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.     

Substrate-dependent conformational dynamics of the Escherichia coli membrane insertase YidC

The binding of Pf3 coat protein to the membrane insertase YidC from Escherichia coli induces a conformational change in the tertiary structure of the insertase, resulting in a quenching of the intrinsic tryptophan (Trp) fluorescence. Tryptophan mutants of YidC were generated to examine such conformational movements in detail with time-resolved and steady-state fluorescence spectroscopy. Ten of the 11 Trp residues within YidC were substituted to phenylalanines generating single Trp mutants either at position 354, 454, or 508. In addition, a double mutant with Trp residues at 332 and 334 was studied. Purified YidC mutants were reconstituted into DOPC/DOPG vesicles and titrated with a Trp-free mutant of Pf3 coat, enabling a detailed conformational study of the periplasmic P1, P2, and P3 domains of YidC before and after binding of substrate. Time-resolved fluorescence anisotropy revealed that the mobility of the residues W332/W334 and W508 was considerably increased after binding of Pf3 coat to the insertase. Furthermore, analysis of the fluorescence emission spectra and the decay times showed that all Trp residues are embedded in an equivalent environment that is a membrane/water interface.     

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.     

The Pco proteins are involved in periplasmic copper handling in Escherichia coli

The interactions between the plasmid-borne copper resistance determinant, pco, and the main copper export system in Escherichia coli have been investigated and no direct interaction has been found. The PcoE and PcoC proteins are periplasmic and PcoC binds one Cu ion per protein molecule. PcoA is also periplasmic and can substitute for the chromosomally encoded CueO protein. The pco determinant is proposed to exert its effect through periplasmic handling of excess copper ions and to increase the level of resistance to copper ions above that conferred by copA alone.     

Two regions of mature periplasmic maltose-binding protein of Escherichia coli involved in secretion.

Six mutations in malE, the structural gene for the periplasmic maltose-binding protein (MBP) from Escherichia coli, prevent growth on maltose as a carbon source, as well as release of the mutant proteins by the cold osmotic-shock procedure. These mutations correspond to insertion of an oligonucleotide linker, concomitant with a deletion. One of the mutations (malE127) affects the N-terminal extension (the signal peptide), whereas the five others lie within the mature protein. As expected, the export of protein MalE127 is blocked at an early stage. This protein is neither processed to maturity nor sensitive to proteinase K in spheroplasts. In contrast, in the five other mutants, the signal peptide is cleaved and the protein is accessible to proteinase K added to spheroplasts. This indicates that the five mutant proteins are, at least in part, exported through the inner membrane. We propose that the corresponding mutations define two regions of the mature protein (between residues 18 and 42 and between residues 280 and 306), which are important for release of the protein from the inner membrane into the periplasm. We discuss the results in terms of possible conformational changes at this late step of export to the periplasm.     

Distinct regions of the colicin A translocation domain are involved in the interaction with TolA and TolB proteins upon import into Escherichia coli

Group A colicins need proteins of the Escherichia coli envelope Tol complex (TolA, TolB, TolQ and TolR) to reach their cellular target. The N-terminal domain of colicins is involved in the import process. The N-terminal domains of colicins A and E1 have been shown to interact with TolA, and the N-terminal domain of colicin E3 has been shown to interact with TolB. We found that a pentapeptide conserved in the N-terminal domain of all group A colicins, the 'TolA box', was important for colicin A import but was not involved in the colicin A–TolA interaction. It was, however, involved in the colicin A–TolB interaction. The interactions of colicin A N-terminal domain deletion mutants with TolA and TolB were investigated. Random mutagenesis was performed on a construct allowing the colicin A N-terminal domain to be exported in the bacteria periplasm. This enabled us to select mutant protein domains unable to compete with the wild-type domain of the entire colicin A for import into the cells. Our results demonstrate that different regions of the colicin A N-terminal domain interact with TolA and TolB. The colicin A N-terminal domain was also shown to form a trimeric complex with TolA and TolB.     

YidC is involved in the biogenesis of anaerobic respiratory complexes in the inner membrane of Escherichia coli

YidC of Escherichia coli belongs to the evolutionarily conserved Oxa1/Alb3/YidC family. Members of this family have all been implicated in membrane protein biogenesis of aerobic respiratory and energy-transducing proteins. YidC is essential for the insertion of subunit c of the F(1)F(0)-ATP synthase and subunit a of cytochrome o oxidase. The aim of this study was to investigate whether YidC plays a role during anaerobic growth of Escherichia coli, specifically when either nitrate or fumarate are used as terminal electron acceptors or under fermentative conditions. The effect of YidC depletion on the growth, enzyme activities, and protein levels in the inner membrane was determined. YidC is essential for all anaerobic growth conditions tested, and this is not because of the decreased levels of F(1)F(0)-ATP synthase in the inner membrane only. The results suggest a role for YidC in the membrane biogenesis of integral membrane parts of the anaerobic respiratory chain.     

Crystal structure of the major periplasmic domain of the bacterial membrane protein assembly facilitator YidC

The essential bacterial membrane protein YidC facilitates insertion and assembly of proteins destined for integration into the inner membrane. It has homologues in both mitochondria and chloroplasts. Here we report the crystal structure of the Escherichia coli YidC major periplasmic domain (YidCECP1) at 2.5A resolution. This domain is present in YidC from Gram-negative bacteria and is more than half the size of the full-length protein. The structure reveals that YidCECP1 is made up of a large twisted beta-sandwich protein fold with a C-terminal alpha-helix that packs against one face of the beta-sandwich. Our structure and sequence analysis reveals that the C-terminal alpha-helix and the beta-sheet that it lays against are the most conserved regions of the domain. The region corresponding to the C-terminal alpha-helix was previously shown to be important for the protein insertase function of YidC and is conserved in other YidC-like proteins. The structure reveals that a region of YidC that was previously shown to be involved in binding to SecF maps to one edge of the beta-sandwich. Electrostatic analysis of the molecular surface for this region of YidC reveals a predominantly charged surface and suggests that the SecF-YidC interaction may be electrostatic in nature. Interestingly, YidCECP1 has significant structural similarity to galactose mutarotase from Lactococcus lactis, suggesting that this domain may have another function besides its role in membrane protein assembly.     

Lipopolysaccharide regions involved in the activation of Escherichia coli outer membrane protease OmpT.

OmpT is an integral outer membrane protease of Escherichia coli. Overexpression of OmpT in E. coli and subsequent in vitro folding of the produced inclusion bodies yielded protein with a native-like structure. However, enzymatically active protease was only obtained after addition of the outer membrane lipid lipopolysaccharide (LPS). OmpT is the first example of an enzyme that requires LPS for activity. In this study, we investigated the nature of this activation. Circular dichroism analysis showed that binding of LPS did not lead to large structural changes. Titration of OmpT with LPS and determining the resulting OmpT activity with a fluorimetric assay yielded a dissociation constant of 10-4 m for E. coli K-12 LPS. Determining the dissociation constants for different LPS chemotypes revealed that a fully acylated lipid A part is minimally required for activation of OmpT. The heptose-bound phosphates in the inner core region were also important for activation. The affinity for LPS was not dependent on the concentration of substrate, neither was affinity for the substrate influenced by the concentration of LPS. This indicated that LPS most likely does not act at the level of substrate binding. We hypothesize that LPS induces a subtle conformational change in the protein that is required for obtaining a native active site geometry.     

Synthesis of the nonconserved dihydroorotase domain of the multifunctional hamster CAD protein in Escherichia coli.

CAD is the multifunctional protein of higher eukaryotes which catalyzes the first three steps of pyrimidine biosynthesis. Its enzymatic activities exist as independent domains in the order: N terminus-carbamylphosphate synthetase II(CPSase)-dihydroorotase(DHOase)-aspartate transcarbamylase(ATCase)-C terminus. To functionally define the minimum hamster cDNA region required to encode an active DHOase, expression constructs were generated. Many such constructs complement Escherichia coli mutants defective not only in DHOase but also in ATCase. Constructs deleted for most of the sequence encoding the ATCase domain continue to complement E. coli mutants defective in DHOase. All of these smaller constructs also lack the region encoding CPSase. Therefore, a 'genetic cassette', containing information for neither the CPSase nor the ATCase domain, can direct the synthesis of a polypeptide with DHOase activity. Interestingly, inclusion of a portion of the DHOase-ATCase interdomain bridge appears to be required for optimum activity.     

A novel periplasmic carrier protein involved in the sorting and transport of Escherichia coli lipoproteins destined for the outer membrane.

Lipoproteins are localized in the outer or inner membrane of Escherichia coli, depending on the species of amino acid located next to the N-terminal fatty acylated Cys. The major outer membrane lipoprotein (Lpp) expressed in spheroplasts was, however, retained in the inner membrane as a mature form. A novel protein that is essential for the release of Lpp from the inner membrane was discovered in the periplasm and purified. The partial amino acid sequence of this 20 kDa protein (p20) was determined and used to clone a gene for p20. Sequencing of the gene revealed that p20 is synthesized as a precursor with a signal sequence. p20 formed a soluble complex only with outer membrane-directed lipoproteins such as Lpp, indicating that p20 plays a critical role in the sorting of lipoproteins. Lpp released from the inner membrane in the presence of p20 was specifically assembled into the outer membrane in vitro. These results indicate that p20 is a periplasmic carrier protein involved in the translocation of lipoproteins from the inner to the outer membrane.     

A hydrophilic microenvironment in the substrate-translocating groove of the YidC membrane insertase is essential for enzyme function

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.     

The large first periplasmic loop of SecD and SecF plays an important role in SecDF functioning

A remarkable feature of proteins of the SecD and SecF family involved in protein translocation is that they possess a very large first periplasmic domain. Here we report that this large first periplasmic domain is not required for the SecD-SecF interaction but that it is important for catalyzing protein translocation.     

Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles

Gram-negative bacteria shed outer membrane vesicles composed of outer membrane and periplasmic components. Since vesicles from pathogenic bacteria contain virulence factors and have been shown to interact with eukaryotic cells, it has been proposed that vesicles behave as delivery vehicles. We wanted to determine whether heterologously expressed proteins would be incorporated into the membrane and lumen of vesicles and whether these altered vesicles would associate with host cells. Ail, an outer membrane adhesin/invasin from Yersinia enterocolitica, was detected in purified outer membrane and in vesicles from Escherichia coli strains DH5alpha, HB101, and MC4100 transformed with plasmid-encoded Ail. In vesicle-host cell co-incubation assays we found that vesicles containing Ail were internalized by eukaryotic cells, unlike vesicles without Ail. To determine whether lumenal vesicle contents could be modified and delivered to host cells, we used periplasmically expressed green fluorescent protein (GFP). GFP fused with the Tat signal sequence was secreted into the periplasm via the twin arginine transporter (Tat) in both the laboratory E. coli strain DH5alpha and the pathogenic enterotoxigenic E. coli ATCC strain 43886. Pronase-resistant fluorescence was detectable in vesicles from Tat-GFP-transformed strains, demonstrating that GFP was inside intact vesicles. Inclusion of GFP cargo increased vesicle density but did not result in morphological changes in vesicles. These studies are the first to demonstrate the incorporation of heterologously expressed outer membrane and periplasmic proteins into bacterial vesicles.     

A conserved domain in Escherichia coli Lon protease is involved in substrate discriminator activity

Lon protease of Escherichia coli regulates a diverse set of physiological responses including cell division, capsule production, plasmid stability, and phage replication. Little is known about the mechanism of substrate recognition by Lon. To examine the interaction of Lon with two of its substrates, RcsA and SulA, we generated point mutations in lon which affected its substrate specificity. The most informative lon mutant overproduced capsular polysaccharide (RcsA stabilized) yet was resistant to DNA-damaging agents (SulA degraded). Immunoblots revealed that RcsA protein persisted in this mutant whereas SulA protein was rapidly degraded. The mutant contains a single-base change within lon leading to a single amino acid change of glutamate 240 to lysine. E240 is conserved among all Lon isolates and resides in a charged domain that has a high probability of adopting a coiled-coil conformation. This conformation, implicated in mediating protein-protein interactions, appears to confer substrate discriminator activity on Lon. We propose a model suggesting that this coiled-coil domain represents the discriminator site of Lon.     

Nuclear magnetic resonance solution structure of the periplasmic signalling domain of the TonB-dependent outer membrane transporter FecA from Escherichia coli

Gram-negative bacteria possess outer membrane receptors that utilize energy provided by the TonB system to take up iron. Several of these receptors participate in extracytoplasmic factor (ECF) signalling through an N-terminal signalling domain that interacts with a periplasmic transmembrane anti-sigma factor protein and a cytoplasmic sigma factor protein. The structures of the intact TonB-dependent outer membrane receptor FecA from Escherichia coli and FpvA from Pseudomonas aeruginosa have recently been solved by protein crystallography; however, no electron density was detected for their periplasmic signalling domains, suggesting that it was either unfolded or flexible with respect to the remainder of the protein. Here we describe the well-defined solution structure of this domain solved by multidimensional nuclear magnetic resonance (NMR) spectroscopy. The monomeric protein construct contains the 79-residue N-terminal domain as well as the next 17 residues that are part of the receptor's plug domain. These form two clearly distinct regions: a highly structured domain at the N-terminal end followed by an extended flexible tail at the C-terminal end, which includes the 'TonB-box' region, and connects it to the plug domain of the receptor. The structured region consists of two alpha-helices that are positioned side by side and are sandwiched in between two small beta-sheets. This is a novel protein fold which appears to be preserved in all the periplasmic signalling domains of bacterial TonB-dependent outer membrane receptors that are involved in ECF signalling, because the hydrophobic residues that make up the core of the protein domain are highly conserved.     

YidC family members are involved in the membrane insertion, lateral integration, folding, and assembly of membrane proteins

Members of the YidC family exist in all three domains of life, where they control the assembly of a large variety of membrane protein complexes that function as transporters, energy devices, or sensor proteins. Recent studies in bacteria have shown that YidC functions on its own as a membrane protein insertase independent of the Sec protein-conducting channel. YidC can also assist in the lateral integration and folding of membrane proteins that insert into the membrane via the Sec pathway.     

SecF stabilizes SecD and SecY, components of the protein translocation machinery of the Escherichia coli cytoplasmic membrane.

The effect of the overproduction of SecF encoded by the tac-secF gene on a plasmid on the synthesis of other Sec proteins was studied in Escherichia coli. SecF overproduction resulted in the simultaneous overproduction of SecD encoded by the tac-secD gene on a plasmid. Deletion of the orf6 gene, located downstream of the secF gene, had no effect on SecD overproduction. A pulse-chase experiment revealed that the overproduction was due to stabilization of SecD with SecF. SecF overproduction also resulted in the overproduction of SecY encoded by the tac-secY gene on a plasmid as well. SecF overproduction also enhanced the level of SecY expressed by the chromosomal secY gene. This SecF effect was not due to its effect on SecD or SecE, since SecF overproduction did not affect the levels of SecD and SecE expressed by the chromosomal secD and secE genes, respectively. SecE-dependent overproduction of SecY has already been demonstrated. It is suggested that SecF interacts with both SecD and SecY. SecE-SecY interaction has been demonstrated. It is likely, therefore, that all Sec proteins in the cytoplasmic membrane interact with each other.     

The ribosome and YidC. New insights into the biogenesis of Escherichia coli inner membrane proteins

In the bacterium Escherichia coli, inner membrane proteins (IMPs) are generally targeted through the signal recognition particle pathway to the Sec translocon, which is capable of both linear transport into the periplasm and lateral transport into the lipid bilayer. Lateral transport seems to be assisted by the IMP YidC. In this article, we discuss recent observations that point to a key role for the ribosome in IMP targeting and to the diverse roles of YidC in IMP assembly.     

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.