Mapping of the ExsA dimer interface.

<p>(A) The shown A/A’ ExsA-NTD interface suggests involvement of helix α-2 in ExsA dimerization. Previously identified interface residues are indicated in the same color as the protein backbone. G124 and L117 are colored violet and yellow in the respective molecules. (B) Shown is a sample gel of measurements testing the impact of the L117R and G124R mutations on the ability of ExsA to activate transcription <i>in vitro</i>. Three concentrations of each protein were tested to ensure that the experiments were conducted in a sensitive range. (C) Graphical representation of the <i>in vitro</i> transcription assays from triplicate experiments. Going from left to right: wtExsA, ExsAG124R, and ExsAL117R.</p

Impact of mutations in the conserved cavity of ExsA on ExsD binding.

<p>Three residues lining the cavity within the beta-sandwich structure of ExsA were mutated with alanine to determine if these residues are involved in ExsD binding. (A) Cartoon depiction of a full-length model of an ExsA-DNA complex. This model was generated by overlaying the structures of ExsA-NTD and a homology model of ExsA-CTD (based on the MarA-DNA crystal structure) onto the structure of ToxT. The mutated residues are depicted as ball-and-stick. (B) Results of <i>in vitro</i> transcription assays measuring the impact of the indicated mutations on ExsA-ExsD interactions. Plotted in the chart is the percent change in obtained transcript level when 10 μM ExsD is added to the reaction. A sample gel showing transcript bands is presented above the chart. Experiments were conducted in duplicate.</p

Cartoon model of a full-length dimeric ExsA-DNA complex.

<p>This model was generated by first overlaying the structure of the ExsA-NTD A/A’ dimer and a homology model of ExsA-CTD (based on the MarA-DNA crystal structure) onto the structure of ToxT. Subsequently, crystallographic two-fold axis was applied to create a model of the full-length protein with a dimer interface corresponding to A/A’ dimer observed in the crystal.</p

Diffraction data and crystal structure refinement statistics.

<p>^aThe values in parentheses relate to the highest resolution shell from 2.589–2.5Å.</p><p>^bR_merge = Σ|I|- 〈I〉/ΣI, where I is the observed intensity, and I is the average intensity obtained from multiple observations of symmetry-related reflections after rejections.</p><p>^cCC_1/2 = Pearson correlation coefficient between random half-datasets</p><p>^dCC* = [(2CC1/2)/(1+CC1/2)]^0.5</p><p>^eR_work = Σ||F_o|—|F_c||/Σ|F_o|, where F_o and F_c are the observed and calculated structure factors, respectively.</p><p>^fR_free defined in Ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136533#pone.0136533.ref072" target="_blank">72</a>].</p><p>Diffraction data and crystal structure refinement statistics.</p

Crystal structure of the ExsA-NTD.

<p><b>(A)</b> Model of a monomer encompassing amino acids 2–166 which produced clearly defined electron density. Blue to red rainbow coloring traces the backbone from the N to the C-terminus. Secondary structure elements are numbered. <b>(B)</b> Packing contacts in the crystal suggest the possible structure of the biological dimer. Chains A and B constitute the asymmetric unit of the crystal. Application of two crystallographic two-fold axes produces two additional pairs of chains labeled with a prime and a double-prime, respectively. Contacts between either chains A and A’ or between chains B and B” are proposed to mediate ExsA dimerization <i>in vivo</i>. (C) Shown in gray are the overlaid backbones traces of chains A and B. Also displayed are the symmetry-related molecules A’ and B” to highlight similarities and differences between the two possible quaternary structures. The B” molecule is rotated by approximately 23° around helix α-3. The rotation is visualized by marking the angle between the P20 residues of A’ and B” in the figure.</p

Can Protein Conformers Be Fractionated by Crystallization?

Molecular crystallization typically singles out a specific conformation, or a set of conformations that are identical over large parts and may show some flexibility, from a mixture of equilibrating conformations in solution. To critically evaluate the selectivity of this process, human lactate dehydrogenase isozyme 1 (LDH-1) microcrystals were separately dissolved and subsequently assayed inside capillaries with electrophoretically mediated microanalysis (EMMA) at both the ensemble and the single-molecule level. While fragments from the same crystal exhibited identical enzyme activities, different crystals, even when grown from the same drop of mother liquor, showed markedly different activities. Activities of individual molecules from a crystal were found to be essentially identical, whereas molecules obtained directly from solution showed a 4-fold variation in activity. Furthermore, after storage at 37 °C, the distribution of single-molecule LDH activities from solutions of individual crystals broadened and approached that of LDH obtained from the original solution. X-ray crystallography also showed distinct conformations for single microcrystals and confirms that crystallization properly selects even small conformational variants of proteins and that the slow equilibration to multiple stable conformations in solution is responsible for the observed single-molecule heterogeneity

Overall structure and schematic view of one subunit of drDDC.

<p><i>A,</i> A schematic representation of the structure of drDDC dimer. <i>B,</i> The schematic view of a monomer. The cofactor (LLP) is included in stick. Three parts, large domain (green), small domain (blue) and N-terminal part (pink) are labeled.</p

The roles of T82 and H192.

<p><i>A,</i> Superposition of drDDC structure onto pig DDC structure shows that T82 of drDDC is a putative substrate binding residue. Residues from pig DDC are colored in cyan and those from drDDC are colored in magenta. <i>B,</i> Alignment of drDDC, pig DDC, <i>Drosophila</i> tyrosine decarboxylase-1 (drTDC-1) and <i>Drosophila</i> tyrosine decarboxylase-2 (drTDC-2). The corresponding residues of T82 and H192 are labeled. <i>C,</i> Superposition of drDDC structure onto pig DDC structure shows the H192 of drDDC is a putative substrate and cofactor binding residue. Residues from pig DDC are colored in cyan and those from drDDC are colored in magenta.</p

Superposition of drDDC structure onto pig DDC structure.

<p>The protein portions within 12 Å of the active center are shown in the schematic representation in stereo. Pig DDC and drDDC chain As are colored in cyan and magenta, respectively; and their chain Bs are colored in deep teal and brown, respectively.</p

The drDDC active site.

<p>A stereo view of the active site in the drDDC structure. The LLP and the protein residues within a 4 Å distance of the cofactor are shown. Only the 2<i>F</i>_o - <i>F</i>_c electron density map covering the LLP is shown contoured at the 1.8 sigma. Hydrogen bonds are shown in dashed lines.</p