The crystal structure of the periplasmic domain of the Escherichia coli membrane protein insertase YidC contains a substrate binding cleft

In bacteria the biogenesis of inner membrane proteins requires targeting and insertion factors such as the signal recognition particle and the Sec translocon. YidC is an essential membrane protein involved in the insertion of inner membrane proteins together with the Sec translocon, but also as a separate entity. YidC of Escherichia coli is a member of the conserved YidC (in bacteria)/Oxa1 (in mitochondria)/Alb3 (in chloroplasts) protein family and contains six transmembrane segments and a large periplasmic domain (P1). We determined the crystal structure of the periplasmic domain of YidC from E. coli (P1D) at 1.8 A resolution. The structure of P1D shows the conserved beta-supersandwich fold of carbohydrate-binding proteins and an alpha-helical linker region at the C terminus that packs against the beta-supersandwich by a highly conserved interface. P1D exhibits an elongated cleft of similar architecture as found in the structural homologs. However, the electrostatic properties and molecular details of the cleft make it unlikely to interact with carbohydrate substrates. The cleft in P1D is occupied by a polyethylene glycol molecule suggesting an elongated peptide or acyl chain as a natural ligand. The region of P1D previously reported to interact with SecF maps to a surface area in the vicinity of the cleft. The conserved C-terminal region of the P1 domain was reported to be essential for the membrane insertase function of YidC. The analysis of this region suggests a role in membrane interaction and/or in the regulation of YidC interaction with binding partners.     

Membrane biogenesis of subunit II of cytochrome bo oxidase: contrasting requirements for insertion of N-terminal and C-terminal domains

The membrane assembly of the respiratory complexes requires the membrane insertases Oxa1 in mitochondria and YidC in bacteria. Oxa1 is responsible for the insertion of the mitochondrial cytochrome c oxidase subunit II (CoxII). Here, we investigated whether YidC, the bacterial Oxa1 homolog, plays a crucial role in the assembly of the bacterial subunit II (CyoA) of cytochrome bo oxidase. CyoA spans the membrane twice and is made with a cleavable signal peptide. We find that translocation of the short N-terminal domain of CyoA is YidC-dependent. In contrast, both the SecA/SecYEG complex and YidC are required for translocation of the large C-terminal domain. By studying the N-terminal domain of CyoA alone, we find that translocation is unaffected when SecE is depleted, suggesting that the YidC insertase on its own catalyzes membrane insertion of the N-terminal region of CyoA. Strikingly, we find that the translocation of the N-terminal domain is a prerequisite for translocation of the C-terminal domain in the full-length CyoA protein because translocation of the large C-terminal domain alone in a truncated CyoA derivative was observed in the absence of YidC. This work shows that the distinct domains of CyoA have different translocation requirements (YidC only and Sec/YidC) and confirms that the membrane biogenesis of subunit II of cytochrome oxidase in bacteria and mitochondria have conserved features.     

The Sec-independent function of Escherichia coli YidC is evolutionary-conserved and essential

YidC plays a role in the integration and assembly of many (if not all) Escherichia coli inner membrane proteins. Strikingly, YidC operates in two distinct pathways: one associated with the Sec translocon that also mediates protein translocation across the inner membrane and one independent from the Sec translocon. YidC is homologous to Alb3 and Oxa1 that function in the integration of proteins into the thylakoid membrane of chloroplasts and inner membrane of mitochondria, respectively. Here, we have expressed the conserved region of yeast Oxa1 in a conditional E. coli yidC mutant. We find that Oxa1 restores growth upon depletion of YidC. Data obtained from in vivo protease protection assays and in vitro cross-linking and folding assays suggest that Oxa1 complements the insertion of Sec-independent proteins but is unable to take over the Sec-associated function of YidC. Together, our data indicate that the Sec-independent function of YidC is conserved and essential for cell growth.     

Pediocin-like antimicrobial peptides (class IIa bacteriocins) and their immunity proteins: biosynthesis, structure, and mode of action.

Pediocin-like antimicrobial peptides (AMPs) form a group of lactic acid bacteria produced, cationic membrane-permeabilizing peptides with 37 to 48 residues. Upon exposure to membrane-mimicking entities, their hydrophilic, cationic, and highly conserved N-terminal region forms a three-stranded antiparallel beta-sheet supported by a conserved disulfide bridge. This N-terminal beta-sheet region is followed by a central amphiphilic alpha-helix and this in most (if not all) of these peptides is followed by a rather extended C-terminal tail that folds back onto the central alpha-helix, thereby creating a hairpin-like structure in the C-terminal half. There is a flexible hinge between the beta-sheet N-terminal region and the hairpin C-terminal region and one thus obtains two domains that may move relative to each other. The cationic N-terminal beta-sheet domain mediates binding of the pediocin-like AMPs to the target-cell surface through electrostatic interactions, while the more hydrophobic and amphiphilic C-terminal hairpin domain penetrates into the hydrophobic part of the target-cell membrane, thereby mediating leakage through the membrane. The hinge provides the structural flexibility that enables the C-terminal hairpin domain to dip into the hydrophobic part of the membrane. Despite extensive sequence similarities, these AMPs differ markedly in their target-cell specificity, and results obtained with hybrid AMPs indicate that the membrane-penetrating hairpin-like C-terminal domain is the major specificity determinant.Bacteria that produce pediocin-like AMPs also produce a 11-kDa cognate immunity protein that protects the producer. The immunity proteins are well-structured, 4-helix bundle cytosolic proteins. They show a high degree of specificity in that they largely recognize and confer immunity only to their cognate AMP and in some cases to a few AMPs that are closely related to their cognate AMP. The C-terminal half of the immunity proteins contains a domain that is involved in specific recognition of the C-terminal membrane-penetrating specificity-determining hairpin domain of the cognate AMP.     

Defining the regions of Escherichia coli YidC that contribute to activity

The YidC/Oxa1/Alb3 family of proteins catalyzes membrane protein insertion in bacteria, mitochondria, and chloroplasts. In this study, we investigated which regions of the bacterial YidC protein are important for its function in membrane protein biogenesis. In Escherichia coli, YidC spans the membrane six times, with a large 319-residue periplasmic domain following the first transmembrane domain. We found that this large periplasmic domain is not required for YidC function and that the residues in the exposed hydrophilic loops or C-terminal tail are not critical for YidC activity. Rather, the five C-terminal transmembrane segments that contain the three consensus sequences in the YidC/Oxa1/Alb3 family are important for its function. However, by systematically replacing all the residues in transmembrane segment (TM) 2, TM3, and TM6 with serine and by swapping TM4 and TM5 with unrelated transmembrane segments, we show that the precise sequence of these transmembrane regions is not essential for in vivo YidC activity. Single serine mutations in TM2, TM3, and TM6 impaired the membrane insertion of the Sec-independent procoat-leader peptidase protein. We propose that the five C-terminal transmembrane segments of YidC function as a platform for the translocating substrate protein to support its insertion into the membrane.     

Nascent Lep inserts into the Escherichia coli inner membrane in the vicinity of YidC, SecY and SecA

Targeting and assembly of the Escherichia coli inner membrane protein leader peptidase (Lep) was studied using a homologous in vitro targeting/translocation assay. Assembly of full-length Lep was efficient in the co-translational presence of membrane vesicles and hardly occurred when membranes were added post-translationally. This is consistent with the signal recognition particle-dependent targeting of Lep. Crosslinking experiments showed that the hydrophilic region P1 of nascent membrane-inserted Lep 100-mer was in the vicinity of SecA and SecY, whereas the first transmembrane domain H1 was in the vicinity of YidC. These results suggested that YidC, together with the Sec translocase, functions in the assembly of Lep. YidC might be a more generic component in the assembly of inner membrane proteins.     

The conserved third transmembrane segment of YidC contacts nascent Escherichia coli inner membrane proteins

Escherichia coli YidC is a polytopic inner membrane protein that plays an essential and versatile role in the biogenesis of inner membrane proteins. YidC functions in Sec-dependent membrane insertion but acts also independently as a separate insertase for certain small membrane proteins. We have used a site-specific cross-linking approach to show that the conserved third transmembrane segment of YidC contacts the transmembrane domains of both nascent Sec-dependent and -independent substrates, indicating a generic recognition of insertion intermediates by YidC. Our data suggest that specific residues of the third YidC transmembrane segment alpha-helix is oriented toward the transmembrane domains of nascent inner membrane proteins that, in contrast, appear quite flexibly positioned at this stage in biogenesis.     

Saccharomyces cerevisiae Cox18 complements the essential Sec-independent function of Escherichia coli YidC

Members of the YidC/Oxa1/Alb3 protein family function in the biogenesis of membrane proteins in bacteria, mitochondria and chloroplasts. In Escherichia coli, YidC plays a key role in the integration and assembly of many inner membrane proteins. Interestingly, YidC functions both in concert with the Sec-translocon and as a separate insertase independent of the translocon. Mitochondria of higher eukaryotes contain two distant homologues of YidC: Oxa1 and Cox18/Oxa2. Oxa1 is required for the insertion of membrane proteins into the mitochondrial inner membrane. Cox18/Oxa2 plays a poorly defined role in the biogenesis of the cytochrome c oxidase complex. Employing a genetic complementation approach by expressing the conserved region of yeast Cox18 in E. coli, we show here that Cox18 is able to complement the essential Sec-independent function of YidC. This identifies Cox18 as a bona fide member of the YidC/Oxa1/Alb3 family.     

Structure of a thrombospondin C-terminal fragment reveals a novel calcium core in the type 3 repeats

Thrombospondins (TSPs) are extracellular regulators of cell-matrix interactions and cell phenotype. The most highly conserved region of all TSPs are the calcium-binding type 3 (T3) repeats and the C-terminal globular domain (CTD). The crystal structure of a cell-binding TSP-1 fragment, spanning three T3 repeats and the CTD, reveals a compact assembly. The T3 repeats lack secondary structure and are organised around a core of calcium ions; two DxDxDGxxDxxD motifs per repeat each encapsulate two calcium ions in a novel arrangement. The CTD forms a lectin-like beta-sandwich and contains four strictly conserved calcium-binding sites. Disruption of the hairpin structure of T3 repeats 6 and 7 decreases protein secretion and stability. The availability for cell attachment of an RGD motif in T3 repeat 7 is modulated by calcium loading. The central architectural role of calcium explains how it is critical for the functions of the TSP C-terminal region. Mutations in the T3 repeats of TSP-5/COMP, which cause two human skeletal disorders, are predicted to disrupt the tertiary structure of the T3-CTD assembly.     

Interaction of Streptococcus mutans YidC1 and YidC2 with Translating and Nontranslating Ribosomes

The YidC/OxaI/Alb3 family of membrane proteins is involved in the biogenesis of integral membrane proteins in bacteria, mitochondria, and chloroplasts. Gram-positive bacteria often contain multiple YidC paralogs that can be subdivided into two major classes, namely, YidC1 and YidC2. The Streptococcus mutans YidC1 and YidC2 proteins possess C-terminal tails that differ in charges (+9 and + 14) and lengths (33 and 61 amino acids). The longer YidC2 C terminus bears a resemblance to the C-terminal ribosome-binding domain of the mitochondrial OxaI protein and, in contrast to the shorter YidC1 C terminus, can mediate the interaction with mitochondrial ribosomes. These observations have led to the suggestion that YidC1 and YidC2 differ in their abilities to interact with ribosomes. However, the interaction with bacterial translating ribosomes has never been addressed. Here we demonstrate that Escherichia coli ribosomes are able to interact with both YidC1 and YidC2. The interaction is stimulated by the presence of a nascent membrane protein substrate and abolished upon deletion of the C-terminal tail, which also abrogates the YidC-dependent membrane insertion of subunit c of the F₁F₀-ATPase into the membrane. It is concluded that both YidC1 and YidC2 interact with ribosomes, suggesting that the modes of membrane insertion by these membrane insertases are similar.     

Evolution of YidC/Oxa1/Alb3 insertases: three independent gene duplications followed by functional specialization in bacteria, mitochondria and chloroplasts

Members of the YidC/Oxa1/Alb3 protein family facilitate the insertion, folding and assembly of proteins of the inner membranes of bacteria and mitochondria and the thylakoid membrane of plastids. All homologs share a conserved hydrophobic core region comprising five transmembrane domains. On the basis of phylogenetic analyses, six subgroups of the family can be distinguished which presumably arose from three independent gene duplications followed by functional specialization. During evolution of bacteria, mitochondria and chloroplasts, subgroup-specific regions were added to the core domain to facilitate the association with ribosomes or other components contributing to the substrate spectrum of YidC/Oxa1/Alb3 proteins.     

Evolution of mitochondrial oxa proteins from bacterial YidC. Inherited and acquired functions of a conserved protein insertion machinery

Members of the Oxa1/YidC family are involved in the biogenesis of membrane proteins. In bacteria, YidC catalyzes the insertion and assembly of proteins of the inner membrane. Mitochondria of animals, fungi, and plants harbor two distant homologues of YidC, Oxa1 and Cox18/Oxa2. Oxa1 plays a pivotal role in the integration of mitochondrial translation products into the inner membrane of mitochondria. It contains a C-terminal ribosome-binding domain that physically interacts with mitochondrial ribosomes to facilitate the co-translational insertion of nascent membrane proteins. The molecular function of Cox18/Oxa2 is not well understood. Employing a functional complementation approach with mitochondria-targeted versions of YidC we show that YidC is able to functionally replace both Oxa1 and Cox18/Oxa2. However, to integrate mitochondrial translation products into the inner membrane of mitochondria, the ribosome-binding domain of Oxa1 has to be appended onto YidC. On the contrary, the fusion of the ribosome-binding domain onto YidC prevents its ability to complement COX18 mutants suggesting an indispensable post-translational activity of Cox18/Oxa2. Our observations suggest that during evolution of mitochondria from their bacterial ancestors the two descendents of YidC functionally segregated to perform two distinct activities, one co-translational and one post-translational.     

The Role of the Strictly Conserved Positively Charged Residue Differs among the Gram-positive,Gram-negative,and Chloroplast YidC Homologs

Recently, the structure of YidC2 from Bacillus halodurans revealed that the conserved positively charged residue within transmembrane segment one (at position 72) is located in a hydrophilic groove that is embedded in the inner leaflet of the lipid bilayer. The arginine residue was essential for the Bacillus subtilis SpoIIIJ (YidC1) to insert MifM and to complement a SpoIIIJ mutant strain. Here, we investigated the importance of the conserved positively charged residue for the function of the Escherichia coli YidC, Streptococcus mutans YidC2, and the chloroplast Arabidopsis thaliana Alb3. Like the Gram-positive B. subtilis SpoIIIJ, the conserved arginine was required for functioning of the Gram-positive S. mutans YidC2 and was necessary to complement the E. coli YidC depletion strain and to promote insertion of a YidC-dependent membrane protein synthesized with one but not two hydrophobic segments. In contrast, the conserved positively charged residue was not required for the E. coli YidC or the A. thaliana Alb3 to functionally complement the E. coli YidC depletion strain or to promote insertion of YidC-dependent membrane proteins. Our results also show that the C-terminal half of the helical hairpin structure in cytoplasmic loop C1 is important for the activity of YidC because various deletions in the region either eliminate or impair YidC function. The results here underscore the importance of the cytoplasmic hairpin region for YidC and show that the arginine is critical for the tested Gram-positive YidC homolog but is not essential for the tested Gram-negative and chloroplast YidC homologs.     

The solution structure of a domain from the Neisseria meningitidis lipoprotein PilP reveals a new beta-sandwich fold

Type IV pili are long, thin fibres, which extend from the surface of the bacterial pathogen Neisseria meningitidis; they play a key role in adhesion and colonisation of host cells. PilP is a lipoprotein, suggested to be involved in the assembly and stabilization of an outer membrane protein, PilQ, which is required for pilus formation. Here we describe the expression of a recombinant fragment of PilP, spanning residues 20 to 181, and determination of the solution structure of a folded domain, spanning residues 85 to 163, by NMR. The N-terminal third of the protein, from residues 20 to 84, is apparently unfolded. Protease digestion yielded a 113 residue fragment that contained the folded domain. The domain adopts a simple beta-sandwich type fold, consisting of a three-stranded beta-sheet packed against a four-stranded beta-sheet. There is also a short segment of 3(10) helix at the N-terminal part of the folded domain. We were unable to identify any other proteins that are closely related in structure to the PilP domain, although the fold appears to be distantly related to the lipocalin family. Over 40 homologues of PilP have been identified in Gram-negative bacteria and the majority of conserved residues lie within the folded domain. The fourth beta-strand and adjacent loop regions contain a high proportion of conserved residues, including three glycine residues, which seem to play a role in linking the two beta-sheets. The two beta-sheets pack together to form a crevice, lined with conserved hydrophobic residues: we suggest that this feature could act as a binding site for a small ligand. The results show that PilP and its homologues have a conserved, folded domain at the C-terminal end of the protein that may be involved in mediating binding to hydrophobic ligands.     

Crystal structure of the RIM1alpha C2B domain at 1.7 A resolution

RIM proteins play critical roles in synaptic vesicle priming and diverse forms of presynaptic plasticity. The C-terminal C2B domain is the only module that is common to all RIMs but is only distantly related to well-studied C2 domains, and its three-dimensional structure and interactions have not been characterized in detail. Using NMR spectroscopy, we now show that N- and C-terminal extensions beyond the predicted C2B domain core sequence are necessary to form a folded, stable RIM1alpha C2B domain. We also find that the isolated RIM1alpha C2B domain is not sufficient for previously described protein-protein interactions involving the RIM1alpha C-terminus, suggesting that additional sequences adjacent to the C2B domain might be required for these interactions. However, analytical ultracentrifugation shows that the RIM1alpha C2B domain forms weak dimers in solution. The crystal structure of the RIM1alpha C2B domain dimer at 1.7 A resolution reveals that it forms a beta-sandwich characteristic of C2 domains and that the unique N- and C-terminal extensions form a small subdomain that packs against the beta-sandwich and mediates dimerization. Our results provide a structural basis to understand the function of RIM C2B domains and suggest that dimerization may be a crucial aspect of RIM function.     

The C Terminus of the Alb3 Membrane Insertase Recruits cpSRP43 to the Thylakoid Membrane

The YidC/Oxa1/Alb3 family of membrane proteins controls the insertion and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here we describe the molecular mechanisms underlying the interaction of Alb3 with the chloroplast signal recognition particle (cpSRP). The Alb3 C-terminal domain (A3CT) is intrinsically disordered and recruits cpSRP to the thylakoid membrane by a coupled binding and folding mechanism. Two conserved, positively charged motifs reminiscent of chromodomain interaction motifs in histone tails are identified in A3CT that are essential for the Alb3-cpSRP43 interaction. They are absent in the C-terminal domain of Alb4, which therefore does not interact with cpSRP43. Chromodomain 2 in cpSRP43 appears as a central binding platform that can interact simultaneously with A3CT and cpSRP54. The observed negative cooperativity of the two binding events provides the first insights into cargo release at the thylakoid membrane. Taken together, our data show how Alb3 participates in cpSRP-dependent membrane targeting, and our data provide a molecular explanation why Alb4 cannot compensate for the loss of Alb3. Oxa1 and YidC utilize their positively charged, C-terminal domains for ribosome interaction in co-translational targeting. Alb3 is adapted for the chloroplast-specific Alb3-cpSRP43 interaction in post-translational targeting by extending the spectrum of chromodomain interactions.     

Identification of Bacillus subtilis YidC Substrates Using a MifM-instructed Translation Arrest-based Reporter

YidC is a member of the YidC/Oxa1/Alb3 protein family that is crucial for membrane protein biogenesis in the bacterial plasma membrane. While YidC facilitates the folding and complex assembly of membrane proteins along with the Sec translocon, it also functions as a Sec-independent membrane protein insertase in the YidC-only pathway. However, little is known about how membrane proteins are recognized and sorted by these pathways, especially in Gram-positive bacteria, for which only a small number of YidC substrates have been identified to date. In this study, we aimed to identify Bacillus subtilis membrane proteins whose membrane insertion depends on SpoIIIJ, the primary YidC homolog in B. subtilis. We took advantage of the translation arrest sequence of MifM, which can monitor YidC-dependent membrane insertion. Our systematic screening identified eight membrane proteins as candidate SpoIIIJ substrates. Results of our genetic study also suggest that the conserved arginine in the hydrophilic groove of SpoIIIJ is crucial for the membrane insertion of the substrates identified here. However, in contrast to MifM, a previously identified YidC substrate, the importance of the negatively charged residue on the substrates for membrane insertion varied depending on the substrate. These results suggest that B. subtilis YidC uses substrate-specific interactions to facilitate membrane insertion.     

Structure of the chromatin binding (chromo) domain from mouse modifier protein 1.

The structure of a chromatin binding domain from mouse chromatin modifier protein 1 (MoMOD1) was determined using nuclear magnetic resonance (NMR) spectroscopy. The protein consists of an N-terminal three-stranded anti-parallel beta-sheet which folds against a C-terminal alpha-helix. The structure reveals an unexpected homology to two archaebacterial DNA binding proteins which are also involved in chromatin structure. Structural comparisons suggest that chromo domains, of which more than 40 are now known, act as protein interaction motifs and that the MoMOD1 protein acts as an adaptor mediating interactions between different proteins.     

Oxal/Alb3/YidC system for insertion of membrane proteins in mitochondria,chloroplasts and bacteria (Review)

Recent studies have shown that there is a pathway that is evolutionarily conserved for the insertion of proteins into the membrane in mitochondria, chloroplasts, and bacteria. In this pathway, the Oxa1/Alb3/YidC proteins are believed to function as membrane insertases that play an important role in the membrane protein biogenesis of respiratory and energy transduction proteins. Additional roles of the Oxa1/Alb3/YidC members may be in the lateral integration of proteins into the lipid bilayer, and in the folding and assembly of proteins into membrane protein complexes.     

Oxa1/Alb3/YidC system for insertion of membrane proteins in mitochondria, chloroplasts and bacteria (review)

Recent studies have shown that there is a pathway that is evolutionarily conserved for the insertion of proteins into the membrane in mitochondria, chloroplasts, and bacteria. In this pathway, the Oxa1/Alb3/YidC proteins are believed to function as membrane insertases that play an important role in the membrane protein biogenesis of respiratory and energy transduction proteins. Additional roles of the Oxa1/Alb3/YidC members may be in the lateral integration of proteins into the lipid bilayer, and in the folding and assembly of proteins into membrane protein complexes.