Contacts by helix 3 in the major groove of Conformation B during the solution simulation.

<p>A) Ribbon diagram looking into helix 3 and the major groove. Asn 51 contacts Ade 3 directly (blue dotted line). Gln 50 makes no direct contact with the DNA. About 20% of the time, water mediated contacts bridge between Gln 50 and Asn 51 (W1) and Gln 50 and DNA bases (in green) (W2). Distances show helix 3 binds farther in the major groove (Asn 51 C-alpha to Ade 3 N7 distance 6.15 Å), and the major groove is slightly wider than in Conformation A (Thy 1 P – Cyt 7* P distance 19.9 Å). B) Asn 51 forms a direct hydrogen bond with the base Ade 3 N7 only in Conformation B (red), not in Conformation A (black). C) and D) The position of the homeodomain differs in Conformation A and Conformation B during the solution simulation. C) Helix 3 binds closer to the DNA in Conformation B (red) than Conformation A (black), as measured by the distance between Asn 51 and Ade 3. D) The major groove is wider in Conformation B than Conformation A during most of the solution simulation, as measured by the Cyt 7* P-Thy 1 P distance. This is consistent with Pdx1 binding deeper in the DNA major groove in Conformation B.</p

Agreement of simulations with the crystal structure.

<p>A) Stability of the crystal simulation. Mass weighted RMSD relative to the crystal structure (PDB 2H1K) (<a href="http://www.rcsb.org" target="_blank">www.rcsb.org</a>) for the eight Pdx1/DNA complexes comprising the unit cell of the 2H1K crystal during unrestrained molecular dynamics in the crystal environment. Conformation A is shown in the top panel and conformation B in the bottom panel. Different colors correspond to the different asymmetric units as specified in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003160#pcbi.1003160.s001" target="_blank">Figure S1</a>. B) Stability of the solution simulation. Mass weighted root mean square deviation of the Pdx1/DNA complex computed with respect to the crystal Conformation A (black) and crystal conformation B (red) starting from Conformation A (top panel) and starting from Conformation B (bottom panel). Both simulations were closer to the crystal Conformation A. The two overhanging DNA bases in the crystal structure were excluded from the simulation.</p

Pdx1 homeodomain/DNA interactions from the crystal structure.

<p>A) Structure of the Pdx1 homeodomain/DNA complex. Pdx1 (blue ribbon) binds the TAAT core DNA sequence (grey). The N-terminal tail binds in the minor groove, and the recognition helix, helix 3, binds in the major groove. Key residues contacting the DNA are shown as stick figures (red): Arg 5 in the minor groove, and Asn 51 in the major groove. Gln 50 contacts the phosphate backbone or the DNA bases through a water-mediated contact. Arg 3 and Arg 43 (black line representation, circled) help stabilize the N-terminal arm, and Lys 2, in the minor groove when helix 3 is properly positioned in the major groove. B) Hydrogen bond contacts with the DNA differ between Conformation A and B in the Pdx1 homeodomain/DNA crystal structure (PDB 2H1K) (<a href="http://www.rcsb.org" target="_blank">www.rcsb.org</a>) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003160#pcbi.1003160-Berman1" target="_blank">[56]</a>. In both conformations (left) Arg 5 contacts Thy 1 and Gua −1* through the minor grove, and Asn 51 contacts Ade 3 through the major grove. The difference in the DNA contacts between Conformations A (orange) and B (blue) is shown on the right. Conformation B makes additional base contacts by Asn 51, by Lys 2 from the ordered N-terminal arm, and a water-mediated contact by Gln 50. Conformation A forms additional phosphate contacts. Arrows represent hydrogen bonds. C) DNA sequence and numbering in the crystal structure. The TAAT core sequence is in bold.</p

Contacts by helix 3 in the major groove of Conformation A during the solution simulation.

<p>A) Ribbon diagram looking into helix 3 and the major groove. Gln 50 and Asn 51 (labeled as Q50 and N51) contact the phosphate backbone only, with Cyt 7* and Ade 2, respectively (blue dashed lines). Gln 50 is within van der Waals contact of Thy 6* C7 (green, connect by a red dotted line). About 7% of the time a water molecule (W3) mediates contact between Asn 51 and Ade 3 (green). The position of helix 3 in the major groove is measured by the distance between Asn 51 C-alpha to Ade 3 N7 (8.4 Å), and the width of the major groove: Thy 1 P – Cyt 7* P (18.3 Å). The structure represents interactions at 30 ns of the simulation. B) Asn 51 contacts the backbone of Ade 2 O2P only in Conformation A (black), not in Conformation B (red). C) Gln 50 contacts the phosphate backbone of Cyt 7* O2P only in Conformation A (black), not in Conformation B (red).</p

Interactions specific to Conformation A and B after 30 ns of the solution simulation.

<p>A) and B) Conformation A. C) and D) Conformation B. The base contacts by Arg 5 are identical in both conformations (red, underlined). Invariant (in Conformation A and B) contacts with the phosphate backbone include (cyan): Lys 46 – Ade 8* O2P, Arg 53 – Thy 6* O2P, Lys 57 – Cyt 5* O2P, Lys 55 – Thy 1 O2P, and Thy6 – Ade 3 O1P. The intramolecular hydrogen bond Arg 53 – Lys 24 is also conserved (circled). A) Hydrogen bond interactions by Pdx1 Conformation A (grey ribbon) with the DNA. Contacts unique to Conformation A (green, underlined) include a phosphate contact by Arg 31 with Ade 8*, in the major groove opposite the N-terminal arm, and the intramolecular contact between Arg 31 and Glu 42. B) Conformation A viewed looking into the minor groove. Residues 1–4 of the N-terminal arm are highly mobile in the solution simulation. Asn 51 and Glu 50 are shown in black lines. C) Hydrogen bonds by Pdx1 Conformation B (grey) with the DNA during the solution simulation, facing helix 3 in the major groove. Contacts unique to Conformation B (green, underlined) include a phosphate contacts by Tyr 25. D) Conformation B viewed looking into the minor groove. Arg 3 and Arg 43 hydrogen bond with the backbone of Thy 4 (green, underlined), assisting in stabilizing the N-terminal arm in the minor groove and the interaction by Lys 2 with the bases of Thy 2* and Ade 3.</p

N-terminal arm contacts in the crystal simulation.

<p>A) In Conformation B, the N-terminal arm is mostly ordered with Lys 2 hydrogen bonding with the base of Thy 2* O2P. In model B4 (blue) the N-terminal arm escapes from the minor groove. B) The N-terminal arm in Conformation A starts the simulation disordered. Lys 2 never enters the minor groove, but Arg 3 enters the minor groove in model A2 (red), A4 (blue), and it seems to do so at the end of the simulation in A3 (green). The colors represent one of the four asymmetric units as depicted in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003160#pcbi.1003160.s001" target="_blank">Figure S1</a>: A1, B1 black; A2, B2 red; A3, B3 green; A4, B4 blue.</p

A Novel DNA Binding Mechanism for maf Basic Region-Leucine Zipper Factors Inferred from a MafA–DNA Complex Structure and Binding Specificities

MafA is a proto-oncoprotein and is critical for insulin gene expression in pancreatic β-cells. Maf proteins belong to the AP1 superfamily of basic region-leucine zipper (bZIP) transcription factors. Residues in the basic helix and an ancillary N-terminal domain, the Extended Homology Region (EHR), endow maf proteins with unique DNA binding properties: binding a 13 bp consensus site consisting of a core AP1 site (TGACTCA) flanked by TGC sequences and binding DNA stably as monomers. To further characterize maf DNA binding, we determined the structure of a MafA–DNA complex. MafA forms base-specific hydrogen bonds with the flanking G_–5C_–4 and central C₀/G₀ bases, but not with the core-TGA bases. However, in vitro binding studies utilizing a pulse–chase electrophoretic mobility shift assay protocol revealed that mutating either the core-TGA or flanking-TGC bases dramatically increases the binding off rate. Comparing the known maf structures, we propose that DNA binding specificity results from positioning the basic helix through unique phosphate contacts. The EHR does not contact DNA directly but stabilizes DNA binding by contacting the basic helix. Collectively, these results suggest a novel multistep DNA binding process involving a conformational change from contacting the core-TGA to contacting the flanking-TGC bases

A High-Resolution Crystal Structure of a Psychrohalophilic α–Carbonic Anhydrase from <i>Photobacterium profundum</i> Reveals a Unique Dimer Interface

<div><p>Bacterial α–carbonic anhydrases (α-CA) are zinc containing metalloenzymes that catalyze the rapid interconversion of CO₂ to bicarbonate and a proton. We report the first crystal structure of a pyschrohalophilic α–CA from a deep-sea bacterium, <i>Photobacterium profundum</i>. Size exclusion chromatography of the purified <i>P</i>. <i>profundum</i> α–CA (PprCA) reveals that the protein is a heterogeneous mix of monomers and dimers. Furthermore, an “in-gel” carbonic anhydrase activity assay, also known as protonography, revealed two distinct bands corresponding to monomeric and dimeric forms of PprCA that are catalytically active. The crystal structure of PprCA was determined in its native form and reveals a highly conserved “knot-topology” that is characteristic of α–CA’s. Similar to other bacterial α–CA’s, PprCA also crystallized as a dimer. Furthermore, dimer interface analysis revealed the presence of a chloride ion (Cl^-) in the interface which is unique to PprCA and has not been observed in any other α–CA’s characterized so far. Molecular dynamics simulation and chloride ion occupancy analysis shows 100% occupancy for the Cl^- ion in the dimer interface. Zinc coordinating triple histidine residues, substrate binding hydrophobic patch residues, and the hydrophilic proton wire residues are highly conserved in PprCA and are identical to other well-studied α–CA’s.</p></div

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Cartoon representation of the PprCA active site and oligomerization status of PprCA.

<p>(<b>A</b>) The highly conserved active site residues of PprCA showing Zinc ion in the center of the active site (gray sphere). The hydrophobic (Val117, Val127, Leu181, Val190 and Trp192), the hydrophilic (Tyr17, Asn69, Gln74, Thr182 and Thr183), and zinc coordinating residues (His96, His98 and His115) are similar to other well characterized α–CA’s. (<b>B</b>) Active site water network involved in proton transfer is shown as red spheres. 2Fo-Fc electron density contoured at 8.0σ (green) and 1.0σ in blue. (<b>C</b>) Purified PprCA was analyzed on a Hiprep 16/60 Sephacryl S-200 size-exclusion column that was equilibrated with 20 mM Tris pH 7.5 and 150 mM NaCl. Peaks A and B correspond to dimer and monomer, respectively. The elution fractions were analyzed by SDS-PAGE and stained with Coomassie Blue. A single ~35 kDa band was observed for both dimeric and monomeric PprCA. MW–Molecular-weight marker, lanes 43 to 51 (elution fractions representing PprCA dimer) and lanes 52 to 64 (elution fractions representing PprCA monomer). (<b>D</b>) Purified PprCA was separated under both reducing and non-reducing conditions on a SDS-PAGE. Lanes were loaded with PprCA from monomer fraction, dimer fraction and from a sample containing a mixture of monomers and dimers. The gels were stained either by Coomassie blue or subjected to protonography. Protonography was performed according to De Luca et al, 2015 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168022#pone.0168022.ref009" target="_blank">9</a>]. The gel was incubated in CO₂ enriched water for 5 to 15 seconds at room temperature. Appearance of distinctive yellow bands in gels subjected to protonography indicates both monomeric (~27 kDa) and dimeric (~58 kDa) PprCA is catalytically active.</p