Heidi A. Ernst

Structure of the conserved domain of ANAC, a member of the NAC family of transcription factors.

The structure of the DNA‐binding NAC domain of Arabidopsis ANAC (abscisic‐acid‐responsive NAC) has been determined by X‐ray crystallography to 1.9 Å resolution (Protein Data Bank codes 1UT4 and 1UT7). This is the first structure determined for a member of the NAC family of plant‐specific transcriptional regulators. NAC proteins are characterized by their conserved N‐terminal NAC domains that can bind both DNA and other proteins. NAC proteins are involved in developmental processes, including formation of the shoot apical meristem, floral organs and lateral shoots, as well as in plant hormonal control and defence. The NAC domain does not possess a classical helix–turn–helix motif; instead it reveals a new transcription factor fold consisting of a twisted β‐sheet surrounded by a few helical elements. The functional dimer formed by the NAC domain was identified in the structure, which will serve as a structural template for understanding NAC protein function at the molecular level.

Preliminary crystallographic analysis of the NAC domain of ANAC, a member of the plant-specific NAC transcription factor family

The NAC domain (residues 1-168) of ANAC, encoded by the abscisic acid-responsive NAC gene from Arabidopsis thaliana, was recombinantly produced in Escherichia coli and crystallized in hanging drops. Three morphologically different crystal forms were obtained within a relatively narrow range of conditions: 10-15% PEG 4000 and 0.1 M imidazole/malic acid buffer pH 7.0 in the reservoir, 3.2-7.7 mg ml(-1) protein stock and a 1:1 ratio of reservoir to protein solution in the hanging drop. One of the crystal forms, designated crystal form III, was found to be suitable for further X-ray analysis. Form III crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 62.0, b = 75.2, c = 80.8 A at 100 K. The unit-cell volume is consistent with two molecules in the asymmetric unit and a peak in the native Patterson map suggests the presence of a non-crystallographic twofold axis parallel to a crystallographic axis. Size-exclusion chromatography of the NAC domain showed that the dimeric state is also the preferred state in solution and probably represents the biologically active form. Data sets were collected from four potential heavy-atom derivatives of the form III crystals. The derivatized crystals are reasonably isomorphous with the non-derivatized crystals and the four data sets are being evaluated for use in structure determination by multiple isomorphous replacement.

Ligand binding analyses of the putative peptide transporter YjdL from E. coli display a significant selectivity towards dipeptides

Proton-dependent oligopeptide transporters (POTs) are secondary active transporters that couple the inwards translocation of di- and tripeptides to inwards proton translocation. Escherichia coli contains four genes encoding the putative POT proteins YhiP, YdgR, YjdL and YbgH. We have over-expressed the previously uncharacterized YjdL and investigated the peptide specificity by means of uptake inhibition. The IC(50) value for the dipeptide Ala-Ala was measured to 22 mM while Ala-Ala-Ala was not able to inhibit uptake. In addition, IC(50) values of 0.3 mM and 1.5 mM were observed for Ala-Lys and Tyr-Ala, respectively, while the alanyl-extended tripeptides Ala-Lys-Ala, Ala-Ala-Lys, Ala-Tyr-Ala and Tyr-Ala-Ala displayed values of 8, >50, 31 and 31 mM, respectively. These results clearly indicate that unlike most POT members characterized to date, including YdgR and YhiP, YjdL shows significantly higher specificity towards dipeptides.

New insights into the substrate specificities of proton-coupled oligopeptide transporters from E. coli by a pH sensitive assay

Proton‐coupled oligopeptide transporters (POTs) are secondary active transporters that facilitate di‐ and tripeptide uptake by coupling it to an inward directed proton electrochemical gradient. Here the substrate specificities of Escherichia coli POTs YdgR, YhiP and YjdL were investigated by means of a label free transport assay using the hydrophilic pH sensitive dye pyranine and POT overexpressing E. coli cells. The results confirm and extend the functional knowledge on E. coli POTs. In contrast to previous assumptions, alanine and trialanine appears to be substrates of YjdL, albeit poor compared to dipeptides. Similarly tetraalanine apparently is a substrate of both YdgR and YhiP.

The Structure of Amylosucrase from Deinococcus Radiodurans Has an Unusual Open Active-Site Topology

Amylosucrases (ASes) catalyze the formation of an α-1,4-glucosidic linkage by transferring a glucosyl unit from sucrose onto an acceptor α-1,4-glucan. To date, several ligand-bound crystal structures of wild-type and mutant ASes from Neisseria polysaccharea and Deinococcus geothermalis have been solved. These structures all display a very similar overall conformation with a deep pocket leading to the site for transglucosylation, subsite -1. This has led to speculation on how sucrose enters the active site during glucan elongation. In contrast to previous studies, the AS structure from D. radiodurans presented here has a completely empty -1 subsite. This structure is strikingly different from other AS structures, as an active-site-lining loop comprising residues Leu214-Asn225 is found in a previously unobserved conformation. In addition, a large loop harbouring the conserved active-site residues Asp133 and Tyr136 is disordered. The result of the changed loop conformations is that the active-site topology is radically changed, leaving subsite -1 exposed and partially dismantled. This structure provides novel insights into the dynamics of ASes and comprises the first structural support for an elongation mechanism that involves considerable conformational changes to modulate accessibility to the sucrose-binding site and thereby allows successive cycles of glucosyl-moiety transfer to a growing glucan chain.

Functional Investigation of Conserved Membrane-Embedded Glutamate Residues in the Proton-Coupled Peptide Transporter YjdL

Proton-dependent oligopeptide transporters (POTs) are secondary active symporters that utilize the proton gradient to drive the inward translocation of di- and tripeptides. We have mutated two highly conserved membraneembedded glutamate residues (Glu20 and Glu388) in the E. coli POT YjdL to probe their possible functional roles, in particular if they were involved/implicated in recognition of the substrate N-terminus. The mutants (Glu20Asp, Glu20Gln, Glu388Asp, and Glu388Gln) were tested for substrate uptake, which indicated that both the negative charge and the side chain length were important for function. The IC50 values of dipeptides with lack of or varying N-terminus (Ac-Lys, Gly- Lys, β-Ala-Lys, and 4-GABA-Lys), showed that Gly-Lys and β-Ala-Lys ranged between ~0.1 to ~1.0 mM for wild type and Glu20 mutants. However, for Glu388Gln the IC50 increased to ~2.0 and > 10 mM for Gly-Lys and β-Ala-Lys, respectively, suggesting that Glu388, and not Glu20, is able to sense the position of the N-terminus and important for the interaction. Furthermore, uptake as a function of pH showed that the optimum at around pH 6.5 for wild type YjdL shifted to 7.0-7.5 for the Glu388Asp/Gln mutants while the Glu20Asp retained the wild type optimum. Uptake by the Glu20Gln on the other hand was completely unaffected by the bulk pH in the range tested, which indicated a possible role of Glu20 in proton translocation.

Crystallization and preliminary X-ray analysis of Alicyclobacillus acidocaldarius endoglucanase CelA

Crystallization of a family 9 beta-1,4-glucanase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius CelA is reported. Thin plates can be obtained by hanging-drop vapour-diffusion crystallization in high concentrations (60%) of MPD. These crystals are unusual in that they do not bind the dye IZIT in the mother liquor and do not appear to dissolve in water after three weeks or in the storage buffer after 2 d. The crystals diffract weakly and the diffraction pattern is compatible with crystal disorder in one direction. After testing several crystals at the ESRF beamlines ID14-1 and ID14-2, a crystal was found which gave ordered diffraction in all directions. A full data set was collected to 3.0 A resolution, which allowed unambiguous determination of the space group as P2(1)2(1)2 and the unit-cell parameters as a = 85, b = 129.7, c = 48.6 A. Initial promising results from molecular-replacement searches are reported.

Salt Bridge Swapping in the EXXERFXYY Motif of Proton-coupled Oligopeptide Transporters.

Background: Proton-coupled oligopeptide transporters (POTs) facilitate di- and tripeptide uptake. Results: Both glutamates of the highly conserved POT motif EXXERFXYY are required simultaneously for substrate accumulation. Arginine swaps interaction between the glutamates and interacts with another conserved motif, FYING, to facilitate larger structural change. Conclusion: Two conserved motifs interact to facilitate structural changes upon substrate and proton binding. Significance: Our results have contributed to understanding the mechanism of POTs. Proton-coupled oligopeptide transporters (POTs) couple the inward transport of di- or tripeptides with an inwardly directed transport of protons. Evidence from several studies of different POTs has pointed toward involvement of a highly conserved sequence motif, E1XXE2RFXYY (from here on referred to as E1XXE2R), located on Helix I, in interactions with the proton. In this study, we investigated the intracellular substrate accumulation by motif variants with all possible combinations of glutamate residues changed to glutamine and arginine changed to a tyrosine, the latter being a natural variant found in the Escherichia coli POT YjdL. We found that YjdL motif variants with E1XXE2R, E1XXE2Y, E1XXQ2Y, or Q1XXE2Y were able to accumulate peptide, whereas those with E1XXQ2R, Q1XXE2R, or Q1XXQ2Y were unable to accumulate peptide, and Q1XXQ2R abolished uptake. These results suggest a mechanism that involves swapping of an intramotif salt bridge, i.e. R-E2 to R-E1, which is consistent with previous structural studies. Molecular dynamics simulations of the motif variants E1XXE2R and E1XXQ2R support this mechanism. The simulations showed that upon changing conformation arginine pushes Helix V, through interactions with the highly conserved FYING motif, further away from the central cavity in what could be a stabilization of an inward facing conformation. As E2 has been suggested to be the primary site for protonation, these novel findings show how protonation may drive conformational changes through interactions of two highly conserved motifs.

Characterization of different crystal forms of the α-glucosidase MalA from Sulfolobus solfataricus

MalA is an alpha-glucosidase from the hyperthermophilic archaeon Sulfolobus solfataricus. It belongs to glycoside hydrolase family 31, which includes several medically interesting alpha-glucosidases. MalA and its selenomethionine derivative have been overproduced in Escherichia coli and crystallized in four different crystal forms. Microseeding was essential for the formation of good-quality crystals of forms 2 and 4. For three of the crystal forms (2, 3 and 4) full data sets could be collected. The most suitable crystals for structure determination are the monoclinic form 4 crystals, belonging to space group P2(1), from which data sets extending to 2.5 A resolution have been collected. Self-rotation functions calculated for this form and for the orthorhombic (P2(1)2(1)2(1)) form 2 indicate the presence of six molecules in the asymmetric unit related by 32 symmetry.

Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus.

The human alanine-serine-cysteine transporter 1 (Asc-1) belongs to the slc7a family of solute carrier transporters. Asc-1 mediates the uptake of d-serine in an exchanger-type fashion, coupling the process to the release of alanine and cysteine. Among the bacterial Asc-1 homologues, one transporter shows a significantly higher sequence identity (35%) than other bacterial homologues. Therefore, this homologue from Gloeobacter violaceus might represent the best bacterial target for structural studies probing the molecular mechanism of Asc-1. We have over-expressed the G. violaceus transporter by auto-induction, and performed purification and biophysical characterization. In addition, growth studies indicate a preference for alanine as nitrogen source in cells expressing the G. violaceus transporter. It was observed that use of the auto-induction method and subsequent optimization of the length of auto-induction was crucial for obtaining high yields and purity of the transporter. The transporter was purified with yields in the range of 0.2-0.4mg per L culture and eluted in a single peak from a size-exclusion column. The circular dichroism spectrum revealed a folded and apparently all-helical protein.