (SRP), while yidC1 and yidC2 are dual paralogs encoding members of the YidC/Oxa/Alb family of membrane-localized chaperone insertases. Our results suggest that the co-translational SRP pathway works in concert with either YidC1 or YidC2 specifically, or with no preference for paralog, in the insertion of most membrane-localized substrates. A few instances were identified in which the SRP pathway alone, or one of the YidCs alone, appeared to be most relevant. These data shed light on underlying reasons for differing phenotypic consequences of ffh, yidC1 or yidC2 deletion. Our data further suggest that many membrane proteins present in a ∆yidC2 background may be non-functional, that ∆yidC1 is better able to adapt physiologically to the loss of this paralog, that shared phenotypic properties of ∆ffh and ∆yidC2 mutants can stem from impacts on different proteins, and that independent binding to ribosomal proteins is not a primary functional activity of YidC2. Lastly, genomic mutations accumulate in a ∆yidC2 background coincident with phenotypic reversion, including an apparent W138R suppressor mutation within yidC1. [ABSTRACT FROM AUTHOR]

As an essential cofactor of photosystem I and photosystem II, chlorophyll plays a fundamental role in oxygenic photosynthesis. Chlorophyll molecules are responsible for both the absorption of visible light and its photochemical conversion during the process of charge separation. The vast majority of chlorophyll molecules located in photosystems is bound to six core subunits that appear to have a common evolutionary origin. Available data indicate that these large transmembrane proteins are synthesized on membrane-bound ribosomes and inserted into the thylakoid membrane with the assistance of SecY translocase and various protein factors. Newly synthesized chlorophyll-proteins associate with small transmembrane subunits, carotenoids, and other cofactors, and assemble in a stepwise manner into the final functional photosystems. This chapter summarizes our current knowledge of the individual events during photosystem biogenesis: apoprotein translation and membrane insertion, loading of chlorophyll molecules into the synthesized apoproteins, formation of assembly modules, and final assembly into the photosystems. [ABSTRACT FROM AUTHOR]

Abstract Different attributes of membrane protein substrates have been proposed and characterized as translocation-pathway determinants. However, several gaps in our understanding of the mechanism of targeting, insertion, and assembly of inner-membrane proteins exist. Specifically, the role played by hydrophilic N-terminal tails in pathway selection is unclear. In this study, we have evaluated length and charge density as translocase determinants using model proteins. Strikingly, the 36-residue N-tail of 2Pf3–Lep translocates independent of YidC–Sec. This is the longest known substrate of this pathway. We confirmed this using a newly constructed YidC–Sec double-depletion strain. Increasing its N-tail length with uncharged spacer peptides led to YidC dependence and eventually YidC–Sec dependence, hence establishing that length has a linear effect on translocase dependence. Tails longer than 60 residues were not inserted; however, an MBP–2Pf3–Lep fusion protein could be ranslocated. This suggests that longer N-tails can be translocated if it can engage SecA. In addition, we have examined how the positioning of charges within the translocated N-tail affects the insertion pathway. Additional charges can be translocated by the Lep TM when the charges are distributed across a longer N-tail. We tested charge density as a translocase determinant and confirmed that the addition of positive or negatives charges led to a greater dependence on YidC–Sec when they were placed close to each other than away. Findings from this work make an important advance in our existing knowledge about the different insertion mechanisms of membrane proteins in Escherichia coli. Graphical Abstract Unlabelled Image Highlights • An increase in substrate N-terminal length or charge density mandates a switch in its translocase requirement from independent to YidC-only and YidC–Sec. • The mature domain of MBP, if followed by a C-terminal TM segment, can be translocated by the Sec channel. • The YidC–Sec-independent substrate 2Pf3–Lep is not promiscuously inserted by either YidC or Sec in the absence of the other, as confirmed by a double-depletion strain. [ABSTRACT FROM AUTHOR]

The insertion and folding of proteins into membranes is crucial for cell viability. Yet, the detailed contributions of insertases remain elusive. Here, we monitor how the insertase YidC guides the folding of the polytopic melibiose permease MelB into membranes. In vivo experiments using conditionally depleted E. coli strains show that MelB can insert in the absence of SecYEG if YidC resides in the cytoplasmic membrane. In vitro single-molecule force spectroscopy reveals that the MelB substrate itself forms two folding cores from which structural segments insert stepwise into the membrane. However, misfolding dominates, particularly in structural regions that interface the pseudo-symmetric α-helical domains of MelB. Here, YidC takes an important role in accelerating and chaperoning the stepwise insertion and folding process of both MelB folding cores. Our findings reveal a great flexibility of the chaperoning and insertase activity of YidC in the multifaceted folding processes of complex polytopic membrane proteins. [Display omitted] • YidC chaperones membrane insertion of MelB in vivo and in vitro • MelB forms two folding cores related to its structural symmetry • Misfolding dominates in the structural region interfacing both folding cores • YidC chaperones weakly hydrophobic structural segments interfacing the folding cores Blaimschein et al. monitors the folding pathways of single melibiose permeases MelB into the membrane. MelB itself forms two folding cores from which it stepwise inserts and folds into the membrane. However, this self-insertion process is prone to misfolding. It is observed how YidC accelerates and chaperones this folding process. [ABSTRACT FROM AUTHOR]

YidC belongs to an evolutionarily conserved family of insertases, YidC/Oxa1/Alb3, in bacteria, mitochondria, and chloroplasts, respectively. Unlike Gram-negative bacteria, Gram-positives including Streptococcus mutans harbor two paralogs of YidC. The mechanism for paralog-specific phenotypes of bacterial YidC1 versus YidC2 has been partially attributed to the differences in their cytoplasmic domains. However, we previously identified a W138R gain-of-function mutation in the YidC1 transmembrane helix 2. YidC1 W138R mostly phenocopied YidC2, yet the mechanism remained unknown. Primary sequence comparison of streptococcal YidCs led us to identify and mutate the YidC1 W138 analog, YidC2 S152 to W/A, which resulted in a loss of YidC2- and acquisition of YidC1-like phenotype. The predicted lipid-facing side chains of YidC1 W138 /YidC2 S152 led us to propose a role for membrane phospholipids in specific-residue dependent phenotypes of S. mutans YidC paralogs. Cardiolipin (CL), a prevalent phospholipid in the S. mutans cytoplasmic membrane during acid stress, is encoded by a single gene, cls . We show a concerted mechanism for cardiolipin and YidC2 under acid stress based on similarly increased promoter activities and similar elimination phenotypes. Using coarse grain molecular dynamics simulations with the Martini2.2 Forcefield, YidC1 and YidC2 wild-type and mutant interactions with CL were assessed in silico . We observed substantially increased CL interaction in dimeric versus monomeric proteins, and variable CL occupancy in YidC1 and YidC2 mutant constructs that mimicked characteristics of the other wild-type paralog. Hence, paralog-specific amino acid- CL interactions contribute to YidC1 and YidC2-associated phenotypes that can be exchanged by point mutation at positions 138 or 152, respectively., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mishra, van Aalst, Wylie and Brady.)

Abstract YidC/Alb3/Oxa1 family proteins are involved in the insertion and assembly of membrane proteins. The core five transmembrane regions of YidC, which are conserved in the protein family, form a positively charged cavity open to the cytoplasmic side. The cavity plays an important role in membrane protein insertion. In all reported structural studies of YidC, the second cytoplasmic loop (C2 loop) was disordered, limiting the understanding of its role. Here, we determined the crystal structure of YidC including the C2 loop at 2.8 Å resolution with R / R free = 21.8/27.5. This structure and subsequent molecular dynamics simulation indicated that the intrinsic flexible C2 loop covered the positively charged cavity. This crystal structure provides the coordinates of the complete core region including the C2 loop, which is valuable for further analyses of YidC. [ABSTRACT FROM AUTHOR]

About 30% of all bacterial proteins execute their function outside of the cytosol and have to be transported into or across the cytoplasmic membrane. Bacteria use multiple protein transport systems in parallel, but the majority of proteins engage two distinct targeting systems. One is the co-translational targeting by two universally conserved GTPases, the signal recognition particle (SRP) and its receptor FtsY, which deliver inner membrane proteins to either the SecYEG translocon or the YidC insertase for membrane insertion. The other targeting system depends on the ATPase SecA, which targets secretory proteins, i.e. periplasmic and outer membrane proteins, to SecYEG for their subsequent ATP-dependent translocation. While SRP selects its substrates already very early during their synthesis, the recognition of secretory proteins by SecA is believed to occur primarily after translation termination, i.e. post-translationally. In this review we highlight recent progress on how SRP recognizes its substrates at the ribosome and how the fidelity of the targeting reaction to SecYEG is maintained.We furthermore discuss similarities and differences in the SRP-dependent targeting to either SecYEG or YidC and summarize recent results that suggest that some membrane proteins are co-translationally targeted by SecA. [ABSTRACT FROM AUTHOR]

The F 0 c subunit of F 0 F 1 ATPase (F 0 -c) possesses two membrane-spanning stretches with N- and C-termini exposed to the periplasmic (extracellular) side of the cytoplasmic membrane of E. coli . Although F 0 -c insertion has been extensively analyzed in vitro by means of protease protection assaying, it is unclear whether such assays allow elucidation of the insertion process faithfully, since the membrane-protected fragment, an index of membrane insertion, is a full-length polypeptide of F 0 -c, which is the same as the protease-resistant conformation without membrane insertion. We found that the protease-resistant conformation could be discriminated from membrane-insertion by including octyl glucoside on protease digestion. By means of this system, we found that F 0 -c insertion depends on MPIase, a glycolipozyme involved in membrane insertion, and is stimulated by YidC. In addition, we found that acidic phospholipids PG and CL transform F 0 -c into a protease-resistant form, while MPIase prevents the acquisition of such a protease-resistant conformation. [ABSTRACT FROM AUTHOR]

Signal-sequence-dependent protein targeting is essential for the spatiotemporal organization of eukaryotic and prokaryotic cells and is facilitated by dedicated protein targeting factors such as the signal recognition particle (SRP). However, targeting signals are not exclusively contained within proteins but can also be present within mRNAs. By in vivo and in vitro assays, we show that mRNA targeting is controlled by the nucleotide content and by secondary structures within mRNAs. mRNA binding to bacterial membranes occurs independently of soluble targeting factors but is dependent on the SecYEG translocon and YidC. Importantly, membrane insertion of proteins translated from membrane-bound mRNAs occurs independently of the SRP pathway, while the latter is strictly required for proteins translated from cytosolic mRNAs. In summary, our data indicate that mRNA targeting acts in parallel to the canonical SRP-dependent protein targeting and serves as an alternative strategy for safeguarding membrane protein insertion when the SRP pathway is compromised. [Display omitted] • Nucleotide composition and secondary structures determine mRNA targeting • The SecYEG translocon and the YidC insertase act as mRNA receptors in bacteria • mRNA targeting enables SRP-independent membrane protein insertion Protein targeting is generally believed to depend on targeting information that is retained within the protein itself. Sarmah et al. now show that mRNA targeting contributes to membrane protein insertion in bacteria and that it provides an alternative strategy when the canonical signal recognition particle-dependent protein-targeting pathway is saturated or inhibited. [ABSTRACT FROM AUTHOR]

The membrane insertase YidC, is an essential bacterial component and functions in the folding and insertion of many membrane proteins during their biogenesis. It is a multispanning protein in the inner (cytoplasmic) membrane of Escherichia coli that binds its substrates in the "greasy slide" through hydrophobic interaction. The hydrophilic part of the substrate transiently localizes in the groove of YidC before it is translocated into the periplasm. The groove, which is flanked by the greasy slide, is within the center of the membrane, and provides a promising target for inhibitors that would block the insertase function of YidC. In addition, since the greasy slide is available for the binding of various substrates, it could also provide a binding site for inhibitory molecules. In this review we discuss in detail the structure and the mechanism of how YidC interacts not only with its substrates, but also with its partner proteins, the SecYEG translocase and the SRP signal recognition particle. Insight into the substrate binding to the YidC catalytic groove is presented. We wind up the review with the idea that the hydrophilic groove would be a potential site for drug binding and the feasibility of YidC-targeted drug development. Adapted from Ref [12]. [Display omitted] • Understanding the essential YidC functions in membrane protein insertion • During insertion, substrate contacts the YidC greasy slide and hydrophilic groove. • Several Celecoxib compounds target the YidC hydrophilic groove killing Gram-positive bacteria. • Down regulation of YidC sensitizes E. coli to antibacterial oils. • YidC structure can provide a template for rational design of novel antibiotics. [ABSTRACT FROM AUTHOR]

Oxa1/Alb3/YidC family members promote the insertion of proteins into the mitochondrial inner membrane, the chloroplast thylakoid membrane, and the bacterial plasma membrane. Remarkably, two recent studies identify new Oxa1 homologs that reside in the endoplasmic reticulum (ER) and function in ER membrane protein biogenesis. [ABSTRACT FROM AUTHOR]

The integration of most membrane proteins into the cytoplasmic membrane of bacteria occurs co-translationally. The universally conserved YidC protein mediates this process either individually as a membrane protein insertase, or in concert with the SecY complex. Here, we present a structural model of YidC based on evolutionary co-variation analysis, lipid-versus-protein-exposure and molecular dynamics simulations. The model suggests a distinctive arrangement of the conserved five transmembrane domains and a helical hairpin between transmembrane segment 2 (TM2) and TM3 on the cytoplasmic membrane surface. The model was used for docking into a cryo-electron microscopy reconstruction of a translating YidC-ribosome complex carrying the YidC substrate FOc. This structure reveals how a single copy of YidC interacts with the ribosome at the ribosomal tunnel exit and identifies a site for membrane protein insertion at the YidC protein-lipid interface. Together, these data suggest a mechanism for the co-translational mode of YidC-mediated membrane protein insertion.

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants. [ABSTRACT FROM AUTHOR]

YidC, the bacterial homologous protein of Oxa1 and Alb3, could insert membrane proteins into the membrane. Rhodobacter sphaeroides is a kind of photobacteria with abundant intracytoplasmic membranes. In this study, the functions of R. sphaeroides YidC and its C-terminus were investigated in the Escherichia coli YidC gene depletion strain FTL10. The results showed that RS_YidC could complement the growth of the strain FTL10, but the RS_YidC last 5 residues (619–623, KKRKP) deletion mutant could not. Interestingly, the site-directed RS_YidC mutants of any one or all of these 5 residues were still active. The deletion mutant of the last 4 residues and even the last 4 residues deletion mutant with substitution of the Ala or Glu for Lys619 still had sufficient activity to complement the growth of the strain FTL10. These results indicated that the length of the C-terminus of Rs_YidC is more important for its function than the amino acid composition or the charges of it, and the presence of an amino acid residue at position 619 is required for Rs_YidC function in E. coli. Our result also suggests that Rs_YidC may function differently as compared to its homologs. [ABSTRACT FROM AUTHOR]

YidC, a member of the YidC/Oxa1/Alb3 family, inserts proteins into the membrane and facilitates membrane-protein folding in bacteria. YidC plays key roles in both Sec-mediated integration and Sec-independent insertion of membrane proteins. Here, Bacillus halodurans YidC2, which has five transmembrane helices conserved among the other family members, was identified as a target protein for structure determination by a fluorescent size-exclusion chromatography analysis. The protein was overexpressed, purified and crystallized in the lipidic cubic phase. The crystals diffracted X-rays to 2.4 Å resolution and belonged to space group P21, with unit-cell parameters a = 43.9, b = 60.6, c = 58.9 Å, β = 100.3°. The experimental phases were determined by the multiwavelength anomalous diffraction method using a mercury-derivatized crystal. [ABSTRACT FROM AUTHOR]