Identification and characterization of half-sites S1 and S2 on DNA that interacts with KdpE_DBD.

<p><b>A.</b> Sequence logo representation to highlight conserved sequences in a 24 bp stretch of <i>kdpFABC_BS</i>. In the logo, the height of the letter represents its frequency of occurrence in a multiple sequence alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone.0030102.s003" target="_blank">Fig. S3</a>) and the error bars indicate the sampling error at individual positions. Two 6 bp imperfect direct repeats (TTTATA and TTTACA) separated by a 5 bp sequence are shown in dashed boxes below the logo. <b>B.</b> Identification of the minimal length of DNA required for binding KdpE. For EMSA, double-stranded DNA molecules with progressive deletions (indicated by Δ) at either 5′, 3′, or both ends were used (the nomenclature for oligonucleotides: 5′Δ2, 3′Δ8 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone-0030102-g004" target="_blank">Fig. 4B</a>, lane 9) refers to deletion of 2 and 8 bp from the 5′ and 3′ ends respectively of the wild-type (30 bp) DNA molecule; oligonucleotides used are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone.0030102.s005" target="_blank">Table S2</a>). The interpretation of EMSA was qualitative: discreet band shifts as observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone-0030102-g004" target="_blank">Fig. 4B</a>, lane 1 were considered a positive reaction (+), whereas no shift (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone-0030102-g004" target="_blank">Fig. 4B</a>, lane 3) was scored negative (−) and smeared bands as exemplified by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone-0030102-g004" target="_blank">Fig. 4B</a>, lane 2 were considered partial binding. <b>C.</b> Effects of changes in DNA sequence on the KdpE_DBD-DNA interaction. A summary of EMSA data (data not shown) using the 30 bp <i>kdpFABC_BS</i> sequence and modified oligonucleotides (only specific two or one nucleotide substitutions are noted) are presented. The scoring of EMSA analysis was as described above. The dashed boxes represent the 6 bp direct repeats that form half-sites S1 and S2.</p

Sedimentation velocity analysis of KdpE_DBD—<i>kdpFABC_BS</i> association.

<p><b>A.</b> Continuous distribution of sedimentation coefficients [c(s)] as a function of increasing concentration of protein against a fixed concentration of <i>kdpFABC_BS</i> DNA (0.5 µM). The protein concentrations used varied between 0.25 and 16 µM as shown. The largest complex with sedimentation coefficient of 4.1 S was observed at protein concentration of 4 to 16 µM. Independent experiments established the sedimentation coefficients of KdpE_DBD and <i>kdpFABC_BS</i> at 1.4 S and 2.8 S respectively (data not shown). <b>B.</b> A plot of the weight average sedimentation coefficients (S_w) against the concentration of KdpE_DBD is shown. Analysis of the isotherm indicated that DNA was saturated beginning at 8-fold molar excess of KdpE_DBD protein. <b>C.</b> SV c(s) distributions comparing binding of KdpE_DBD to the S1 and S2 sites individually and to the both sites simultaneously. Wild-type DNA with both sites intact (<i>kdpFABC_BS</i>), functional S1 (<i>kdpFABC_BS —</i>7) and S2 (<i>kdpFABC_BS —</i>1) sites were analyzed with a 16-fold molar excess of Kdp_DBD. Complexes with DNA possessing single sites have sedimentation coefficients of 3.5 S whereas when both sites were occupied a 4.1 S species was formed.</p

Effects of mutation of residues conserved in <i>kdpE_DBD</i>.

<p><b>A.</b> Comparison of β-galactosidase activities of KdpE mutants and wild-type KdpE in the <i>kdpFABC_Pro-lacZ</i> fusion strain HAK003. Residues located in the α-8 (R193 and R200) and β-hairpin (T215) of KdpE (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone-0030102-g002" target="_blank">Fig. 2</a>) were targeted for mutagenesis to alanine. β-galactosidase (a reporter for <i>kdpFABC</i> expression) was measured in cells grown in media containing either K10 (white bar, 10 mM K⁺) or K0 (gray bar, 0 mM K⁺). <b>B.</b> EMSA showing effects of mutations in KdpE on interaction with the 30 bp DNA fragment representing its binding site. The triangles represent increasing molar ratios of 1∶0, 1∶1, 1∶2, 1∶4, and 1∶8 of DNA to purified mutants as indicated and wild-type KdpE_DBD.</p

Comparison of molecular masses calculated from sequence and sedimentation equilibrium analysis of KdpE_DBD, its DNA recognition sequence, and their complexes.

<p><i>kdpFABC_BS</i> represents the wild-type DNA sequence, whereas <i>kdpFABC_BS—1</i> and <i>kdpFABC_BS—7</i> DNA have mutations that abolish binding at half-sites S1 and S2, respectively. All DNAs are 30 bp in length.</p

<i>Binding analysis of the half-sites of kdpFABC_BS</i>.

<p>SE analysis of binding of KdpE_DBD to S1 (<i>kdpFABC_BS—7</i>) (<b>A</b>)and S2 (<i>kdpFABC_BS</i>—<i>1</i>) (<b>B</b>) half-sites revealed a 1∶1 stoichiometry. Mixtures of KdpE_DBD and DNA were spun at 9,000 (•), 19,800 (□) and 34,000 (Δ) rpm. The <i>K_d</i>s obtained for KdpE_DBD binding at half-sites S1 was 350±100 nM and for S2 was 200±100 nM using a one site binding model (AB) in SEDPHAT. The molecular weights calculated from the SE data were 30,000±1,500 for <i>kdpFABC_BS—1</i> and 30,000±2,500 for <i>kdpFABC_BS—7</i>.</p

Biochemical and functional characterization of KdpE_DBD.

<p><b>A.</b> Sedimentation velocity analysis of the KdpE_DBD to detect self-association. The c(s) distribution of the KdpE_DBD at 21 (dots), 42 (solid line), and 84 µM (dashes) shows a single species of 1.4 S. No concentration-dependent formation of higher-order species was observed. <b>B.</b> Interaction of KdpE_DBD protein with <i>kdpFABC_BS</i> and <i>ompF_Pro</i> DNA sequences analyzed by EMSA. The triangles represent increasing molar ratios of 1∶0, 1∶1, 1∶2, and 1∶3 of DNA to purified KdpE_DBD. The lower and upper bands represent free DNA and DNA-KdpE_DBD complex, respectively. <b>C. </b><i>In vivo</i> analysis of expression of the β-galactosidase gene fused to <i>kdpFABC_Pro</i>. <i>E. coli</i> RH003 cells lacking the histidine kinase (<i>kdpD</i>) and RR (<i>kdpE</i>) were used to express full-length KdpD alone as well as KdpD combined with KdpE or KdpE_DBD. As described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#s2" target="_blank">methods</a>, the cells were grown in K0 (▪) and K10 (□) media prior to analysis of gene expression. Growth in K0 medium mimics stresses resulting from external K⁺ depletion. The β-galactosidase activity expressed as Miller units represents the mean of three independent experiments; error bars represent standard error.</p

Structure of KdpE_DBD.

<p><b>A.</b> A cartoon representation of a molecule showing the wHTH motif in progressive coloring; the rest is in gray. To maintain continuity with the structure of the N-terminal receiver domain of KdpE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone.0030102-ToroRoman1" target="_blank">[25]</a>, the β-strands and α-helices of KdpE_DBD are labeled starting with β-6 and α-6. The side chains shown in stick representation are residues R193 and R200 in α8 and T215 in β11 targeted for mutagenesis. N and C refer to the amino- and carboxyl- termini. <b>B.</b> Conservation of the sequence in the wHTH motif across members of the OmpR/PhoB family (upper panel) and between KdpE orthologs (lower panel) presented in logo format derived from multiple sequence alignments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone.0030102-Crooks1" target="_blank">[61]</a>. The Y-axis represents sequence conservation in bits. The residues targeted for mutagenesis in KdpE are boxed, the triangles represent residues involved in base specific interactions in PhoB-DNA complex (PDB code: 1GXP), and the residue numbering is that of KdpE sequence. Shown below the logo representation are the sequences of the wHTH motif of KdpE and PhoB (upper panel) and that of KdpE in the lower panel. The gap in the lower panel represents a three residue insertion in few of the KdpE orthologs used in sequence alignment. The schematic of the secondary structure was derived from the structure of KdpE_DBD. <b>C.</b> Superposition of KdpE_DBD onto the structure of PhoB bound to DNA (PDB code: 1GXP). Only wHTH motifs of KdpE_DBD and chain A of PhoB in 1GXP and part of the DNA are shown. The coloring scheme: green, KdpE_DBD; purple, PhoB and yellow/orange, DNA strands. The following side chains of residues of PhoB (and in parenthesis equivalent residues in KdpE_DBD labeled in blue) are shown as sticks: T194 (Y191), V197 (I194), R201 (H198) and R219 (T217, not shown), R203 (R200) and T217 (T215) and D196 (R193). Residues T194, V197, R201 and R219 (that penetrates the minor groove is labeled in red) of PhoB have been shown to be form base specific interactions.</p

Binding affinities of KdpE_DBD to wild-type and mutant DNA molecules determined by Sedimentation Equilibrium analysis.

a<p>The apparent <i>K_d</i> values assigned to S1 and S2 are based on values obtained using <i>kdpFABC_BS-1</i>and <i>kdpFABC_BS-7</i> that have single functional binding sites at S2 and S1 respectively. Error limits were generated using F-statistics with a confidence interval of 1σ.</p

Crystallographic data and results of refinement.

a<p>.</p>b<p><i>R</i>_work = ∑‖<i>F</i>_o|−|<i>F</i>_c‖/∑|<i>F</i>_o| for reflections contained in the working set, and <i>R-</i>free = ∑‖<i>F</i>_o|−|<i>F</i>_c‖/∑|<i>F</i>_o| for reflections contained in the test set held aside during refinement. |<i>F</i>_o| and |<i>F</i>_c| are the observed and calculated structure factor amplitudes, respectively.</p

Structural Investigation of a Novel N-Acetyl Glucosamine Binding Chi-Lectin Which Reveals Evolutionary Relationship with Class III Chitinases

<div><p>The glycosyl hydrolase 18 (GH18) family consists of active chitinases as well as chitinase like lectins/proteins (CLPs). The CLPs share significant sequence and structural similarities with active chitinases, however, do not display chitinase activity. Some of these proteins are reported to have specific functions and carbohydrate binding property. In the present study, we report a novel chitinase like lectin (TCLL) from <i>Tamarindus indica</i>. The crystal structures of native TCLL and its complex with N-acetyl glucosamine were determined. Similar to the other CLPs of the GH18 members, TCLL lacks chitinase activity due to mutations of key active site residues. Comparison of TCLL with chitinases and other chitin binding CLPs shows that TCLL has substitution of some chitin binding site residues and more open binding cleft due to major differences in the loop region. Interestingly, the biochemical studies suggest that TCLL is an N-acetyl glucosamine specific chi-lectin, which is further confirmed by the complex structure of TCLL with N-acetyl glucosamine complex. TCLL has two distinct N-acetyl glucosamine binding sites S1 and S2 that contain similar polar residues, although interaction pattern with N-acetyl glucosamine varies extensively among them. Moreover, TCLL structure depicts that how plants utilize existing structural scaffolds ingenuously to attain new functions. To date, this is the first structural investigation of a chi-lectin from plants that explore novel carbohydrate binding sites other than chitin binding groove observed in GH18 family members. Consequently, TCLL structure confers evidence for evolutionary link of lectins with chitinases.</p></div