Sequences determining the cytoplasmic localization of a chemoreceptor domain.

The Escherichia coli serine chemoreceptor (Tsr) is a protein with a simple topology consisting of two membrane-spanning sequences (TM1 and TM2) separating a large periplasmic domain from N-terminal and C-terminal cytoplasmic regions. We analyzed the contributions of several sequence elements to the cytoplasmic localization of the C-terminal domain by using chemoreceptor-alkaline phosphatase gene fusions. The principal findings were as follows. (i) The cytoplasmic localization of the C-terminal domain depended on TM2 but was quite tolerant of mutations partially deleting or introducing charged residues into the sequence. (ii) The basal level of C-terminal domain export was significantly higher in proteins with the wild-type periplasmic domain than in derivatives with a shortened periplasmic domain, suggesting that the large size of the wild-type domain promotes partial membrane misinsertion. (iii) The membrane insertion of deletion derivatives with a single spanning segment (TM1 or TM2) could be controlled by either an adjacent positively charged sequence or an adjacent amphipathic sequence. The results provide evidence that the generation of the Tsr membrane topology is an overdetermined process directed by an interplay of sequences promoting and opposing establishment of the normal structure.     

A C-terminal region of Yersinia pestis YscD binds the outer membrane secretin YscC

YscD is an essential component of the plasmid pCD1-encoded type III secretion system (T3SS) of Yersinia pestis. YscD has a single transmembrane (TM) domain that connects a small N-terminal cytoplasmic region (residues 1 to 121) to a larger periplasmic region (residues 143 to 419). Deletion analyses established that both the N-terminal cytoplasmic region and the C-terminal periplasmic region are required for YscD function. Smaller targeted deletions demonstrated that a predicted cytoplasmic forkhead-associated (FHA) domain is also required to assemble a functional T3SS; in contrast, a predicted periplasmic phospholipid binding (BON) domain and a putative periplasmic "ring-building motif" domain of YscD could be deleted with no significant effect on the T3S process. Although deletion of the putative "ring-building motif" domain did not disrupt T3S activity per se, the calcium-dependent regulation of the T3S apparatus was affected. The extreme C-terminal region of YscD (residues 354 to 419) was essential for secretion activity and had a strong dominant-negative effect on the T3S process when exported to the periplasm of the wild-type parent strain. Coimmunoprecipitation studies demonstrated that this region of YscD mediates the interaction of YscD with the outer membrane YscC secretin complex. Finally, replacement of the YscD TM domain with a TM domain of dissimilar sequence had no effect on the T3S process, indicating that the TM domain has no sequence-specific function in the assembly or function of the T3SS.     

Effects of photosynthetic reaction center H protein domain mutations on photosynthetic properties and reaction center assembly in Rhodobacter sphaeroides

Purple bacterial photosynthetic reaction center (RC) H proteins comprise three cellular domains: an 11 amino acid N-terminal sequence on the periplasmic side of the inner membrane; a single transmembrane alpha-helix; and a large C-terminal, globular cytoplasmic domain. We studied the roles of these domains in Rhodobacter sphaeroides RC function and assembly, using a mutagenesis approach that included domain swapping with Blastochloris viridis RC H segments and a periplasmic domain deletion. All mutations that affected photosynthesis reduced the amount of the RC complex. The RC H periplasmic domain is shown to be involved in the accumulation of the RC H protein in the cell membrane, while the transmembrane domain has an additional role in RC complex assembly, perhaps through interactions with RC M. The RC H cytoplasmic domain also functions in RC complex assembly. There is a correlation between the amounts of membrane-associated RC H and RC L, whereas RC M is found in the cell membrane independently of RC H and RC L. Furthermore, substantial amounts of RC M and RC L are found in the soluble fraction of cells only when RC H is present in the membrane. We suggest that RC M provides a nucleus for RC complex assembly, and that a RC H/M/L assemblage results in a cytoplasmic pool of soluble RC M and RC L proteins to provide precursors for maximal production of the RC complex.     

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.     

Distinct requirements for translocation of the N-tail and C-tail of the Escherichia coli inner membrane protein CyoA

Inner membrane proteins (IMPs) of Escherichia coli use different pathways for membrane targeting and integration. YidC plays an essential but poorly defined role in the integration and folding of IMPs both in conjunction with the Sec translocon and as a Sec-independent insertase. Depletion of YidC only marginally affects the insertion of Sec-dependent IMPs, whereas it blocks the insertion of a subset of Sec-independent IMPs. Substrates of this latter "YidC-only" pathway include the relatively small IMPs M13 procoat, Pf3 coat protein, and subunit c of the F(1)F(0) ATPase. Recently, it has been shown that the steady state level of the larger and more complex CyoA subunit of the cytochrome o oxidase is also severely affected upon depletion of YidC. In the present study we have analyzed the biogenesis of the integral lipoprotein CyoA. Collectively, our data suggest that the first transmembrane segment of CyoA rather than the signal sequence recruits the signal recognition particle for membrane targeting. Membrane integration and assembly appear to occur in two distinct sequential steps. YidC is sufficient to catalyze insertion of the N-terminal domain consisting of the signal sequence, transmembrane segment 1, and the small periplasmic domain in between. Translocation of the large C-terminal periplasmic domain requires the Sec translocon and SecA, suggesting that for this particular IMP the Sec translocon might operate downstream of YidC.     

Thermodynamic aspects of DsbD-mediated electron transport

DsbD from Escherichia coli transports electrons from cytoplasmic thioredoxin across the inner membrane to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of three domains: a periplasmic N-terminal domain, a central transmembrane domain (tmDsbD) and a periplasmic C-terminal domain. Each domain contains two essential cysteine residues that are required for electron transport. In contrast to the quinone reductase DsbB, HPLC analysis of the methanol/hexane extracts of purified DsbD revealed no presence of quinones, suggesting that the tmDsbD interacts with thioredoxin and the periplasmic C-terminal domain exclusively via disulfide exchange. We also demonstrate that a DsbD variant containing only the redox-active cysteine pair C163 and C285 in tmDsbD, reconstituted into liposomes, has a redox potential of − 0.246 V. The results show that all steps in the DsbD-mediated electron flow are thermodynamically favorable.     

The solution structure of the periplasmic domain of the TonB system ExbD protein reveals an unexpected structural homology with siderophore-binding proteins

The transport of iron complexes through outer membrane transporters from Gram-negative bacteria is highly dependent on the TonB system. Together, the three components of the system, TonB, ExbB and ExbD, energize the transport of iron complexes through the outer membrane by utilizing the proton motive force across the cytoplasmic membrane. The three-dimensional (3D) structure of the periplasmic domain of TonB has previously been determined. However, no detailed structural information for the other two components of the TonB system is currently available and their role in the iron-uptake process is not yet clearly understood. ExbD from Escherichia coli contains 141 residues distributed in three domains: a small N-terminal cytoplasmic region, a single transmembrane helix and a C-terminal periplasmic domain. Here we describe the first well-defined solution structure of the periplasmic domain of ExbD (residues 44-141) solved by multidimensional nuclear magnetic resonance (NMR) spectroscopy. The monomeric structure presents three clearly distinct regions: an N-terminal flexible tail (residues 44-63), a well-defined folded region (residues 64-133) followed by a small C-terminal flexible region (residues 134-141). The folded region is formed by two alpha-helices that are located on one side of a single beta-sheet. The central beta-sheet is composed of five beta-strands, with a mixed parallel and antiparallel arrangement. Unexpectedly, this fold closely resembles that found in the C-terminal lobe of the siderophore-binding proteins FhuD and CeuE. The ExbD periplasmic domain has a strong tendency to aggregate in vitro and 3D-TROSY (transverse relaxation optimized spectroscopy) NMR experiments of the deuterated protein indicate that the multimeric protein has nearly identical secondary structure to that of the monomeric form. Chemical shift perturbation studies suggest that the Glu-Pro region (residues 70-83) of TonB can bind weakly to the surface and the flexible C-terminal region of ExbD. At the same time the Lys-Pro region (residues 84-102) and the folded C-terminal domain (residues 150-239) of TonB do not show significant binding to ExbD, suggesting that the main interactions forming the TonB complex occur in the cytoplasmic membrane.     

The N-terminal region of the transmembrane domain of human erythrocyte band 3. Residues critical for membrane insertion and transport activity

We studied the role of the N-terminal region of the transmembrane domain of the human erythrocyte anion exchanger (band 3; residues 361-408) in the insertion, folding, and assembly of the first transmembrane span (TM1) to give rise to a transport-active molecule. We focused on the sequence around the 9-amino acid region deleted in Southeast Asian ovalocytosis (Ala-400 to Ala-408), which gives rise to nonfunctional band 3, and also on the portion of the protein N-terminal to the transmembrane domain (amino acids 361-396). We examined the effects of mutations in these regions on endoplasmic reticulum insertion (using cell-free translation), chloride transport, and cell-surface movement in Xenopus oocytes. We found that the hydrophobic length of TM1 was critical for membrane insertion and that formation of a transport-active structure also depended on the presence of specific amino acid sequences in TM1. Deletions of 2 or 3 amino acids including Pro-403 retained transport activity provided that a polar residue was located 2 or 3 amino acids on the C-terminal side of Asp-399. Finally, deletion of the cytoplasmic surface sequence G(381)LVRD abolished chloride transport, but not surface expression, indicating that this sequence makes an essential structural contribution to the anion transport site of band 3.     

Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ.

Chemoreceptor Trg and osmosensor EnvZ of Escherichia coli share a common transmembrane organization but have essentially unrelated primary structures. We created a hybrid gene coding for a protein in which Trg contributed its periplasmic and transmembrane domains as well as a short cytoplasmic segment and EnvZ contributed its cytoplasmic kinase/phosphatase domain. Trz1 transduced recognition of sugar-occupied, ribose-binding protein by its periplasmic domain into activation of its cytoplasmic kinase/phosphatase domain as assessed in vivo by using an ompC-lacZ fusion gene. Functional coupling of sugar-binding protein recognition to kinase/phosphatase activity indicates shared features of intramolecular signalling in the two parent proteins. In combination with previous documentation of transduction of aspartate recognition by an analogous fusion protein created from chemoreceptor Tar and EnvZ, the data indicate a common mechanism of transmembrane signal transduction by chemoreceptors and EnvZ. Signalling through the fusion proteins implies functional interaction between heterologous domains, but the minimal sequence identity among relevant segments of EnvZ, Tar, and Trg indicates that the link does not require extensive, specific interactions among side chains. The few positions of identity in those three sequences cluster in transmembrane segment 1 and the short chemoreceptor sequence in the cytoplasmic part of the hybrid proteins. These regions may be particularly important in physical and functional coupling. The specific cellular conditions necessary to observe ligand-dependent activation of Trz1 can be understood in the context of the importance of phosphatase control in EnvZ signalling and limitations on maximal receptor occupancy in binding protein-mediated recognition.     

Topological characterization of the essential Escherichia coli cell division protein FtsN.

Genetic and biochemical approaches were used to analyze a topological model for FtsN, a 36-kDa protein with a putative transmembrane segment near the N terminus, and to ascertain the requirements of the putative cytoplasmic and membrane-spanning domains for the function of this protein. Analysis of FtsN-PhoA fusions revealed that the putative transmembrane segment of FtsN could act as a translocation signal. Protease accessibility studies of FtsN in spheroblasts and inverted membrane vesicles confirmed that FtsN had a simple bitopic topology with a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large periplasmic carboxy terminus. To ascertain the functional requirements of the N-terminal segments of FtsN, various constructs were made. Deletion of the N-terminal cytoplasmic and membrane-spanning domains led to intracellular localization of the carboxy domain, instability,and loss of function. Replacement of the N-terminal cytoplasmic and membrane-spanning domains with a membrane-spanning domain from MalG restored subcellular localization and function. These N-terminal domains of FtsN could also be replaced by the cleavable MalE signal sequence with restoration of subcellular localization and function. It is concluded that the N-terminal, cytoplasmic, and transmembrane domains of FtsN are not required for function of the carboxy domain other than to transport it to the periplasm. FtsQ and FtsI were also analyzed.     

Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) – a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide – to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB’s two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45–50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.     

Modeling the transmembrane domain of bacterial chemoreceptors

Bacterial chemoreceptors signal across the membrane by conformational changes that traverse a four-helix transmembrane domain. High-resolution structures are available for the chemoreceptor periplasmic domain and part of the cytoplasmic domain but not for the transmembrane domain. Thus, we constructed molecular models of the transmembrane domains of chemoreceptors Trg and Tar, using coordinates of an unrelated four-helix coiled coil as a template and the X-ray structure of a chemoreceptor periplasmic domain to establish register and positioning. We tested the models using the extensive data for cross-linking propensities between cysteines introduced into adjacent transmembrane helices, and we found that many aspects of the models corresponded with experimental observations. The one striking disparity, the register of transmembrane helix 2 (TM2) relative to its partner transmembrane helix 1, could be corrected by sliding TM2 along its long axis toward the periplasm. The correction implied that axial sliding of TM2, the signaling movement indicated by a large body of data, was of greater magnitude than previously thought. The refined models were used to assess effects of inter-helical disulfides on the two ligand-induced conformational changes observed in alternative crystal structures of periplasmic domains: axial sliding within a subunit and subunit rotation. Analyses using a measure of disulfide potential energy provided strong support for the helical sliding model of transmembrane signaling but indicated that subunit rotation could be involved in other ligand-induced effects. Those analyses plus modeled distances between diagnostic cysteine pairs indicated a magnitude for TM2 sliding in transmembrane signaling of several angstroms.     

Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin

We describe the isolation, characterization, and sequence of cDNA clones encoding one subunit of the complex of membrane glycoproteins that forms part of the transmembrane connection between the extracellular matrix and the cytoskeleton. The cDNA sequence encodes a polypeptide of 89 kd that has features strongly suggesting the presence of a large N-terminal extracellular domain, a single transmembrane segment, and a small C-terminal cytoplasmic domain. The extracellular domain contains a threefold repeat of a novel 40 residue cysteine-rich segment, and the cytoplasmic domain contains a tyrosine residue that is a potential site for phosphorylation by tyrosine kinases. We propose the name integrin for this protein complex to denote its role as an integral membrane complex involved in the transmembrane association between the extracellular matrix and the cytoskeleton.     

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.     

Bioinformatic analysis of the TonB protein family

TonB is a protein prevalent in a large number of Gram-negative bacteria that is believed to be responsible for the energy transduction component in the import of ferric iron complexes and vitamin B₁₂ across the outer membrane. We have analyzed all the TonB proteins that are currently contained in the Entrez database and have identified nine different clusters based on its conserved 90-residue C-terminal domain amino acid sequence. The vast majority of the proteins contained a single predicted cytoplasmic transmembrane domain; however, nine of the TonB proteins encompass a ∼290 amino acid N-terminal extension homologous to the MecR1 protein, which is composed of three additional predicted transmembrane helices. The periplasmic linker region, which is located between the N-terminal domain and the C-terminal domain, is extremely variable both in length (22–283 amino acids) and in proline content, indicating that a Pro-rich domain is not a required feature for all TonB proteins. The secondary structure of the C-terminal domain is found to be well preserved across all families, with the most variable region being between the second α-helix and the third β-strand of the antiparallel β-sheet. The fourth β-strand found in the solution structure of the Escherichia coli TonB C-terminal domain is not a well conserved feature in TonB proteins in most of the clusters. Interestingly, several of the TonB proteins contained two C-terminal domains in series. This analysis provides a framework for future structure-function studies of TonB, and it draws attention to the unusual features of several TonB proteins. Byron C. H. Chu and R. Sean Peacock contributed equally to this work.     

Membrane topology of the beta-subunit of the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae.

The topology of the beta-subunit of the oxaloacetate Na+ pump (OadB) was probed with the alkaline phosphatase (PhoA) and beta-galactosidase (lacZ) fusion technique. Additional evidence for the topology was derived from amino acid alignments and comparative hydropathy profiles of OadB with related proteins. Consistent results were obtained for the three N-terminal and the six C-terminal membrane-spanning alpha-helices. However, the two additional helices that were predicted by hydropathy analyses between the N-terminal and C-terminal blocks did not conform with the fusion results. The analyses were therefore extended by probing the sideness of various engineered cysteine residues with the membrane-impermeant reagent 4-acetamido-4'-maleimidylstilbene-2, 2'-disulfonate. The results were in accord with those of the fusion analyses, suggesting that the protein folds within the membrane by a block of three N-terminal transmembrane segments and another one with six C-terminal transmembrane segments. The mainly hydrophobic connecting segment is predicted not to traverse the membrane fully, but to insert in an undefined manner from the periplasmic face. According to our model, the N-terminus is at the cytoplasmic face and the C-terminus is at the periplasmic face of the membrane.     

Topology-informed strategies for the overexpression and purification of membrane proteins

Membrane proteins represent a significant fraction of all genomes and play key roles in many aspects of biology, but their structural analysis has been hampered by difficulties in large-scale production and crystallisation. To overcome the first of these hurdles, we present here a systematic approach for expression and affinity-tagging which takes into account transmembrane topology. Using a set of bacterial transporters with known topologies, we tested the efficacy of a panel of conventional and Gateway recombinational cloning vectors designed for protein expression under the control of the tac promoter, and for the addition of differing N- and C-terminal affinity tags. For transporters in which both termini are cytoplasmic, C-terminal oligohistidine tagging by recombinational cloning typically yielded functional protein at levels equivalent to or greater than those achieved by conventional cloning. In contrast, it was not effective for examples of the substantial minority of proteins that have one or both termini located on the periplasmic side of the membrane, possibly because of impairment of membrane insertion by the tag and/or att-site-encoded sequences. However, fusion either of an oligohistidine tag to cytoplasmic (but not periplasmic) termini, or of a Strep-tag II peptide to periplasmic termini using conventional cloning vectors did not interfere with membrane insertion, enabling high-level expression of such proteins. In conjunction with use of a C-terminal Lumio fluorescence tag, which we found to be compatible with both periplasmic and cytoplasmic locations, these findings offer a system for strategic planning of construct design for high throughput expression of membrane proteins for structural genomics projects.