Mutations in subunits of the Escherichia coli twin-arginine translocase block function via differing effects on translocation activity or tat complex structure

We have used a combination of blue-native (BN) gel electrophoresis and protein purification to analyze the effects of TatA or TatC mutations on the structures of the primary TatABC and multimeric TatA complexes in Escherichia coli. Expression of wild-type TatABC leads to the production of a single major TatABC complex of 370 kDa and a heterogeneous set of TatA complexes of <100 kDa to approximately 500 kDa. Two TatC mutations that block translocation have different effects on complex structures. P48A causes massive defects in TatABC assembly, including a marked separation of the TatBC subunits and the production of TatB and TatC aggregates. In contrast, TatABC complexes from the inactive TatC F94A mutant are structurally intact, suggesting that this mutation affects translocation activity rather than assembly. Neither TatC mutation affects the separate TatA complexes, showing that assembly of the TatA complexes is independent of TatABC assembly or activity. In contrast, three TatA mutations affect both the TatA and TatABC complexes. F39A assembles into smaller, incorrectly organized TatA complexes and the TatABC complexes contain an incorrect TatB:TatC ratio and unusually large amounts of TatA. A triple mutant in the amphipathic region forms slightly larger TatA complexes that are likewise disorganized, and a mutant containing three glycine substitutions in the transmembrane (TM) span assembles as grossly affected TatA complexes that are much larger than wild-type complexes. These mutants lead to a partial failure of TatB to assemble correctly. The data show that the amphipathic and TM regions play critical roles in TatA complex assembly. All of the TatA mutations lead to partial or substantial defects in TatABC complex formation, demonstrating that the properties of TatA can have a marked influence on the TatABC complex.     

Expression of the bifunctional <Emphasis Type="Italic">Bacillus subtilis</Emphasis> TatAd protein in <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> reveals distinct TatA/B-family and TatB-specific domains

In the Tat protein export pathway of Gram-negative bacteria, TatA and TatB are homologous proteins that carry out distinct and essential functions in separate sub-complexes. In contrast, Gram-positive Tat systems usually lack TatB and the TatA protein is bifunctional. We have used a mutagenesis approach to delineate TatA/B-type domains in the bifunctional TatAd protein from Bacillus subtilis. This involved expression of mutated TatAd variants in Escherichia coli and tests to determine whether the variants could function as TatA or TatB by complementing E. coli tatA and/or tatB mutants. We show that mutations in the C-terminal half of the transmembrane span and the subsequent FGP ‘hinge’ motif are critical for TatAd function with its partner TatCd subunit, and the same determinants are required for complementation of either tatA or tatB mutants in Escherichia coli. This is thus a critical domain in both TatA and TatB proteins. In contrast, substitution of a series of residues at the N-terminus specifically blocks the ability of TatAd to substitute for E. coli TatB. The results point to the presence of a universally conserved domain in the TatA/B-family, together with a separate N-terminal domain that is linked to the TatB-type function in Gram-negative bacteria.     

TatA and TatB generate a hydrophobic mismatch important for the function and assembly of the Tat translocon in Escherichia coli

The twin-arginine translocation (Tat) system serves to translocate folded proteins across energy-transducing membranes in bacteria, archaea, plastids, and some mitochondria. In Escherichia coli, TatA, TatB, and TatC constitute functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) followed by an amphipathic helix. The TMHs of TatA and TatB generate a hydrophobic mismatch with the membrane, as the helices comprise only 12 consecutive hydrophobic residues; however, the purpose of this mismatch is unclear. Here, we shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both and analyzed effects on translocon function and assembly. We found the WT length helices functioned best, but some variation was clearly tolerated. Defects in function were exacerbated by simultaneous mutations in TatA and TatB, indicating partial compensation of mutations in each by the other. Furthermore, length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residue-length is important but not essential for this interaction and translocon assembly. To also address potential effects of helix length on TatA interactions, we characterized these interactions by molecular dynamics simulations, after having characterized the TatA assemblies by metal-tagging transmission electron microscopy. In these simulations, we found that interacting short TMHs of larger TatA assemblies were thinning the membrane and—together with laterally-aligned tilted amphipathic helices—generated a deep V-shaped membrane groove. We propose the 12 consecutive hydrophobic residues may thus serve to destabilize the membrane during Tat transport, and their conservation could represent a delicate compromise between functionality and minimization of proton leakage.     

Mutational analysis of the oligomer assembly domain in the transmembrane subunit of the Rous sarcoma virus glycoprotein.

The transmembrane (TM) subunits of retroviral envelope glycoproteins appear to direct the assembly of the glycoprotein precursor into a discrete oligomeric structure. We have examined mutant Rous sarcoma virus envelope proteins with truncations or deletions within the ectodomain of TM for their ability to oligomerize in a functional manner. Envelope proteins containing an intact surface (SU) domain and a TM domain truncated after residue 120 or 129 formed intracellular trimers in a manner similar to that of proteins that had an intact ectodomain and were efficiently secreted. Whereas independent expression of the SU domain yielded an efficiently transported molecule, proteins containing SU and 17, 29, 37, 59, 73, 88, and 105 residues of TM were defective in intracellular transport. With the exception of a protein truncated after residue 88 of TM, the truncated proteins were also defective in formation of stable trimers that could be detected on sucrose gradients. Deletion mutations within the N-terminal 120 amino acids of TM also disrupted transport to the Golgi complex, but a majority of these mutant glycoproteins were still able to assemble trimers. Deletion of residues 60 to 74 of TM caused the protein to remain monomeric, while a deletion C terminal of residue 88 that removed two cysteine residues resulted in nonspecific aggregation. Thus, it appears that amino acids throughout the N-terminal 120 residues of TM contribute to assembly of a transport-competent trimer. This region of TM contains two amino acid domains capable of forming alpha helices, separated by a potential disulfide-bonded loop. While the N-terminal helical sequence, which extends to residue 85 of TM, may be capable of mediating the formation of Env trimers if C-terminal sequences are deleted, our results show that the putative disulfide-linked loop and C-terminal alpha-helical sequence play a key role in directing the formation of a stable trimer that is competent for intracellular transport.     

Functional Analysis of Yeast bcs1 Mutants Highlights the Role of Bcs1p-Specific Amino Acids in the AAA Domain

The mitochondrial protein Bcs1p is conserved from Saccharomyces cerevisiae to humans and its C-terminal region exhibits an AAA (ATPases associated with diverse cellular activities) domain. The absence of the yeast Bcs1p leads to an assembly defect of the iron-sulfur protein (ISP) subunit within the mitochondrial respiratory complex III, whereas human point mutations located all along the protein cause various pathologies. We have performed a structure-function analysis of the yeast Bcs1p by randomly generating a collection of respiratory-deficient point mutants. We showed that most mutations are in the C-terminal region of Bcs1p and have localized them on a theoretical three-dimensional model based on the structure of several AAA proteins. The mutations can be grouped into classes according to their respiratory competence and their location on the three-dimensional model. We have further characterized five mutants, each substituting an amino acid conserved in yeast and mammalian Bcs1 proteins but not in other AAA proteins. The effects on respiratory complex assembly and Bcs1p accumulation were analyzed. Intragenic and extragenic compensatory mutations able to restore complex III assembly to the mutants affecting the AAA domain were isolated. Our results bring new insights into the role of specific residues in critical regions that are also conserved in the human Bcs1p. We show that (1) residues located at the junction between the Bcs1p-specific and the AAA domains are important for the activity and stability of the protein and (2) the residue F342 is important for interactions with other partners or substrate proteins.     

Evidence for interactions between domains of TatA and TatB from mutagenesis of the TatABC subunits of the twin-arginine translocase

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane. Three subunits, TatA, B and C, are known to be involved but their modes of action are poorly understood, as are the inter-subunit interactions occurring within Tat complexes. We have generated mutations in the single transmembrane (TM) spans of TatA and TatB, with the aim of generating structural distortions. We show that substitution in TatB of three residues by glycine, or a single residue by proline, has no detectable effect on translocation, whereas the presence of three glycines in the TatA TM span completely blocks Tat translocation activity. The results show that the integrity of the TatA TM span is vital for Tat activity, whereas that of TatB can accommodate large-scale distortions. Near-complete restoration of activity in TatA mutants is achieved by the simultaneous presence of a V12P mutation in the TatB TM span, strongly implying a direct functional interaction between the TatA/B TM spans. We also analyzed the predicted amphipathic regions in TatA and TatB and again find evidence of direct interaction; benign mutations in either subunit completely blocked translocation of two Tat substrates when present in combination. Finally, we have re-examined the effects of previously analyzed TatABC mutations under conditions of high translocation activity. Among numerous TatA or TatB mutations tested, TatA F39A alone blocked translocation, and only substitutions of P48 and F94 in TatC blocked translocation activity.     

Molecular determinants of the yeast Arc1p-aminoacyl-tRNA synthetase complex assembly

Eukaryotic aminoacyl-tRNA synthetases are usually organized into high-molecular-weight complexes, the structure and function of which are poorly understood. We have previously described a yeast complex containing two aminoacyl-tRNA synthetases, methionyl-tRNA synthetase and glutamyl-tRNA synthetase, and one noncatalytic protein, Arc1p, which can stimulate the catalytic efficiency of the two synthetases. To understand the complex assembly mechanism and its relevance to the function of its components, we have generated specific mutations in residues predicted by a recent structural model to be located at the interaction interfaces of the N-terminal domains of all three proteins. Recombinant wild-type or mutant forms of the proteins, as well as the isolated N-terminal domains of the two synthetases, were overexpressed in bacteria, purified and used for complex formation in vitro and for determination of binding affinities using surface plasmon resonance. Moreover, mutant proteins were expressed as PtA or green fluorescent protein fusion polypeptides in yeast strains lacking the endogenous proteins in order to monitor in vivo complex assembly and their subcellular localization. Our results show that the assembly of the Arc1p-synthetase complex is mediated exclusively by the N-terminal domains of the synthetases and that the two enzymes bind to largely independent sites on Arc1p. Analysis of single-amino-acid substitutions identified residues that are directly involved in the formation of the complex in yeast cells and suggested that complex assembly is mediated predominantly by van der Waals and hydrophobic interactions, rather than by electrostatic forces. Furthermore, mutations that abolish the interaction of methionyl-tRNA synthetase with Arc1p cause entry of the enzyme into the nucleus, proving that complex association regulates its subcellular distribution. The relevance of these findings to the evolution and function of the multienzyme complexes of eukaryotic aminoacyl-tRNA synthetases is discussed.     

The Platform Protein Is Essential for Type IV Pilus Biogenesis

A systematic genetic analysis was performed to identify the inner membrane proteins essential for type IV pilus (T4P) expression in Pseudomonas aeruginosa. By inactivating the retraction aspect of pilus function, genes essential for T4P assembly were discriminated. In contrast to previous studies in the T4P system of Neisseria spp., we found that components of the inner membrane subcomplex consisting of PilMNOP were not essential for surface pilus expression, whereas the highly conserved inner membrane protein PilC was essential. Here, we present data that PilC may coordinate the activity of cytoplasmic polymerization (PilB) and depolymerization (PilT) ATPases via their interactions with its two cytoplasmic domains. Using in vitro co-affinity purification, we show that PilB interacts with the N-terminal cytoplasmic domain of PilC. We hypothesized that PilT similarly interacts with the PilC C-terminal cytoplasmic domain. Overexpression of that domain in the wild-type protein reduced twitching motility by ∼50% compared with the vector control. Site-directed mutagenesis of conserved T4P-specific residues in the PilC C-terminal domain yielded mutant proteins that supported wild-type pilus assembly but had a reduced capacity to support twitching motility, suggesting impairment of putative PilC-PilT interactions. Taken together, our results show that PilC is an essential inner membrane component of the T4P system, controlling both pilus assembly and disassembly.     

Mim1 functions in an oligomeric form to facilitate the integration of Tom20 into the mitochondrial outer membrane

The translocase of the outer mitochondrial membrane (TOM) complex is the general entry site into the organelle for newly synthesized proteins. Despite its central role in the biogenesis of mitochondria, the assembly process of this complex is not completely understood. Mim1 (mitochondrial import protein 1) is a mitochondrial outer membrane protein with an undefined role in the assembly of the TOM complex. The protein is composed of an N-terminal cytosolic domain, a central putative transmembrane segment (TMS) and a C-terminal domain facing the intermembrane space. Here we show that Mim1 is required for the integration of the import receptor Tom20 into the outer membrane. We further investigated what the structural characteristics allowing Mim1 to fulfil its function are. The N- and C-terminal domains of Mim1 are crucial neither for the function of the protein nor for its biogenesis. Thus, the TMS of Mim1 is the minimal functional domain of the protein. We show that Mim1 forms homo-oligomeric structures via its TMS, which contains two helix-dimerization GXXXG motifs. Mim1 with mutated GXXXG motifs did not form oligomeric structures and was inactive. With all these data taken together, we propose that the homo-oligomerization of Mim1 allows it to fulfil its function in promoting the integration of Tom20 into the mitochondrial outer membrane.     

Dimerization of the erythropoietin receptor transmembrane domain in micelles

Erythropoietin receptor (EpoR) homodimerization is an initial regulatory step in erythrocyte formation. Receptor dimers form before ligand binding, suggesting that association between receptor proteins is dependent on the receptor itself. EpoR dimerization is an essential step in erythropoiesis, and misregulation of this dimerization has been implicated in several disease states, including multi-lineage leukemias; nevertheless, how EpoR regulates its own dimerization is unclear. In vivo experiments suggest the single-pass transmembrane helix is the strongest candidate for driving ligand-independent association. To address the self-association potential of this transmembrane segment, we studied its interaction energetics in micelles by utilizing a previously successful Staphylococcal nuclease (SN-EpoR TM) fusion protein. This fusion protein strategy allows expression of the EpoR transmembrane domain in Escherichia coli independent of the other EpoR domains. Sedimentation equilibrium analytical ultracentrifugation of the detergent-solubilized SN-EpoR TM demonstrated that the murine EpoR transmembrane domain self-associates to form dimers. Although this interaction is not as stable as the dimerization of the well-studied glycophorin A transmembrane dimer, the murine EpoR transmembrane domain dimer is more stable than the interactions of the colon carcinoma kinase 4 transmembrane domain. The same experiments with the human EpoR transmembrane domain, which differs from the mouse sequence by only three residues, revealed a less favorable interaction than that of the murine sequence and is only slightly more favorable than that expected for non-preferential binding. These results suggest that the mouse and human receptor proteins may differ in the roles they play in signaling.     

Identification and functional characterization of the zeta-chain dimerization motif for TCR surface expression.

We recognized a common dimerization motif between the transmembrane (TM) domain of zeta-chain family members and glycophorin A. We have shown that a glycine within the zeta-dimerization motif is critical for zeta-homodimerization and also for its association with the TCR/CD3 complex. Similarly, two residues within the CD3 delta gamma TM domains have proven to be critical for their interaction with the zeta-homodimer. A three-dimensional homology model of the zeta-chain TM domain highlights potential residues preferentially involved either in the zeta 2-CD3 or zeta 2-TCR alpha beta association, confirming our experimental findings. These results indicate that, for symmetrical reasons, the zeta-homodimer participates in the TCR/CD3 complex assembly by interacting with CD3 gamma delta TM domains, thereby masking their degradation signals located in the cytoplasmic tails.     

Synthesis and initial characterization of FGFR3 transmembrane domain: consequences of sequence modifications

Receptor Tyrosine Kinases (RTKs) conduct biochemical signals via lateral dimerization in the plasma membrane, and defects in their dimerization lead to unregulated signaling and disease. RTK transmembrane (TM) domains are proposed to play an important role in the process, underscored by the finding that single amino acids mutations in the TM domains can induce pathological phenotypes. Therefore, many important questions pertaining to the mode of signal transduction and the mechanism of pathology induction could be answered by studying the chemical-physical basis behind RTK TM domain dimerization and the interactions of RTK TM domains with lipids in model bilayer systems. As a first step towards this goal, here we report the synthesis of the TM domain of fibroblast growth factor receptor 3 (FGFR3), an RTK that is crucial for skeletal development. We have used solid phase peptide synthesis to produce two peptides: one corresponding to the membrane embedded segment and the naturally occurring flanking residues at the N- and C-termini (TM_wt), and a second one in which the flanking residues have been substituted with diLysines at the termini (TM_KK). We have demonstrated that the hydrophobic FGFR3 TM domain can be synthesized for biophysical studies with high yield. The protocol presented in the paper can be applied to the synthesis of other RTK TM domains. As expected, the Lys flanks decrease the hydrophobicity of the TM domain, such that TM_KK elutes much earlier than TM_wt during reverse phase HPLC purification. The Lysines have no effect on peptide solubility in SDS and on peptide secondary structure, but they abolish peptide dimerization on SDS gels. These results suggest that caution should be exercised when modifying RTK TM domains to render them more manageable for biophysical studies.     

The role of individual amino acids in the dimerization of CR4 and ACR4 transmembrane domains

CRINKLY4 is a growth factor-like plant receptor kinase designated as CR4 in Zea mays and ACR4 in Arabidopsis. Using the TOXCAT system, a genetic assay that measures helix interactions in a natural membrane environment, we have previously demonstrated that the dimerization potential of the ACR4 transmembrane (TM) domain is significantly weaker than that of CR4 TM domain, even though 13 of the 24 residues are identical. Neither of the TM domains contain the GxxxG motif that has been shown to be important for the dimerization of the TM segments of several receptors. To further investigate the relationship between protein sequence and dimerization potential, we (a) mutated each of the 11 differing residues in the CR4 TM domain to the corresponding residue of ACR4 (b) made reciprocal mutations in ACR4 and (c) made hybrids consisting of half CR4 and half ACR4 TM domains. Our results suggest that most mutations in ACR4 or CR4 TM domains have low to moderate effects on the dimerization potential and that residues in the N-terminal half of the CR4 TM domain are important for dimerization.     

Alteration of the C-Terminal Ligand Specificity of the Erbin PDZ Domain by Allosteric Mutational Effects

Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain. We constructed a library of PDZ domains by randomizing residues in direct contact with position 0 and in a loop that is close to but does not contact position 0. We used phage display to select for PDZ variants that bind to 19 peptide ligands differing only at position 0. To verify that each obtained PDZ domain exhibited the correct binding specificity, we selected peptide ligands for each domain. Despite intensive efforts, we were only able to evolve Erbin PDZ domain variants with selectivity for the aliphatic C-terminal side chains Val, Ile and Leu. Interestingly, many PDZ domains with these three distinct specificities contained identical amino acids at positions that directly contact position 0 but differed in the loop that does not contact position 0. Computational modeling of the selected PDZ domains shows how slight conformational changes in the loop region propagate to the binding site and result in different binding specificities. Our results demonstrate that second-sphere residues could be crucial in determining protein binding specificity.     

Characterization of a Dual-Function Domain That Mediates Membrane Insertion and Excision of Ff Filamentous Bacteriophage

The filamentous phage Ff (f1, fd, or M13) of Escherichia coli is assembled at the cell membranes by a process that is morphologically similar to that of pilus assembly. The release of the filament virion is mediated by excision from the membrane; conversely, entry into a host cell is mediated by insertion of the virion coat proteins into the membrane. The N-terminal domains of the minor virion protein pIII have the sole role of binding to host receptors during infection. In contrast, the C domain of pIII is required for two opposite functions: insertion of the virion into the membrane during infection and excision at the termination step of assembly/secretion. We identified a 28-residue-long segment in the pIII C domain, which is required for phage entry but dispensable for release from the membrane at the end of assembly. This segment, which we named the infection-competence segment (ICS), works only in cis with the N-terminal receptor-binding domains and does not require the equivalent ICS sequences in other subunits within the virion cap. The ICS contains a predicted amphipathic α-helix and is rich in small amino acids, Gly, Ala, and Ser, which are arranged as a [small]XXX[small]XX[small]XXX[small]XXX[small] motif. Scanning Ala/Gly mutagenesis of ICS showed that small residues are compatible with infection. Overall, organization of the C domain is reminiscent of α-helical pore-forming toxins' membrane insertion domains. The unique ability of pIII to mediate both membrane insertion and excision allowed us to compare these two fundamental membrane transactions and to show that receptor-triggered insertion is a more complex process than excision from membranes.     

Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein

ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease.     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H-V-ATPase

Vacuolar (H⁺)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an α-helical conformation for peptide MTM7 and in DMSO three α-helical regions are identified by 2D ¹H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an α-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

A Chimeric Fusion Protein Engineered with Disparate Functionalities—Enzymatic Activity and Self-assembly

The fusion of protein domains is an important mechanism in molecular evolution and a valuable strategy for protein engineering. We are interested in creating fusion proteins containing both globular and structural domains so that the final chimeric protein can be utilized to create novel bioactive biomaterials. Interactions between fused domains can be desirable in some fusion protein applications, but in this case the optimal configuration will enable the bioactivity to be unaffected by the structural cross-linking. To explore this concept, we have created a fusion consisting of a thermostable aldo-keto reductase, two α-helical leucine zipper domains, and a randomly coiled domain. The resulting protein is bifunctional in that (1) it can self-assemble into a hydrogel material as the terminal leucine zipper domains form interprotein coiled-coil cross-links, and (2) it expresses alcohol dehydrogenase and aldo-keto reductase activity native to AdhD from Pyrococcus furiosus. The kinetic parameters of the enzyme are minimally affected by the addition of the helical appendages, and rheological studies demonstrate that a supramolecular assembly of the bifunctional protein building blocks forms a hydrogel. An active hydrogel is produced at temperatures up to 60 °C, and we demonstrate the functionality of the biomaterial by monitoring the oxidation and reduction of the native substrates by the gel. The design of chimeric fusion proteins with both globular and structural domains is an important advancement for the creation of bioactive biomaterials for biotechnology applications such as tissue engineering, bioelectrocatalysis, and biosensing and for the study of native assembled enzyme structures and clustered enzyme systems such as metabolons.     

Conserved Salt Bridges Facilitate Assembly of the Helical Core Export Apparatus of a Salmonella enterica Type III Secretion System

Virulence-associated type III secretion systems (T3SS) are utilized by Gram negative bacterial pathogens for injection of effector proteins into eukaryotic host cells. The transmembrane export apparatus at the core of T3SS is composed of a unique helical complex of the hydrophobic proteins SctR, SctS, SctT, and SctU. These components comprise a number of highly conserved charged residues within their hydrophobic domains. The structure of the closed state of the core complex SctR₅S₄T₁ revealed that several of these residues form inter- and intramolecular salt bridges, some of which have to be broken for pore opening. Mutagenesis of individual residues was shown to compromise assembly or secretion of both, the virulence-associated and the related flagellar T3SS. However, the exact role of these conserved charged residues in the assembly and function of T3SS remains elusive. Here we performed an in-depth mutagenesis analysis of these residues in the T3SS of Salmonella Typhimurium, coupled to blue native PAGE, in vivo photocrosslinking and luciferase-based secretion assays. Our data show that these conserved salt bridges are not critical for assembly of the respective protein but rather facilitate the incorporation of the following subunit into the assembling complex. Our data also indicate that these conserved charged residues are critical for type III-dependent secretion and reveal a functional link between SctS_E44 and SctT_R204 and the cytoplasmic domain of SctU in gating the T3SS injectisome. Overall, our analysis provides an unprecedented insight into the delicate requirements for the assembly and function of the machinery at the core of T3SS.