Conserved Asp Residues Research Articles

Characterization of the Deep-Sea Streptomyces sp. SCSIO 02999 Derived VapC/VapB Toxin-Antitoxin System in Escherichia coli.

Toxin-antitoxin (TA) systems are small genetic elements that are ubiquitous in prokaryotes. Most studies on TA systems have focused on commensal and pathogenic bacteria; yet very few studies have focused on TAs in marine bacteria, especially those isolated from a deep sea environment. Here, we characterized a type II VapC/VapB TA system from the deep-sea derived Streptomyces sp. SCSIO 02999. The VapC (virulence-associated protein) protein belongs to the PIN (PilT N-terminal) superfamily. Overproduction of VapC strongly inhibited cell growth and resulted in a bleb-containing morphology in E. coli. The toxicity of VapC was neutralized through direct protein–protein interaction by a small protein antitoxin VapB encoded by a neighboring gene. Antitoxin VapB alone or the VapB/VapC complex negatively regulated the vapBC promoter activity. We further revealed that three conserved Asp residues in the PIN domain were essential for the toxic effect of VapC. Additionally, the VapC/VapB TA system stabilized plasmid in E. coli. Furthermore, VapC cross-activated transcription of several TA operons via a partially Lon-dependent mechanism in E. coli, and the activated toxins accumulated more preferentially than their antitoxin partners. Collectively, we identified and characterized a new deep sea TA system in the deep sea Streptomyces sp. and demonstrated that the VapC toxin in this system can cross-activate TA operons in E. coli.

RGG boxes within the TET/FET family of RNA-binding proteins are functionally distinct

ABSTRACTThe multi-functional TET (TAF15/EWS/TLS) or FET (FUS/EWS/TLS) protein family of higher organisms harbor a transcriptional-activation domain (EAD) and an RNA-binding domain (RBD). The transcriptional activation function is, however, only revealed in oncogenic TET-fusion proteins because in native TET proteins it is auto-repressed by RGG-boxes within the TET RBD. Auto-repression is suggested to involve direct cation–pi interactions between multiple Arg residues within RGG boxes and EAD aromatics. Via analysis of TET transcriptional activity in different organisms, we report herein that repression is not autonomous but instead requires additional trans-acting factors. This finding is not supportive of a proposed model whereby repression occurs via a simple intramolecular EAD/RGG-box interaction. We also show that RGG-boxes present within reiterated YGGDRGG repeats that are unique to TAF15, are defective for repression due to the conserved Asp residue. Thus, RGG boxes within TET proteins can be functionally distinguished. While our results show that YGGDRGG repeats are not involved in TAF15 auto-repression, their remarkable number and conservation strongly suggest that they may confer specialized properties to TAF15 and thus contribute to functional differentiation within the TET/FET protein family.

Transcriptional Regulation, Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans

ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn(2+) and Mn(2+) from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn(2+) or Mn(2+) specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties, and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn(2+) over Mn(2+) specificity, the Zn(2+) ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn(2+)-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn(2+)-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn(2+)-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn(2+) specificity.

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

Most enzymes present a catalytic mechanism where one or more proton transfer events occur coupled tightly together with the enzymatic chemical reaction. We show here that inactivating mutations decouple this proton transfer step from the phosphate cleavage reaction in dUTPase. Homotrimeric dUTPase enzymes catalyze the hydrolysis of dUTP to dUMP and pyrophosphate, using largely similar structural and functional groups as most AAA+ enzymes. dUTPases typically use a single Mg2+ ion as a cofactor in the active site that is formed by direct protein–protein contacts including all three protomers. Here we focus on the C-terminal arm structural motif, which has sequence and functional similarities to P-loop motifs and is required for catalysis. In this work, we have studied the functional roles of the C-terminal arm in ligand binding and catalysis by using QM/MM (quantum mechanics/molecular mechanics) calculations in conjunction with site-directed mutagenesis experiments. We also present a new method to assess the metal ion coordination symmetry during the catalytic reaction. Using this new implementation, we identified that the coordination symmetry follows a consistent pattern in the three systems studied, reaching the most symmetrical state near the transition states. We found that the phosphate cleavage proceeds with a concerted bimolecular (ANDN) mechanism with a loose dissociative transition state and that it is coupled with a proton transfer step involving a unanimously conserved Asp residue. We show that the main mechanistic effect of the lack of the C-terminal arm is to decouple the phosphate cleavage from the subsequent proton transfer step, resulting in a high-barrier altered reaction pathway.

The VieB auxiliary protein negatively regulates the VieSA signal transduction system in Vibrio cholerae.

BackgroundVibrio cholerae is a facultative pathogen that lives in the aquatic environment and the human host. The ability of V. cholerae to monitor environmental changes as it transitions between these diverse environments is vital to its pathogenic lifestyle. One way V. cholerae senses changing external stimuli is through the three-component signal transduction system, VieSAB, which is encoded by the vieSAB operon. The VieSAB system plays a role in the inverse regulation of biofilm and virulence genes by controlling the concentration of the secondary messenger, cyclic-di-GMP. While the sensor kinase, VieS, and the response regulator, VieA, behave similar to typical two-component phosphorelay systems, the role of the auxiliary protein, VieB, is unclear.ResultsHere we show that VieB binds to VieS and inhibits its autophosphorylation and phosphotransfer activity thus preventing phosphorylation of VieA. Additionally, we show that phosphorylation of the highly conserved Asp residue in the receiver domain of VieB regulates the inhibitory activity of VieB.ConclusionTaken together, these data point to an inhibitory role of VieB on the VieSA phosphorelay, allowing for additional control over the signal output. Insight into the function and regulatory mechanism of the VieSAB system improves our understanding of how V. cholerae controls gene expression as it transitions between the aquatic environment and human host.

Membrane Topology of Hedgehog Acyltransferase

Hedgehog acyltransferase (Hhat) is a multipass transmembrane enzyme that mediates the covalent attachment of the 16-carbon fatty acid palmitate to the N-terminal cysteine of Sonic Hedgehog (Shh). Palmitoylation of Shh by Hhat is critical for short and long range signaling. Knowledge of the topological organization of Hhat transmembrane helices would enhance our understanding of Hhat-mediated Shh palmitoylation. Bioinformatics analysis of transmembrane domains within human Hhat using 10 different algorithms resulted in highly consistent predictions in the C-terminal, but not in the N-terminal, region of Hhat. To empirically determine the topology of Hhat, we designed and exploited Hhat constructs containing either terminal or 12 different internal epitope tags. We used selective permeabilization coupled with immunofluorescence as well as a protease protection assay to demonstrate that Hhat contains 10 transmembrane domains and 2 re-entrant loops. The invariant His and highly conserved Asp residues within the membrane-bound O-acyltransferase (MBOAT) homology domain are segregated on opposite sides of the endoplasmic reticulum membrane. The localization of His-379 on the lumenal membrane surface is consistent with a role for this invariant residue in catalysis. Analysis of the activity and stability of the Hhat constructs revealed that the C-terminal MBOAT domain is especially sensitive to manipulation. Moreover, there was remarkable similarity in the overall topological organization of Hhat and ghrelin O-acyltransferase, another MBOAT family member. Knowledge of the topological organization of Hhat could serve as an important tool for further design of selective Hhat inhibitors.

Characterization of recombinant biosynthetic precursors of the cysteine tryptophylquinone cofactors of l-lysine-epsilon-oxidase and glycine oxidase from Marinomonas mediterranea

The lysine-ε-oxidase, LodA, and glycine oxidase, GoxA, from Marinomonas mediteranea each possesses a cysteine tryptophylquinone (CTQ) cofactor. This cofactor is derived from posttranslational modifications which are covalent crosslinking of tryptophan and cysteine residues and incorporation of two oxygen atoms into the indole ring of Trp. In this manuscript, it is shown that the recombinant synthesis of LodA and GoxA containing a fully synthesized CTQ cofactor requires coexpression of a partner flavoprotein, LodB for LodA and GoxB for GoxA, which are not interchangeable. An inactive precursor of LodA or GoxA which contained a monohydroxylated Trp residue and no crosslink to the Cys was isolated from the soluble fraction when they were expressed alone. The structure of LodA revealed an Asp residue close to the cofactor which is conserved in quinohemoprotein amine dehydrogenase (QHNDH), containing CTQ, and methylamine dehydrogenase (MADH) containing tryptophan tryptophylquinone (TTQ) as cofactor. To study the role of this residue in the synthesis of the LodA precursor, Asp-512 was mutated to Ala. When the mutant protein was coexpressed with LodB an inactive protein was isolated which was soluble and contained no modifications at all, suggesting a role for this Asp in the initial LodB-independent hydroxylation of Trp. A similar role had been proposed for this conserved Asp residue in MADH. It is noteworthy that the formation of TTQ in MADH from the precursor also requires an accessory enzyme for its biosynthesis but it is a diheme enzyme MauG and not a flavoprotein. The results presented reveal novel mechanisms of post-translational modification involved in the generation of protein-derived cofactors. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Structure of a backtracked state reveals conformational changes similar to the state following nucleotide incorporation in human norovirus polymerase.

The RNA-dependent RNA polymerase (RdRP) from norovirus (NV) genogroup II has previously been crystallized as an apoenzyme (APO1) in multiple crystal forms, as well as as a pre-incorporation ternary complex (PRE1) bound to Mn(2+), various nucleoside triphosphates and an RNA primer-template duplex in an orthorhombic crystal form. When crystallized under near-identical conditions with a slightly different RNA primer/template duplex, however, the enzyme-RNA complex forms tetragonal crystals (anisotropic data, dmin ≃ 1.9 Å) containing a complex with the primer/template bound in a backtracked state (BACK1) similar to a post-incorporation complex (POST1) in a step of the enzymatic cycle immediately following nucleotidyl transfer. The BACK1 conformation shows that the terminal nucleotide of the primer binds in a manner similar to the nucleoside triphosphate seen in the PRE1 complex, even though the terminal two phosphoryl groups in the triphosphate moiety are absent and a covalent bond is present between the α-phosphoryl group of the terminal nucleotide and the 3'-oxygen of the penultimate nucleotide residue. The two manganese ions bound at the active site coordinate to conserved Asp residues and the bridging phosphoryl group of the terminal nucleotide. Surprisingly, the conformation of the thumb domain in BACK1 resembles the open APO1 state more than the closed conformation seen in PRE1. The BACK1 complex thus reveals a hybrid state in which the active site is closed while the thumb domain is open. Comparison of the APO1, PRE1 and BACK1 structures of NV polymerase helps to reveal a more complete and complex pathway of conformational changes within a single RdRP enzyme system. These conformational changes lend insight into the mechanism of RNA translocation following nucleotidyl transfer and suggest novel approaches for the development of antiviral inhibitors.

Atypical OmpR/PhoB Subfamily Response Regulator GlnR of Actinomycetes Functions as a Homodimer, Stabilized by the Unphosphorylated Conserved Asp-focused Charge Interactions

The OmpR/PhoB subfamily protein GlnR of actinomycetes is an orphan response regulator that globally coordinates the expression of genes related to nitrogen metabolism. Biochemical and genetic analyses reveal that the functional GlnR from Amycolatopsis mediterranei is unphosphorylated at the potential phosphorylation Asp(50) residue in the N-terminal receiver domain. The crystal structure of this receiver domain demonstrates that it forms a homodimer through the α4-β5-α5 dimer interface highly similar to the phosphorylated typical response regulator, whereas the so-called "phosphorylation pocket" is not conserved, with its space being occupied by an Arg(52) from the β3-α3 loop. Both in vitro and in vivo experiments confirm that GlnR forms a functional homodimer via its receiver domain and suggest that the charge interactions of Asp(50) with the highly conserved Arg(52) and Thr(9) in the receiver domain may be crucial in maintaining the proper conformation for homodimerization, as also supported by molecular dynamics simulations of the wild type GlnR versus the deficient mutant GlnR(D50A). This model is backed by the distinct phenotypes of the total deficient GlnR(R52A/T9A) double mutant versus the single mutants of GlnR (i.e. D50N, D50E, R52A and T9A), which have only minor effects upon both dimerization and physiological function of GlnR in vivo, albeit their DNA binding ability is weakened compared with that of the wild type. By integrating the supportive data of GlnRs from the model Streptomyces coelicolor and the pathogenic Mycobacterium tuberculosis, we conclude that the actinomycete GlnR is atypical with respect to its unphosphorylated conserved Asp residue being involved in the critical Arg/Asp/Thr charge interactions, which is essential for maintaining the biologically active homodimer conformation.

A Cyclic Nucleotide-Gated Channel Mutation Associated with Canine Daylight Blindness Provides Insight into a Role for the S2 Segment Tri-Asp motif in Channel Biogenesis

Cone cyclic nucleotide-gated channels are tetramers formed by CNGA3 and CNGB3 subunits; CNGA3 subunits function as homotetrameric channels but CNGB3 exhibits channel function only when co-expressed with CNGA3. An aspartatic acid (Asp) to asparagine (Asn) missense mutation at position 262 in the canine CNGB3 (D262N) subunit results in loss of cone function (daylight blindness), suggesting an important role for this aspartic acid residue in channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. This motif is conserved in all CNG channels. Here we examine mutations in canine CNGA3 homomeric channels using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in the absence of nucleotide-activated currents in heterologous expression. A fluorescent tag on CNGA3 shows mislocalization of mutant channels. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in some functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 channels. This failure to record heteromeric currents suggests that Asp/Asn mutations affect heteromeric subunit assembly. A homology model of S1–S6 of the CNGA3 channel was generated and relaxed in a membrane using molecular dynamics simulations. The model predicts that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3/S4. We propose that the D262N mutation in dogs with CNGB3-day blindness results in the loss of these inter-helical interactions altering the electrostatic equilibrium within in the S1–S4 bundle. Because residues analogous to Tri-Asp in the voltage-gated Shaker potassium channel family were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions impairs the monomer folding state in D262N mutant CNG channels during biogenesis.

A Conserved Aspartic Acid Is Important for Agonist (VUAA1) and Odorant/Tuning Receptor-Dependent Activation of the Insect Odorant Co-Receptor (Orco)

Insect odorant receptors function as heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor, and a conserved co-receptor (Orco). An Orco agonist, VUAA1, is able to activate both heteromeric and homomeric Orco-containing channels. Very little is known about specific residues in Orco that contribute to cation permeability and gating. We investigated the importance of two conserved Asp residues, one in each of transmembrane domains 5 and 7, for channel function by mutagenesis. Drosophila melanogaster Orco and its substitution mutants were expressed in HEK cells and VUAA1-stimulated channel activity was determined by Ca2+ influx and whole-cell patch clamp electrophysiology. Substitution of D466 in transmembrane 7 with amino acids other than glutamic acid resulted in a substantial reduction in channel activity. The D466E Orco substitution mutant was ∼2 times more sensitive to VUAA1. The permeability of the D466E Orco mutant to cations was unchanged relative to wild-type Orco. When D466E Orco is co-expressed with a conventional tuning odorant receptor, the heteromeric complex also shows increased sensitivity to an odorant. Thus, the effect of the D466E mutation is not specific to VUAA1 agonism or dependent on homomeric Orco assembly. We suggest the gain-of-activation characteristic of the D466E mutant identifies an amino acid that is likely to be important for activation of both heteromeric and homomeric insect odorant receptor channels.

Structure‐based engineering of an infrared fluorescent protein marker

Infrared fluorescent proteins (IFPs) emit a signal less prone to absorbance by hemoglobin and water, making them ideal candidates for mammalian imaging. Bacteriophytochromes (Bphs), bacterial red‐light photoreceptors, are an optimal source of IFPs. The required cofactor for Bph photochemistry, biliverdin (BV), exists naturally in mammalian cells as a product of heme metabolism. Recently, RpBphP2 (P2) from Rhodopseudomonas palustris was successfully engineered as an IFP marker for noninvasive mammalian imaging. RpBphP3 (P3), which shares a 52% sequence identity with P2, is naturally fluorescent due to an unusually long excited‐state lifetime. Comparative sequence and structural analyses of Bphs identified a highly conserved Asp residue essential for stabilization of BV in the chromophore‐binding domain. Using site‐directed mutagenesis we introduced a single amino acid substitution, D216H, effectively disrupting the photochemistry of P3 as observed by UV‐vis absorption spectroscopy. In addition, the P3 D216H mutant displays a higher fluorescence quantum yield relative to wild type P3. We cloned P3 D216H into the mammalian expression vector, Adtrack‐CMV. Stability of this protein marker will be characterized using in vitro comparison studies of protein maturation with the P2‐derived IFP. Subsequent research goals include expression and visualization of the P3 mutant in HEK293 and HeLa cell lines.

The abp gene in Geobacillus stearothermophilus T-6 encodes a GH27 β-l-arabinopyranosidase

In this study we demonstrate that the abp gene in Geobacillus stearothermophilus T-6 encodes a family 27 glycoside hydrolase β-l-arabinopyranosidase. The catalytic constants towards the chromogenic substrate pNP-β-l-arabinopyranoside were 0.8±0.1mM, 6.6±0.3s−1, and 8.2±0.3s−1mM−1 for Km, kcat and kcat/Km, respectively. 13C NMR spectroscopy unequivocally showed that Abp is capable of removing β-l-arabinopyranose residues from the natural arabino-polysaccharide, larch arabinogalactan. Most family 27 enzymes are active on galactose and contain a conserved Asp residue, whereas in Abp this residue is Ile67, which shifts the specificity of the enzyme towards arabinopyranoside.

Critical roles of Asp270 and Trp273 in the α-repeat of the carbohydrate-binding module of endo-1,3-β-glucanase for laminarin-binding avidity

A carbohydrate-binding module from family 13 (CBM13), appended to the catalytic domain of endo-1,3-β-glucanase from Cellulosimicrobium cellulans, was overexpressed in E. coli, and its interactions with β-glucans, laminarin and laminarioligosaccharides, were analyzed using surface plasmon resonance biosensor and isothermal titration calorimetry. The association constants for laminarin and laminarioligosaccharides were determined to be approximately 10(6) M(-1) and 10(4) M(-1), respectively, indicating that 2 or 3 binding sites in the α-, β-, and γ-repeats of CBM13 are involved in laminarin binding in a cooperative manner. The binding avidity is approximately 2-orders higher than the monovalent binding affinity. Mutational analysis of the conserved Asp residues in the respective repeats showed that the α-repeat primarily contributes to β-glucan binding. A Trp residue is predicted to be exposed to the solvent only in the α-repeat and would contribute to β-glucan binding. The α-repeat bound β-glucan with an affinity of approximately 10(4) M(-1), and the other repeats additionally bound laminarin, resulting in the increased binding avidity. This binding is unique compared to the recognition mode of another CBM13 from Streptomyces lividans xylanase.

A D262n Mutation in Canine CNGB3 in a Highly Conserved Acidic S2 Region Causes Complete Colorblindness in a Canine Model

Cyclic nucleotide-gated (CNG) channels in cone photoreceptors are composed of CNGA3 (A3) and CNGB3 (B3) subunits. In a canine achromatopsia model, loss of cone ERG function and color vision are associated with the mutation of Asp (D) 262 to Asn (N) in the B3 subunit. This Asp residue is conserved in all CNG channels. We examined the D/N mutation in the human (h) B3 channel co-expressed with a wild-type canine (c) A3 and in a mutant cA3 homomeric channel in which the conserved Asp residue was substituted with Asn. In patch clamp experiments, both heteromeric wild type cA3 co-expressed with D/N hB3 and mutant homomeric D/N cA3 CNG channels had no cGMP-activated currents providing molecular insights into the cause of canine achromatopsia. D262 of the cB3 is located in the S2 transmembrane domain and, along with D252 and D256, these residues comprise an acidic motif conserved in the CNG channel family and extending to some members of the Shaker K superfamily. Hints about the role of this Asp motif have been gained from studies of related channels reporting interactions between the negatively-charged residues in S2 and the conserved positively-charged residues in other transmembrane domains. We generated mutant A3 subunits by substituting each of the three conserved S2 Asp residues with Asn, Cys, or Glu. Patch clamp analysis showed all of the mutant homomeric A3 channels lost function. These YFP-tagged A3 mutant channels exhibited changes in cellular localization compared to wild type channels suggesting that any change in the Asp motif alters subunit trafficking. Future studies will focus on understanding the role of this Asp motif in intra-subunit helical contacts and proper trafficking and assembly of cone CNG channels.

Functional roles of aspartate residues of the proton-coupled folate transporter (PCFT-SLC46A1); a D156Y mutation causing hereditary folate malabsorption

AbstractThe proton-coupled folate transporter (PCFT; SLC46A1) mediates folate transport into enterocytes in the proximal small intestine; pcft loss-of-function mutations are the basis for hereditary folate malabsorption. The current study explored the roles of Asp residues in PCFT function. A novel, homozygous, loss-of-function mutation, D156Y, was identified in a child of Pakistani origin with hereditary folate malabsorption. Of the 6 other conserved Asp residues, only one, D109, is shown to be required for function. D156Y, along with a variety of other substitutions at this site (Trp, Phe, Val, Asn, or Lys), lacked function due to instability of the PCFT protein. Substantial function was preserved with Glu, Gly, and, to a lesser extent, with Ser, Thr, and Ala substitutions. This correlated with PCFT bio-tinylated at the cell surface. In contrast, all D109 mutants, including D109E, lacked function irrespective of pH (4.5, 5.5, and 7.4) or substrate concentration (0.5-100μM), despite surface expression comparable to wild-type PCFT. Hence, D156 plays a critical role in PCFT protein stability, and D109, located in the first intracellular loop between the second and third transmembrane domains, is absolutely required for PCFT function.

Deceleration of the E1P-E2P Transition and Ion Transport by Mutation of Potentially Salt Bridge-forming Residues Lys-791 and Glu-820 in Gastric H+/K+-ATPase

A lysine residue within the highly conserved center of the fifth transmembrane segment in P(IIC)-type ATPase α-subunits is uniquely found in H,K-ATPases instead of a serine in all Na,K-ATPase isoforms. Because previous studies suggested a prominent role of this residue in determining the electrogenicity of non-gastric H,K-ATPase and in pK(a) modulation of the proton-translocating residues in the gastric H,K-ATPases as well, we investigated its functional significance for ion transport by expressing several Lys-791 variants of the gastric H,K-ATPase in Xenopus oocytes. Although the mutant proteins were all detected at the cell surface, none of the investigated mutants displayed any measurable K(+)-induced stationary currents. In Rb(+) uptake measurements, replacement of Lys-791 by Arg, Ala, Ser, and Glu substantially impaired transport activity and reduced the sensitivity toward the E(2)-specific inhibitor SCH28080. Furthermore, voltage clamp fluorometry using a reporter site in the TM5/TM6 loop for labeling with tetra-methylrhodamine-6-maleimide revealed markedly changed fluorescence signals. All four investigated mutants exhibited a strong shift toward the E(1)P state, in agreement with their reduced SCH28080 sensitivity, and an about 5-10-fold decreased forward rate constant of the E(1)P ↔ E(2)P conformational transition, thus explaining the E(1)P shift and the reduced Rb(+) transport activity. When Glu-820 in TM6 adjacent to Lys-791 was replaced by non-charged or positively charged amino acids, severe effects on fluorescence signals and Rb(+) transport were also observed, whereas substitution by aspartate was less disturbing. These results suggest that formation of an E(2)P-stabilizing interhelical salt bridge is essential to prevent futile proton exchange cycles of H(+) pumping P-type ATPases.

Acceptor Substrate Discrimination in Phosphatidyl-myo-inositol Mannoside Synthesis: STRUCTURAL AND MUTATIONAL ANALYSIS OF MANNOSYLTRANSFERASE CORYNEBACTERIUM GLUTAMICUM PimB′

Long term survival of the pathogen Mycobacterium tuberculosis in humans is linked to the immunomodulatory potential of its complex cell wall glycolipids, which include the phosphatidylinositol mannoside (PIM) series as well as the related lipomannan and lipoarabinomannan glycoconjugates. PIM biosynthesis is initiated by a set of cytosolic α-mannosyltransferases, catalyzing glycosyl transfer from the activated saccharide donor GDP-α-d-mannopyranose to the acceptor phosphatidyl-myo-inositol (PI) in an ordered and regio-specific fashion. Herein, we report the crystal structure of mannosyltransferase Corynebacterium glutamicum PimB′ in complex with nucleotide to a resolution of 2.0 Å. PimB′ attaches mannosyl selectively to the 6-OH of the inositol moiety of PI. Two crystal forms and GDP- versus GDP-α-d-mannopyranose-bound complexes reveal flexibility of the nucleotide conformation as well as of the structural framework of the active site. Structural comparison, docking of the saccharide acceptor, and site-directed mutagenesis pin regio-selectivity to a conserved Asp residue in the N-terminal domain that forces presentation of the correct inositol hydroxyl to the saccharide donor.

Crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase from the hyperthermophile Thermotoga maritima for insights into the coordination of conformational changes and an inhibitor binding

Isopentenyl diphosphate is a precursor of various isoprenoids and is produced by the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids of plants, protozoa and many eubacteria. A key enzyme in the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), has been shown to be the target of fosmidomycin, which works as an antimalarial, antibacterial and herbicidal compound. In this paper, we report studies of kinetics and the crystal structures of the thermostable DXR from the hyperthermophile Thermotoga maritima. Unlike the mesophilic DXRs, Thermotoga DXR (tDXR) showed activity only with Mg(2+) at its growth temperature. We solved the crystal structures of tDXR with and without fosmidomycin. The structure without fosmidomycin but unexpectedly bound with 2-methyl-2,4-pentanediol (MPD), revealing a new extra space available for potential drug design. This structure adopted the closed form by rigid domain rotation but without the flexible loop over the active site, which was considered as a novel conformation. Further, the conserved Asp residue responsible for cation binding seemed to play an important role in adjusting the position of fosmidomycin. Taken together, our kinetic and the crystal structures illustrate the binding mode of fosmidomycin that leads to its slow, tight binding according to the conformational changes of DXR.

Metallochaperone-like genes in Arabidopsis thaliana

A complete inventory of metallochaperone-like proteins containing a predicted HMA domain in Arabidopsis revealed a large family of 67 proteins. 45 proteins, the HIPPs, have a predicted isoprenylation site while 22 proteins, the HPPs, do not. Sequence comparisons divided the proteins into seven major clusters (I-VII). Cluster IV is notable for the presence of a conserved Asp residue before the CysXXCys, metal binding motif, analogous to the Zn binding motif in E. coli ZntA. HIPP20, HIPP21, HIPP22, HIPP26 and HIPP27 in Cluster IV were studied in more detail. All but HIPP21 could rescue the Cd-sensitive, ycf1 yeast mutant but failed to rescue the growth of zrt1zrt2, zrc1cot1 and atx1 mutants. In Arabidopsis, single and double mutants did not show a phenotype but the hipp20/21/22 triple mutant was more sensitive to Cd and accumulated less Cd than the wild-type suggesting the HIPPs can have a role in Cd-detoxification, possibly by binding Cd. Promoter-GUS reporter expression studies indicated variable expression of these HIPPs. For example, in roots, HIPP22 and HIPP26 are only expressed in lateral root tips while HIPP20 and HIPP25 show strong expression in the root vasculature.