Structural and functional studies on the N-terminal domain of the Shigella type III secretion protein MxiG

MxiG is a single-pass membrane protein that oligomerizes within the inner membrane ring of the Shigella flexneri type III secretion system (T3SS). The MxiG N-terminal domain (MxiG-N) is the predominant cytoplasmic structure; however, its role in T3SS assembly and secretion is largely uncharacterized. We have determined the solution structure of MxiG-N residues 6-112 (MxiG-N(6-112)), representing the first published structure of this T3SS domain. The structure shows strong structural homology to forkhead-associated (FHA) domains. Canonically, these cell-signaling modules bind phosphothreonine (Thr(P)) via highly conserved residues. However, the putative phosphate-binding pocket of MxiG-N(6-112) does not align with other FHA domain structures or interact with Thr(P). Furthermore, mutagenesis of potential phosphate-binding residues has no effect on S. flexneri T3SS assembly and function. Therefore, MxiG-N has a novel function for an FHA domain. Positioning of MxiG-N(6-112) within the EM density of the S. flexneri needle complex gives insight into the ambiguous stoichiometry of the T3SS, supporting models with 24 MxiG subunits in the inner membrane ring.     

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.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C resonance assignments and secondary structure of PulG,the major pseudopilin from <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> type 2 secretion system

Bacteria use complex transporters to secrete functionally relevant proteins to the extracellular medium. The type 2 secretion system (T2SS) translocates folded proteins involved in bacterial nutrient acquisition, virulence and adaptation. The T2SS pseudopilus is a periplasmic filament, assembled by the polymerization of PulG subunits, the major pseudopilin. Pseudopilin proteins have a conserved N-terminal hydrophobic segment followed by a more variable C-terminal periplasmic and globular domain. To better understand the mechanism of assembly and function of the T2SS, we have been studying the structure and dynamics of PulG by NMR, as well as its interaction with other components of the secretion machinery. As a first step on this study, here we reported the chemical shift assignments of PulG C-terminal domain and its secondary structure prediction based on NMR data.     

Tandem translation generates a chaperone for the Salmonella type III secretion system protein SsaQ

Type III secretion systems (T3SSs) of bacterial pathogens involve the assembly of a surface-localized needle complex, through which translocon proteins are secreted to form a pore in the eukaryotic cell membrane. This enables the transfer of effector proteins from the bacterial cytoplasm to the host cell. A structure known as the C-ring is thought to have a crucial role in secretion by acting as a cytoplasmic sorting platform at the base of the T3SS. Here, we studied SsaQ, an FliN-like putative C-ring protein of the Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS. ssaQ produces two proteins by tandem translation: a long form (SsaQ(L)) composed of 322 amino acids and a shorter protein (SsaQ(S)) comprising the C-terminal 106 residues of SsaQ(L). SsaQ(L) is essential for SPI-2 T3SS function. Loss of SsaQ(S) impairs the function of the T3SS both ex vivo and in vivo. SsaQ(S) binds to its corresponding region within SsaQ(L) and stabilizes the larger protein. Therefore, SsaQ(L) function is optimized by a novel chaperone-like protein, produced by tandem translation from its own mRNA species.     

The extended structure of the periplasmic region of CdsD,a structural protein of the type III secretion system of Chlamydia trachomatis

The type III secretion system (T3SS) is required for the virulence of many gram‐negative bacterial human pathogens. It is composed of several structural proteins, forming the secretion needle and its basis, the basal body. In Chlamydia spp., the T3SS inner membrane ring (IM‐ring) of the basal body is formed by the periplasmic part of CdsD (outer ring) and CdsJ (inner ring). Here we describe the crystal structure of the C‐terminal, periplasmic part of CdsD, not including the last 60 residues. Two crystal forms were obtained, grown in three different crystallization conditions. In both crystal forms there is one molecule per asymmetric unit adopting a similar extended structure. The structures consist of three periplasmic domains (PDs) of similar αββαβ topology as seen also in the structures of the homologous PrgH (Salmonella typhimurium) and YscD (Yersinia enterocolitica). Only in the C2 crystal form, there is a C‐terminal additional helix after the PD3 domain. The relative orientation of the three subsequent CdsD PD domains with respect to each other is more extended than in PrgH but less extended than in YscD. Small‐angle X‐ray scattering data show that also in solution this CdsD construct adopts the same elongated shape. In both crystal forms the CdsD molecules are packed in a parallel fashion, using translational crystallographic symmetry. The most extensive crystal contacts are preserved in both crystal forms, suggesting a possible mode of assembly of the CdsD periplasmic part into a 24‐mer complex forming the outer ring of the IM‐ring of the T3SS.     

Structure and Interactions of the Cytoplasmic Domain of the Yersinia Type III Secretion Protein YscD

The virulence of a large number of Gram-negative bacterial pathogens depends on the type III secretion (T3S) system, which transports select bacterial proteins into host cells. An essential component of the Yersinia T3S system is YscD, a single-pass inner membrane protein. We report here the 2.52-Å resolution structure of the cytoplasmic domain of YscD, called YscDc. The structure confirms that YscDc consists of a forkhead-associated (FHA) fold, which in many but not all cases specifies binding to phosphothreonine. YscDc, however, lacks the structural properties associated with phosphothreonine binding and thus most likely interacts with partners in a phosphorylation-independent manner. Structural comparison highlighted two loop regions, L3 and L4, as potential sites of interactions. Alanine substitutions at L3 and L4 had no deleterious effects on protein structure or stability but abrogated T3S in a dominant negative manner. To gain insight into the function of L3 and L4, we identified proteins associated with YscD by affinity purification coupled to mass spectrometry. The lipoprotein YscJ was found associated with wild-type YscD, as was the effector YopH. Notably, the L3 and L4 substitution mutants interacted with more YopH than did wild-type YscD. These substitution mutants also interacted with SycH (the specific chaperone for YopH), the putative C-ring component YscQ, and the ruler component YscP, whereas wild-type YscD did not. These results suggest that substitutions in the L3 and L4 loops of YscD disrupted the dissociation of SycH from YopH, leading to the accumulation of a large protein complex that stalled the T3S apparatus.     

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.     

Structural and functional studies on the interaction of GspC and GspD in the type II secretion system

Type II secretion systems (T2SSs) are critical for secretion of many proteins from Gram-negative bacteria. In the T2SS, the outer membrane secretin GspD forms a multimeric pore for translocation of secreted proteins. GspD and the inner membrane protein GspC interact with each other via periplasmic domains. Three different crystal structures of the homology region domain of GspC (GspC(HR)) in complex with either two or three domains of the N-terminal region of GspD from enterotoxigenic Escherichia coli show that GspC(HR) adopts an all-β topology. N-terminal β-strands of GspC and the N0 domain of GspD are major components of the interface between these inner and outer membrane proteins from the T2SS. The biological relevance of the observed GspC-GspD interface is shown by analysis of variant proteins in two-hybrid studies and by the effect of mutations in homologous genes on extracellular secretion and subcellular distribution of GspC in Vibrio cholerae. Substitutions of interface residues of GspD have a dramatic effect on the focal distribution of GspC in V. cholerae. These studies indicate that the GspC(HR)-GspD(N0) interactions observed in the crystal structure are essential for T2SS function. Possible implications of our structures for the stoichiometry of the T2SS and exoprotein secretion are discussed.     

Cysteine-scanning mutagenesis around transmembrane segments 1 and 11 and their flanking loop regions of Tn10-encoded metal-Tetracycline/H+ antiporter

Putative transmembrane helices (TM) 1 and 11 in the metal-tetracycline/H(+) antiporter are predicted to be close to each other on the basis of disulfide cross-linking experiments of the double-cysteine mutants in the periplasmic loop regions (Kubo, Y., Konishi, S., Kawabe, T., Nada, S., and Yamaguchi, A. (2000) J. Biol. Chem. 275, 5270-5274). In this study, each amino acid from Asn-2 to Gly-44 in the putative TM1 and loop1-2 regions or that from Ser-328 to Gly-366 in TM11 and its flanking regions was individually replaced with cysteine. With respect to the TM1 region, 10 mutants, from T5C to L14C, were all not reactive with N-ethylmaleimide (NEM), and from D15C to I22C, NEM-reactive and non-reactive mutations periodically appeared every two residues. Three mutants, M23C to V25C, were all NEM-reactive, but the degree of the latter two mutants was very low. Seven mutants, from L26C to E32C, were all highly reactive with NEM. Therefore, the region of TM1 is composed of the 21 amino acid residues from Thr-5 to Val-25. It is a partially amphiphilic helix, that is, the N-terminal (cytoplasmic) half is embedded in the hydrophobic interior, and the C-terminal (periplasmic) half faces a water-filled channel. With respect to TM11, nine mutants, from S328C to G336C, and six mutants, from L361C to G366C, were all reactive with NEM. On the other hand, out of the 24 mutants, from L337C to S360C, 17 were not reactive with NEM, and the 7 NEM-reactive mutants were scattered, indicating that this region is a transmembrane segment. The 7 residues from Val-347 to Phe-353 including Pro-350 formed a central hydrophobic core, and the 7 NEM-reactive mutations were periodically distributed in its flanking regions, indicating that both ends of TM11 face a water-filled channel. Ala-354 is located at about 1/3 of the length from the periplasmic end of TM11. Disulfide cross-linking experiments on double-cysteine mutants having the combination of A354C and a cysteine-scanning mutation in the loop1-2 region indicated that loop1-2 is very flexible and close to the periplasmic end of TM11. Tetracycline prevented the cross-linking formation between the periplasmic ends of TM1 and TM11; however, it did not affect the cross-linking between loop1-2 and TM11, indicating that the substrate-induced conformational change involves a shift in the relative locations of TM1 and TM11.     

Structure and function of transmembrane segment XII in osmosensor and osmoprotectant transporter ProP of Escherichia coli

Escherichia coli transporter ProP acts as both an osmosensor and an osmoregulator. As medium osmolality rises, ProP is activated and mediates H+-coupled uptake of osmolytes like proline. A homology model of ProP with 12-transmembrane (TM) helices and cytoplasmic termini was created, and the protein's topology was substantiated experimentally. Residues 468-497, at the end of the C-terminal domain and linked to TM XII, form an intermolecular, homodimeric alpha-helical coiled-coil that tunes the transporter's response to osmolality. We aim to further define the structure and function of ProP residues Q415-E440, predicted to include TM XII. Each residue was replaced with cysteine (Cys) in a histidine-tagged, Cys-less ProP variant (ProP*). Cys at positions 415-418 and 438-440 were most reactive with Oregon Green Maleimide (OGM), suggesting that residues 419 through 437 are in the membrane. Except for V429-I433, reactivity of those Cys varied with helical periodicity. Cys predicted to face the interior of ProP were more reactive than Cys predicted to face the lipid. The former may be exposed to hydrated polar residues in the protein interior, particularly on the periplasmic side. Intermolecular cross-links formed when ProP* variants with Cys at positions 419, 420, 422, and 439 were treated with DTME. Thus TM XII can participate, along its entire length, in the dimer interface of ProP. Cys substitution E440C rendered ProP* inactive. All other variants retained more than 30% of the proline uptake activity of ProP* at high osmolality. Most variants with Cys substitutions in the periplasmic half of TM XII activated at lower osmolalities than ProP*. Variants with Cys substitutions on one face of the cytoplasmic half of TM XII required a higher osmolality to activate. They included elements of a GXXXG motif that are predicted to form the interface of TM XII with TM VII. These studies define the position of ProP TM XII within the membrane, further support the predicted structure of ProP, reveal the dimerization interface, and show that the structure of TM XII influences the osmolality at which ProP activates.     

Functional domains of NosR, a novel transmembrane iron-sulfur flavoprotein necessary for nitrous oxide respiration

Bacterial nitrous oxide (N2O) respiration depends on the polytopic membrane protein NosR for the expression of N2O reductase from the nosZ gene. We constructed His-tagged NosR and purified it from detergent-solubilized membranes of Pseudomonas stutzeri ATCC 14405. NosR is an iron-sulfur flavoprotein with redox centers positioned at opposite sides of the cytoplasmic membrane. The flavin cofactor is presumably bound covalently to an invariant threonine residue of the periplasmic domain. NosR also features conserved CX3CP motifs, located C-terminally of the transmembrane helices TM4 and TM6. We genetically manipulated nosR with respect to these different domains and putative functional centers and expressed recombinant derivatives in a nosR null mutant, MK418nosR::Tn5. NosR's function was studied by its effects on N2O respiration, NosZ synthesis, and the properties of purified NosZ proteins. Although all recombinant NosR proteins allowed the synthesis of NosZ, a loss of N2O respiration was observed upon deletion of most of the periplasmic domain or of the C-terminal parts beyond TM2 or upon modification of the cysteine residues in a highly conserved motif, CGWLCP, following TM4. Nonetheless, NosZ purified from the recombinant NosR background exhibited in vitro catalytic activity. Certain NosR derivatives caused an increase in NosZ of the spectral contribution from a modified catalytic Cu site. In addition to its role in nosZ expression, NosR supports in vivo N2O respiration. We also discuss its putative functions in electron donation and redox activation.     

A novel human glycosyltransferase: primary structure and characterization of the gene and transcripts

We report the identification and primary structure of a novel human glycosyltransferase, B3GTL (beta3-glycosyltransferase-like). The 498 residue protein consists of a short cytoplasmic N-terminal "tail" (residues 1-4), a single transmembrane domain with type II topology (residues 5-28), a "stem" region (residues 29-260), and a catalytic domain (residues 261-498). The genomes of Anopheles gambiae, Drosophila melanogaster, and Caenorhabditis elegans encode potential orthologs which share 31-39% sequence identity with B3GTL, as well as the following features: a conserved catalytic domain containing a triple aspartate motif (DDD) at its core, a conserved pattern of cysteine residues, a C-terminal KDEL-like motif, and conserved residues and motifs that affiliate this novel group with a family of beta3-glycosyltransferases (GT31 in the CAZY classification). The B3GTL gene lacks canonical TATA and CAAT boxes and contains three functional polyadenylation sites. It is transcribed in a wide range of tissues and in TGF-beta-treated T84 epithelial cells.     

Nearest neighbor analysis of the SecYEG complex. 1. Identification of a SecY-SecG interface

Integral membrane components SecY, SecE, and SecG of protein translocase form a complex in the Escherichia coli plasma membrane. To characterize subunit interactions of the SecYEG complex, a series of SecY variants having a single cysteine in its cytoplasmic (C1-C6) or periplasmic (P1-P5) domain were subjected to site-specific cross-linking experiments using bifunctional agents with thiol-amine reactivity. Experiments using inverted membrane vesicles revealed specific cross-linkings between a cysteine residue placed in the C2 or C3 domain of SecY and the cytosolic lysine (Lys26) near the first transmembrane segment of SecG. These SecY Cys residues also formed a disulfide bond with an engineered cytosolic cysteine at position 28 of SecG. Thus, the C2-C3 region of SecY is in the proximity of the N-terminal half of the SecG cytoplasmic loop. Experiments using spheroplasts revealed the physical proximity of P2 (SecY) and the C-terminal periplasmic region of SecG. In addition, mutations in secG were isolated as suppressors against a cold-sensitive mutation (secY104) affecting the TM4-C3 boundary of SecY. These results collectively suggest that a C2-TM3-P2-TM4-C3 region of SecY serves as an interface with SecG.     

Genetic Dissection of T4 Lysis

t is the holin gene for coliphage T4, encoding a 218-amino-acid (aa) protein essential for the inner membrane hole formation that initiates lysis and terminates the phage infection cycle. T is predicted to be an integral membrane protein that adopts an Nⁱⁿ-C^out topology with a single transmembrane domain (TMD). This holin topology is different from those of the well-studied holins S105 (3 TMDs; N^out-Cⁱⁿ) of the coliphage lambda and S68 (2 TMDs; Nⁱⁿ-Cⁱⁿ) of the lambdoid phage 21. Here, we used random mutagenesis to construct a library of lysis-defective alleles of t to discern residues and domains important for holin function and for the inhibition of lysis by the T4 antiholin, RI. The results show that mutations in all 3 topological domains (N-terminal cytoplasmic, TMD, and C-terminal periplasmic) can abrogate holin function. Additionally, several lysis-defective alleles in the C-terminal domain are no longer competent in binding RI. Taken together, these results shed light on the roles of the previously uncharacterized N-terminal and C-terminal domains in lysis and its real-time regulation.     

Solution structure and backbone dynamics of the cysteine 103 to serine mutant of the N-terminal domain of DsbD from Neisseria meningitidis

The DsbD protein is essential for electron transfer from the cytoplasm to the periplasm of Gram-negative bacteria. Its N-terminal domain dispatches electrons coming from cytoplasmic thioredoxin (Trx), via its central transmembrane and C-terminal domains, to its periplasmic partners: DsbC, DsbE/CcmG, and DsbG. Previous structural studies described the latter proteins as Trx-like folds possessing a characteristic C-X-X-C motif able to generate a disulfide bond upon oxidation. The Escherichia coli nDsbD displays an immunoglobulin-like fold in which two cysteine residues (Cys103 and Cys109) allow a disulfide bond exchange with its biological partners.We have determined the structure in solution and the backbone dynamics of the C103S mutant of the N-terminal domain of DsbD from Neisseria meningitidis. Our results highlight significant structural changes concerning the beta-sheets and the local topology of the active site compared with the oxidized form of the E. coli nDsbD. The structure reveals a "cap loop" covering the active site, similar to the oxidized E. coli nDsbD X-ray structure. However, regions featuring enhanced mobility were observed both near to and distant from the active site, revealing a capacity of structural adjustments in the active site and in putative interaction areas with nDsbD biological partners. Results are discussed in terms of functional consequences.     

C-terminal hydrophobic region in human bone marrow stromal cell antigen 2 (BST-2)/tetherin protein functions as second transmembrane motif

BST-2/CD317/HM1.24/tetherin is a host factor that inhibits the release of HIV-1 and other enveloped viruses. Structurally, tetherin consists of an N-terminal transmembrane (TM) region, a central coiled coil motif, and a putative C-terminal glycosylphosphatidylinositol (GPI) anchor motif. A current working model proposes that BST-2 inhibits virus release by physically tethering viral particles to the cell surface via its TM motif and GPI anchor. Here we analyzed the functional importance of the C-terminal GPI anchor motif in BST-2. We replaced the GPI anchor motif in BST-2 with the TM regions of several surface markers and found that the TM motifs of CD40 and transferrin receptor, but not that of CD45, could functionally substitute for a GPI anchor in BST-2. Conversely, replacing the TM region of CD4 by the putative GPI anchor signal of human BST-2 resulted in proper membrane targeting and surface expression of the chimeric protein, indicating that the BST-2 GPI anchor signal can function as a bona fide TM region. In fact, attempts to demonstrate GPI anchor modification of human BST-2 by biochemical methods failed. Our results demonstrate that the putative C-terminal GPI anchor motif in human BST-2 fulfills the requirements of a bona fide TM motif, leading us to propose that human BST-2 may in fact contain a second TM segment rather than a GPI anchor.     

Structural basis for the secretion of EvpC: a key type VI secretion system protein from Edwardsiella tarda

The recently identified type VI secretion system (T6SS) is implicated in the virulence of many Gram-negative bacteria. Edwardsiella tarda is an important cause of hemorrhagic septicemia in fish and also gastro- and extra-intestinal infections in humans. The E . tarda virulent protein (EVP) gene cluster encodes a conserved T6SS which contains 16 open reading frames. EvpC is one of the three major EVP secreted proteins and shares high sequence similarity with Hcp1, a key T6SS virulence factor from Pseudomonas aeruginosa. EvpC contributes to the virulence of E. tarda by playing an essential role in functional T6SS. Here, we report the crystal structure of EvpC from E. tarda PPD130/91 at a 2.8 Å resolution, along with functional studies of the protein. EvpC has a β-barrel domain with extended loops. The β-barrel consists of 11 anti-parallel β-strands with an α-helix located on one side. In solution, EvpC exists as a dimer at low concentration and as a hexamer at higher concentration. In the crystal, the symmetry related EvpC molecules form hexameric rings which stack together to form a tube similar to Hcp1. Structure based mutagenesis revealed that N-terminal negatively charged residues, Asp4, Glu15 and Glu26, and C-terminal positively charged residues, Lys161, Lys162 and Lys163, played crucial roles in the secretion of EvpC. Moreover, the localization study indicates the presence of wild type EvpC in cytoplasm, periplasm and secreted fractions, whereas the N-terminal and C-terminal mutants were found mostly in the periplasmic region and was completely absent in the secreted fraction. Results reported here provide insight into the structure, assembly and function of EvpC. Further, these findings can be extended to other EvpC homologs for understanding the mechanism of T6SS and targeting T6SS mediated virulence in Gram-negative pathogens.     

Structure of the C-terminal phosphotyrosine interaction domain of Fe65L1 complexed with the cytoplasmic tail of amyloid precursor protein reveals a novel peptide binding mode

Fe65L1, a member of the Fe65 family, is an adaptor protein that interacts with the cytoplasmic domain of Alzheimer amyloid precursor protein (APP) through its C-terminal phosphotyrosine interaction/phosphotyrosine binding (PID/PTB) domain. In the present study, the solution structures of the C-terminal PID domain of mouse Fe65L1, alone and in complex with a 32-mer peptide (DAAVTPEERHLSKMQQNGYENPTYKFFEQMQN) derived from the cytoplasmic domain of APP, were determined using NMR spectroscopy. The C-terminal PID domain of Fe65L1 alone exhibits a canonical PID/PTB fold, whereas the complex structure reveals a novel mode of peptide binding. In the complex structure, the NPTY motif forms a type-I beta-turn, and the residues immediately N-terminal to the NPTY motif form an antiparallel beta-sheet with the beta5 strand of the PID domain, the binding mode typically observed in the PID/PTB.peptide complex. On the other hand, the N-terminal region of the peptide forms a 2.5-turn alpha-helix and interacts extensively with the C-terminal alpha-helix and the peripheral regions of the PID domain, representing a novel mode of peptide binding that has not been reported previously for the PID/PTB.peptide complex. The indispensability of the N-terminal region of the peptide for the high affinity of the PID-peptide interaction is consistent with NMR titration and isothermal calorimetry data. The extensive binding features of the PID domain of Fe65L1 with the cytoplasmic domain of APP provide a framework for further understanding of the function, trafficking, and processing of APP modulated by adapter proteins.     

Structural and functional characterization of SiiA,an auxiliary protein from the SPI4‐encoded type 1 secretion system from Salmonella enterica

Salmonella invasion is mediated by a concerted action of the Salmonella pathogenicity island 4 (SPI4)‐encoded type one secretion system (T1SS) and the SPI1‐encoded type three secretion system (T3SS‐1). The SPI4‐encoded T1SS consists of five proteins (SiiABCDF) and secretes the giant adhesin SiiE. Here, we investigated structure–function relationships in SiiA, a non‐canonical T1SS subunit. We show that SiiA consists of a membrane domain, an intrinsically disordered periplasmic linker region and a folded globular periplasmic domain (SiiA‐PD). The crystal structure of SiiA‐PD displays homology to that of MotB and other peptidoglycan (PG)‐binding domains. SiiA‐PD binds PG in vitro, albeit at an acidic pH, only. Mutation of Arg162 impedes PG binding of SiiA and reduces Salmonella invasion efficacy. SiiA forms a complex with SiiB at the inner membrane (IM), and the observed SiiA‐MotB homology is paralleled by a predicted SiiB‐MotA homology. We show that, similar to MotAB, SiiAB translocates protons across the IM. Mutating Asp13 in SiiA impairs proton translocation. Overall, SiiA shares numerous properties with MotB. However, MotAB uses the proton motif force (PMF) to energize the bacterial flagellum, it remains to be shown how usage of the PMF by SiiAB assists T1SS function and Salmonella invasion.     

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.