Structure and function of the juxtamembrane GAF domain of potassium biosensor KdpD

KdpD/KdpE two‐component signaling system regulates expression of a high affinity potassium transporter responsible for potassium homeostasis. The C‐terminal module of KdpD consists of a GAF domain linked to a histidine kinase domain. Whereas certain GAF domains act as regulators by binding cyclic nucleotides, the role of the juxtamembrane GAF domain in KdpD is unknown. We report the high‐resolution crystal structure of KdpD GAF domain (KdpD^G) consisting of five α‐helices, four β‐sheets and two large loops. KdpD^G forms a symmetry‐related dimer, wherein parallelly arranged monomers contribute to a four‐helix bundle at the dimer‐interface, SAXS analysis of KdpD C‐terminal module reveals an elongated structure that is a dimer in solution. Substitution of conserved residues with various residues that disrupt the dimer interface produce a range of effects on gene expression demonstrating the importance of the interface in inactive to active transitions during signaling. Comparison of ligand binding site of the classic cyclic nucleotide‐binding GAF domains to KdpD^G reveals structural differences arising from naturally occurring substitutions in primary sequence of KdpD^G that modifies the canonical NKFDE sequence motif required for cyclic nucleotide binding. Together these results suggest a structural role for KdpD^G in dimerization and transmission of signal to the kinase domain.     

Crystallographic and mutational studies on the tRNA thiouridine synthetase TtuA

In thermophilic bacteria, specific 2‐thiolation occurs on the conserved ribothymidine at position 54 (T54) in tRNAs, which is necessary for survival at high temperatures. T54 2‐thiolation is achieved by the tRNA thiouridine synthetase TtuA and sulfur‐carrier proteins. TtuA has five conserved CXXC/H motifs and the signature PP motif, and belongs to the TtcA family of tRNA 2‐thiolation enzymes, for which there is currently no structural information. In this study, we determined the crystal structure of a TtuA homolog from the hyperthermophilic archeon Pyrococcus horikoshii at 2.1 Å resolution. The P. horikoshii TtuA forms a homodimer, and each subunit contains a catalytic domain and unique N‐ and C‐terminal zinc fingers. The catalytic domain has much higher structural similarity to that of another tRNA modification enzyme, TilS (tRNA^Ile₂ lysidine synthetase), than to the other type of tRNA 2‐thiolation enzyme, MnmA. Three conserved cysteine residues are clustered in the putative catalytic site, which is not present in TilS. An in vivo mutational analysis in the bacterium Thermus thermophilus demonstrated that the three conserved cysteine residues and the putative ATP‐binding residues in the catalytic domain are important for the TtuA activity. A positively charged surface that includes the catalytic site and the two zinc fingers is likely to provide the tRNA‐binding site. Proteins 2013; 81:1232–1244. © 2013 Wiley Periodicals, Inc.     

Dimerization of Proline Dehydrogenase from Thermus thermophilus Is Crucial for Its Thermostability

Thermus thermophilus proline dehydrogenase ( TtProDH) catalyzes the first step in proline catabolism. The thermostable flavoenzyme consists of a distorted triosephosphate isomerase (TIM) barrel and three N‐terminal helices: αA, αB, and αC. Using maltose‐binding protein (MBP) fused constructs, it has been recently demonstrated that helix αC is crucial for TtProDH catalysis and for tetramerization through positioning of helix α8. Here, the structural features that determine the thermostability of TtProDH are reported. Selective disruption of two ion pairs in the dimerization interface of several MBP‐TtProDH variants result in the formation of monomers. The newly created monomers have improved catalytic properties but their melting temperatures are decreased by more than 20 °C. Sequence comparison suggests that one of the ion‐pairs involved in dimerization is unique for ProDHs from Thermus species. In summary, intermolecular ion‐pairs improve the thermostability of TtProDH and a trade‐off is made between thermostability and catalytic activity.     

Crystal structure of Cas1 in complex with branched DNA

Cas1 is a key component of the CRISPR adaptation complex, which captures and integrates foreign DNA into the CRISPR array,resulting in the generation of new spacers. We have determined crystal structures of Thermus thermophilus Cas1 involved in new spacer acquisition both in complex with branched DNA and in the free state. Cas1 forms an asymmetric dimer without DNA.Conversely, two asymmetrical dimers bound to two branched DNAs result in the formation of a DNA-mediated tetramer, dimer of structurally asymmetrical dimers, in which the two subunits markedly present different conformations. In the DNA binding complex, the N-terminal domain adopts different orientations with respect to the C-terminal domain in the two monomers that form the dimer. Substrate binding triggers a conformational change in the loop 164–177 segment. This loop is also involved in the 3′ fork arm and 5′ fork arm strand recognition in monomer A and B, respectively. This study provides important insights into the molecular mechanism of new spacer adaptation.     

Crystal structure of the catalytic domain of PigE: A transaminase involved in the biosynthesis of 2-methyl-3-n-amyl-pyrrole (MAP) from Serratia sp. FS14

Prodigiosin, a tripyrrole red pigment synthesized by Serratia and some other microbes through a bifurcated biosynthesis pathway, MBC (4-methoxy-2,2′-bipyrrole-5-carbaldehyde) and MAP (2-methyl-3-n-amyl-pyrrole) are synthesized separately and then condensed by PigC to form prodigiosin. MAP is synthesized sequentially by PigD, PigE and PigB. PigE catalyzes the transamination of an amino group to the aldehyde group of 3-acetyloctanal, resulting in an aminoketone, which spontaneously cyclizes to form H₂MAP. Here we report the crystal structure of the catalytic domain of PigE which involved in the biosynthesis of prodigiosin precursor MAP for the first time to a resolution of 2.3 Å with a homodimer in the asymmetric unit. The monomer of PigE catalytic domain is composed of three domains with PLP as cofactor: a small N-terminal domain connecting the catalytic domain with the front part of PigE, a large PLP-binding domain and a C-terminal domain. The residues from both monomers build the PLP binding site at the interface of the dimer which resembles the other PLP-dependent enzymes. Structural comparison of PigE with Thermus thermophilus AcOAT showed a higher hydrophobic and smaller active site of PigE, these differences may be the reason for substrate specificity.     

A comparative analysis of extreme thermophilic bacteria belonging to the genus Thermus

Several extreme thermophilic Gram negative bacteria found in a thermally polluted river in Belgium have been compared with Thermus strains isolated from widely distant geographical areas. This analysis has become possible after the design of a new culture medium (162).All strains examined (including the isolate successively denominated Flavobacterium thermophilum and Thermus thermophilus) were found to be morphologically identical with strain YT-1 of Thermus aquaticus. The cells are immotile, rod-like, strictly aerobic, catalase and oxidase positive. They produce amylase, hydrolyze gelatin and are confirmed to be highly sensitive towards penicillin.The nutritional pattern of all strains has been analysed extensively, by testing a broad spectrum of possible substrates.The strains display a uniform response to the microbiological tests applied and most probably belong to the same species: Thermus aquaticus.Abbreviations GC guanosine cytosine - ATCC American Type Culture Collection - DSM Deutsche Sammlung von Mikroorganismen     

Crystal structure of Helicobacter pylori MinE,a cell division topological specificity factor

In Gram‐negative bacteria, proper placement of the FtsZ ring, mediated by nucleoid occlusion and the activities of the dynamic oscillating Min proteins MinC, MinD and MinE, is required for correct positioning of the cell division septum. MinE is a topological specificity factor that counters the activity of MinCD division inhibitor at the mid‐cell division site. Its structure consists of an anti‐MinCD domain and a topology specificity domain (TSD). Previous NMR analysis of truncated Escherichia coli MinE showed that the TSD domain contains a long α‐helix and two anti‐parallel β‐strands, which mediate formation of a homodimeric α/β structure. Here we report the crystal structure of full‐length Helicobacter pylori MinE and redefine its TSD based on that structure. The N‐terminal region of the TSD (residues 19–26), previously defined as part of the anti‐MinCD domain, forms a β‐strand (βA) and participates in TSD folding. In addition, H. pylori MinE forms a dimer through the interaction of anti‐parallel βA‐strands. Moreover, we observed serial dimer–dimer interactions within the crystal packing, resulting in the formation of a multimeric structure. We therefore redefine the functional domain of MinE and propose that a multimeric filamentous structure is formed through anti‐parallel β‐strand interactions.     

The dimeric transmembrane domain of prolyl dipeptidase DPP-IV contributes to its quaternary structure and enzymatic activities

Dipeptidyl peptidase IV (DPP‐IV) is a drug target in the treatment of human type II diabetes. It is a type II membrane protein with a single transmembrane domain (TMD) anchoring the extracellular catalytic domain to the membrane. DPP‐IV is active as a dimer, with two dimer interacting surfaces located extracellularly. In this study, we demonstrate that the TM of DPP‐IV promotes DPP‐IV dimerization and rescues monomeric DPP‐IV mutants into partial dimers, which is specific and irreplaceable by TMs of other type II membrane proteins. By bioluminescence resonance energy transfer (BRET) and peptide electrophoresis, we found that the TM domain of DPP‐IV is dimerized in mammalian cells and in vitro. The TM dimer interaction is very stable, based on our results with TM site‐directed mutagenesis. None of the mutations, including the introduction of two prolines, resulted in their complete disruption to monomers. However, these TM proline mutations result in a significant reduction of DPP‐IV enzymatic activity, comparable to what is found with mutations near the active site. A systematic analysis of TM structures deposited in the Protein Data Bank showed that prolines in the TM generally produce much bigger kinking angles than occur in nonproline‐containing TMs. Thus, the proline‐dependent reduction in enzyme activity may result from propagated conformational changes from the TM to the extracellular active site. Our results demonstrate that TM dimerization and conformation contribute significantly to the structure and activity of DPP‐IV. Optimal enzymatic activity of DPP‐IV requires an optimal interaction of all three dimer interfaces, including its TM.     

Crystal structure studies of NADP dependent isocitrate dehydrogenase from Thermus thermophilus exhibiting a novel terminal domain

NADP⁺ dependent isocitrate dehydrogenase (IDH) is an enzyme catalyzing oxidative decarboxylation of isocitrate into oxalosuccinate (intermediate) and finally the product α-ketoglutarate. The crystal structure of Thermus thermophilus isocitrate dehydrogenase (TtIDH) ternary complex with citrate and cofactor NADP⁺ was determined using X-ray diffraction method to a resolution of 1.80 Å. The overall fold of this protein was resolved into large domain, small domain and a clasp domain. The monomeric structure reveals a novel terminal domain involved in dimerization, very unique and novel domain when compared to other IDH’s. And, small domain and clasp domain showing significant differences when compared to other IDH’s of the same sub-family. The structure of TtIDH reveals the absence of helix at the clasp domain, which is mainly involved in oligomerization in other IDH’s. Also, helices/beta sheets are absent in the small domain, when compared to other IDH’s of the same sub family. The overall TtIDH structure exhibits closed conformation with catalytic triad residues, Tyr144-Asp248-Lys191 are conserved. Oligomerization of the protein is quantized using interface area and subunit–subunit interactions between protomers. Overall, the TtIDH structure with novel terminal domain may be categorized as a first structure of subfamily of type IV.     

Structure of dystrophia myotonica protein kinase

Dystrophia myotonica protein kinase (DMPK) is a serine/threonine kinase composed of a kinase domain and a coiled‐coil domain involved in the multimerization. The crystal structure of the kinase domain of DMPK bound to the inhibitor bisindolylmaleimide VIII (BIM‐8) revealed a dimeric enzyme associated by a conserved dimerization domain. The affinity of dimerisation suggested that the kinase domain alone is insufficient for dimerisation in vivo and that the coiled‐coil domains are required for stable dimer formation. The kinase domain is in an active conformation, with a fully‐ordered and correctly positioned αC helix, and catalytic residues in a conformation competent for catalysis. The conserved hydrophobic motif at the C‐terminal extension of the kinase domain is bound to the N‐terminal lobe of the kinase domain, despite being unphosphorylated. Differences in the arrangement of the C‐terminal extension compared to the closely related Rho‐associated kinases include an altered PXXP motif, a different conformation and binding arrangement for the turn motif, and a different location for the conserved NFD motif. The BIM‐8 inhibitor occupies the ATP site and has similar binding mode as observed in PDK1.     

Localized synthesis of the outer envelope from Thermus thermophilus

In agreement with its distinct phylogenetic origin, the envelope of Thermus thermophilus consists of a complex pattern of layers with properties intermediate between those of Gram positives and Proteobacteria. Its cell wall of Gram positive composition is surrounded by an outer envelope that includes a crystalline layer scaffold built up by the SlpA protein, lipids and polysaccharides. The synthesis of this outer envelope has been studied by confocal microscopy. Available amino groups from the cell surface, mainly belonging to the SlpA protein, were covalently labelled in vivo with fluorescent dyes. Stained cells were able to grow without any apparent loss of viability, allowing the localization of the regions of new synthesis as dark nonfluorescent spots. Our results demonstrate that the outer envelope of T. thermophilus is synthesized from a central point in the cells, likely following a helical pattern. Cell poles and subpolar regions are basically inert and retain their label for generations.     

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.     

Formation and carbon monoxide‐dependent dissociation of Allochromatium vinosum cytochrome c′ oligomers using domain‐swapped dimers

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c′ from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four‐helix bundle structure containing a covalently bound five‐coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer–monomer transition according to the absence and presence of CO. Herein, domain‐swapped dimeric AVCP was constructed and utilized to form a tetramer and high‐order oligomers. The X‐ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain‐swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain‐swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit–subunit interactions at the interfaces of the domain‐swapped dimer and monomer subunits in the tetramer were also similar to the subunit–subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain‐swapped dimer and two monomers upon CO binding. Without monomers, the domain‐swapped dimers formed tetramers, hexamers, and higher‐order oligomers in the absence of CO, whereas the oligomers dissociated to domain‐swapped dimers in the presence of CO, demonstrating that the domain‐swapped dimer maintains the CO‐induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer–monomer transition protein.     

Identification of residues within ligand‐binding domain 1 (LBD1) of the Borrelia burgdorferi OspC protein required for function in the mammalian environment

Borrelia burgdorferi outer surface protein C (ospC) is required for the establishment of infection in mammals. However, its precise function remains controversial. The biologically active form of OspC appears to be a homodimer. Alpha helix 1 and 1′ of the apposing monomers form a solvent‐accessible pocket at the dimeric interface that presents a putative ligand‐binding domain (LBD1). Here we employ site‐directed and allelic‐exchange mutagenesis to test the hypothesis that LBD1 is a determinant of OspC function in the mammalian environment. Substitution of residues K60, E61 and E63 which line LBD1 resulted in the loss of infectivity or influenced dissemination. Analyses of the corresponding recombinant proteins demonstrated that the loss of function was not due to structural perturbation, impaired dimer formation or the loss of plasminogen binding. This study is the first to assess the involvement of individual residues and domains of OspC in its in vivo function. The data support the hypothesis that OspC interacts with a mammalian derived ligand that is critical for survival during early infection. These results shed new light on the structure–functions relationships of OspC and challenge existing hypotheses regarding OspC function in mammals.     

The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae

The type 2 secretion system (T2SS), a multi-protein machinery that spans both the inner and the outer membranes of Gram-negative bacteria, is used for the secretion of several critically important proteins across the outer membrane. Here we report the crystal structure of the N-terminal cytoplasmic domain of EpsF, an inner membrane spanning T2SS protein from Vibrio cholerae. This domain consists of a bundle of six anti-parallel helices and adopts a fold that has not been described before. The long C-terminal helix α6 protrudes from the body of the domain and most likely continues as the first transmembrane helix of EpsF. Two N-terminal EpsF domains form a tight dimer with a conserved interface, suggesting that the observed dimer occurs in the T2SS of many bacteria. Two calcium binding sites are present in the dimer interface with ligands provided for each site by both subunits. Based on this new structure, sequence comparisons of EpsF homologs and localization studies of GFP fused with EpsF, we propose that the second cytoplasmic domain of EpsF adopts a similar fold as the first cytoplasmic domain and that full-length EpsF, and its T2SS homologs, have a three-transmembrane helix topology.     

Molecular Analyses of the Natural Transformation Machinery and Identification of Pilus Structures in the Extremely Thermophilic Bacterium Thermus thermophilus Strain HB27

Thermus thermophilus HB27, an extremely thermophilic bacterium, exhibits high competence for natural transformation. To identify genes of the natural transformation machinery of T. thermophilus HB27, we performed homology searches in the partially completed T. thermophilus genomic sequence for conserved competence genes. These analyses resulted in the detection of 28 open reading frames (ORFs) exhibiting significant similarities to known competence proteins of gram-negative and gram-positive bacteria. Disruption of 15 selected potential competence genes led to the identification of 8 noncompetent mutants and one transformation-deficient mutant with a 100-fold reduced transformation frequency. One competence protein is similar to DprA of Haemophilus influenzae, seven are similar to type IV pilus proteins of Pseudomonas aeruginosa or Neisseria gonorrhoeae (PilM, PilN, PilO, PilQ, PilF, PilC, PilD), and another deduced protein (PilW) is similar to a protein of unknown function in Deinococcus radiodurans R1. Analysis of the piliation phenotype of T. thermophilus HB27 revealed the presence of single pilus structures on the surface of the wild-type cells, whereas the noncompetent pil mutants of Thermus, with the exception of the pilF mutant, were devoid of pilus structures. These results suggest that pili and natural transformation in T. thermophilus HB27 are functionally linked.     

The structure of the C-terminal actin-binding domain of talin

Talin is a large dimeric protein that couples integrins to cytoskeletal actin. Here, we report the structure of the C-terminal actin-binding domain of talin, the core of which is a five-helix bundle linked to a C-terminal helix responsible for dimerisation. The NMR structure of the bundle reveals a conserved surface-exposed hydrophobic patch surrounded by positively charged groups. We have mapped the actin-binding site to this surface and shown that helix 1 on the opposite side of the bundle negatively regulates actin binding. The crystal structure of the dimerisation helix reveals an antiparallel coiled-coil with conserved residues clustered on the solvent-exposed face. Mutagenesis shows that dimerisation is essential for filamentous actin (F-actin) binding and indicates that the dimerisation helix itself contributes to binding. We have used these structures together with small angle X-ray scattering to derive a model of the entire domain. Electron microscopy provides direct evidence for binding of the dimer to F-actin and indicates that it binds to three monomers along the long-pitch helix of the actin filament.     

Biophysical Analysis of the MHR Motif in Folding and Domain Swapping of the HIV Capsid Protein C-Terminal Domain

Infection by human immunodeficiency virus (HIV) depends on the function, in virion morphogenesis and other stages of the viral cycle, of a highly conserved structural element, the major homology region (MHR), within the carboxyterminal domain (CTD) of the capsid protein. In a modified CTD dimer, MHR is swapped between monomers. While no evidence for MHR swapping has been provided by structural models of retroviral capsids, it is unknown whether it may occur transiently along the virus assembly pathway. Whatever the case, the MHR-swapped dimer does provide a novel target for the development of anti-HIV drugs based on the concept of trapping a nonnative capsid protein conformation. We have carried out a thermodynamic and kinetic characterization of the domain-swapped CTD dimer in solution. The analysis includes a dissection of the role of conserved MHR residues and other amino acids at the dimerization interface in CTD folding, stability, and dimerization by domain swapping. The results revealed some energetic hotspots at the domain-swapped interface. In addition, many MHR residues that are not in the protein hydrophobic core were nevertheless found to be critical for folding and stability of the CTD monomer, which may dramatically slow down the swapping reaction. Conservation of MHR residues in retroviruses did not correlate with their contribution to domain swapping, but it did correlate with their importance for stable CTD folding. Because folding is required for capsid protein function, this remarkable MHR-mediated conformational stabilization of CTD may help to explain the functional roles of MHR not only during immature capsid assembly but in other processes associated with retrovirus infection. This energetic dissection of the dimerization interface in MHR-swapped CTD may also facilitate the design of anti-HIV compounds that inhibit capsid assembly by conformational trapping of swapped CTD dimers.     

Crystal structure of N-domain of FKBP22 from Shewanella sp. SIB1: dimer dissociation by disruption of Val-Leu knot

FK506‐binding protein 22 (FKBP22) from the psychrotophic bacterium Shewanella sp. SIB1 (SIB1 FKBP22) is a homodimeric protein with peptidyl prolyl cis‐trans isomerase (PPIase) activity. Each monomer consists of the N‐terminal domain responsible for dimerization and C‐terminal catalytic domain. To reveal interactions at the dimer interface of SIB1 FKBP22, the crystal structure of the N‐domain of SIB1 FKBP22 (SN‐FKBP22, residues 1‐68) was determined at 1.9 Å resolution. SN‐FKBP22 forms a dimer, in which each monomer consists of three helices (α1, α2, and α3N). In the dimer, two monomers have head‐to‐head interactions, in which residues 8–64 of one monomer form tight interface with the corresponding residues of the other. The interface is featured by the presence of a Val‐Leu knot, in which Val37 and Leu41 of one monomer interact with Val41 and Leu37 of the other, respectively. To examine whether SIB1 FKBP22 is dissociated into the monomers by disruption of this knot, the mutant protein V37R/L41R‐FKBP22, in which Val37 and Leu41 of SIB1 FKBP22 are simultaneously replaced by Arg, was constructed and biochemically characterized. This mutant protein was indistinguishable from the SIB1 FKBP22 derivative lacking the N‐domain in oligomeric state, far‐UV CD spectrum, thermal denaturation curve, PPIase activity, and binding ability to a folding intermediate of protein, suggesting that the N‐domain of V37R/L41R‐FKBP22 is disordered. We propose that a Val‐Leu knot at the dimer interface of SIB1 FKBP22 is important for dimerization and dimerization is required for folding of the N‐domain.