Effect of mutations on Lhx3.

<p>(<b>A</b>) Overlay of CD spectra from Ldb1-Lhx3 (<i>solid black squares</i>) and Ldb1-Lhx3(Y114C) (<i>open magenta circles</i>). Spectra were collected with 30 μM samples in 20 mM Na₂HPO₄, 40 mM NaCl, 1 mM DTT, pH 6.8 at 310K and were buffer baseline corrected. (<b>B</b>) Chemical denaturation of Ldb1-Lhx3 (<i>solid black squares</i>) and Ldb1-Lhx3(Y114C) (<i>open magenta circles</i>). Fraction folded was estimated using tryptophan fluorescence intensities (excitation wavelength 295 nm and emission wavelength 334 nm). Lines show the fits to a sigmoidal function. Proteins were at concentrations of 2.5 μM (<b>C</b>) ¹⁵N-HSQC spectra of Ldb1-Lhx3 (<i>black</i>, ∼800 μM)) and Ldb1-Lhx3(Y114C) (<i>magenta</i>, ∼200 μM) in 20 mM Na₂HPO₄, 40 mM NaCl, 1 mM DTT, pH 6.8 at 310K. (<b>D</b>) Analysis of chemical shift differences from panel C based on assignments for the wildtype protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040719#pone.0040719-Lee3" target="_blank">[25]</a> and inferred assignments for the mutant protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040719#pone.0040719.s006" target="_blank">Table S3</a>). Peaks were identical for the C-terminal half of Ldb1_LID, and Lhx3_LIM1 which make direct contacts in the structure of the complex, but were significantly different for the N-terminal half of Ldb1_LID, and Lhx3_LIM2.</p

Solution structure of Ldb1-Lhx3.

<p>(<b>A</b>) NMR structure of Ldb1-Lhx3 (pdb accession code: 2JTN) with Ldb1_LID in yellow and Lhx3 in blue; zinc ions are depicted as grey spheres. The 20 lowest energy models are aligned over the backbone atoms of both LIM domains. Only the structured regions are shown. The positions of the N and C termini of the interacting domains of Lhx3 and Ldb1, and the position of the two LIM domains from Lhx3 are indicated. (<b>B</b>) Ldb1-Lhx3 with the same 20 conformers from (A) aligned over the backbone atoms of Lhx3_LIM1 and the corresponding region of Ldb1. The unstructured linker and tail from each model is shown in grey. (<b>C</b>) Scattering data for Ldb1-Lhx3 (<i>grey circles</i>) shown as <i>I</i>(<i>q</i>) versus <i>q</i> with the corresponding Guinier plot (ln(<i>I</i>(<i>q</i>)) versus <i>q</i>²) in the inset. The fit is for Model 17 from the Ldb1-Lhx3 NMR ensemble as generated by CRYSOL (<i>black line</i>). The black line in the inset is the fit to the data generated by GNOM. (<b>D</b>) <i>P</i>(<i>r</i>) profiles from experimental scattering data for Ldb1-Lhx3 (<i>grey circles</i>) and calculated scattering profiles from Model 17 of the NMR ensemble (<i>blue line</i>) and the generated BUNCH model (<i>dashed magenta line</i>). (<b>E</b>) Alignment of Model 17 of the Ldb1-Lhx3 NMR ensemble (<i>coloured as in Panel B</i>), BUNCH model (<i>magenta</i>) and the <i>ab initio</i> DAMMIF reconstruction from the scattering data (<i>transparent white surface</i>). (<b>F</b>) Alignment of the NMR ensemble (<i>coloured as in Panel B</i>) with the same DAMMIF consensus model. (<b>G</b>) Most disparate models from the 2JTN NMR structure. Model 1 (blue/yellow) has an angle between the Cα atoms of the first zinc-coordinating residue (C34), the hinge residue (F89) and the last zinc coordinating residue (D147) of 163.7° (solid black line). Model 9 (cyan/light yellow) has an angle over the same atoms of 135.1° (dashed black line). Models are aligned using the backbone atoms of the LIM1 domain. Images of structures were created in Pymol.</p

Effect of mutations in Lhx3 on interactions with Ldb1 and Isl1.

<p>Yeast two-hybrid data for Lhx3 (constructs comprised both LIM domains), the Lhx3 phosphomimic mutants Lhx3(S71D) and Lhx3(S71E) and the CPHDS mutation Lhx3(Y114C) against Ldb1_LID and Isl1_LBD, and. Interaction data for both bait/prey orientations are shown; DBD and AD designate the plasmids pGBT9 and pGAD10, respectively. Serial dilutions of culture (A_600nm  = 0.2, 0.02, and 0.002) were spotted onto each column of the plate with the highest concentration at the top. Two different selection conditions (moderate stringency  =  SD -L-W-H +3-AT; high stringency  =  SD-L-W-H-A). Data are representative of 2­ or 3 separate experiments. In all cases transformation control plates (SD-L-W) showed strong growth of yeast indicating successful transformations, and negative controls of each constructs versus the corresponding empty plasmid showed no yeast growth under the conditions tested.</p

Schematics of Lhx3, Isl1 and Ldb1 proteins and tethered complexes.

<p>(<b>A</b>) Schematics of mouse proteins as indicated. Numbers refer to the mouse proteins with the domain boundaries as defined in SWISS-PROT. (<b>B</b>) Arrangement of domains in the tethered complexes Lhx3-Isl1 and Ldb1-Lhx3. The N and C termini are indicated. HD, homeodomain; LBD, Lhx3-binding domain; LID, LIM interaction domain; LIM1, N-terminal LIM domain; LIM2, C-terminal LIM domain. The linker and the locations of the hinge (located in residues between the two LIM domains) and spacer (corresponding region in the LBD/LID domains) in each construct are shown.</p

Potential roles of Lhx3-S74 and Lhx3-Y114.

<p>Views of murine Lhx3 (<i>white semi-transparent surfaces</i>) showing the position of S74, Y114 and S140 (<i>black sticks</i>). (<b>A</b>) Ldb1_LID (<i>yellow sticks</i>) and (<b>B</b>) Isl1_LBD (<i>green sticks</i>) are shown to indicate the positions of binding faces. Residues from Ldb1 and Isl1 that lie close to Lhx3-S74 are also shown. Zinc ions are shown as grey spheres. Model 17 from the NMR structure of Ldb1-Lhx3 (pdb accession code: 2JTN) and chain B from the X-ray structure of Lhx3-Isl1 (pdb accession code: 2RGT) were used to make these figures in Pymol.</p

Ligation of zinc by histidine sidechains in Ldb1-Lhx3 is not affected by the Y114C mutation.

<p>(<b>A</b>) Schematic representation of chemical shift patterns arising from histidine protonation and expected zinc-ligation pattern adapted from ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040719#pone.0040719-Pelton1" target="_blank">[29]</a>. (<b>B</b>) ¹⁵N-HSQC spectra showing histidine sidechains from wildtype Ldb1-Lhx3 (<i>black</i>, labelled) and mutant Ldb1-Lhx3(Y114C) (<i>magenta</i>)<b>.</b> Arrows indicate the chemical shift movement of H115.</p

Structural parameters for Ldb1-Lhx3 from small-angle X-ray scattering data.

<p>We use the convention for reporting SAXS data as outlined in ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040719#pone.0040719-Jacques1" target="_blank">[42]</a>.</p>a<p>error is 1 S.D.</p>b<p>MM_exp  =  <i>I</i>(0)N_A/[protein]Δρ_M².</p>*<p><i>I</i>(0) and <i>R</i>_g were derived from <i>P</i>(<i>r</i>) using GNOM.</p

Cross-linking of oligomers and lytic activity of endolysins CD27L and CTP1L.

<p>A. SDS-PAGE of the C-terminal domain of wild-type CTP1L with the light sensitive cross-linker pBpa incorporated at positions Y212 or Y260 respectively, as well as the double mutant Y212pBpa/D215A. B. SDS-PAGE of the full-length endolysin with the Y212pBpa cross-linker mutant, showing the presence of 1) a full-length dimer, 2) an oligomer consisting of one C-terminal domain and a full-length CTP1L endolysin molecule. C, D. Lysis assays of 10 µg of recombinant CD27L applied to a culture of <i>C. difficile</i> showing that the autocleavage mutants do not affect lysis <i>in vitro</i> and that amidase active site mutants are not active. E. Lysis assays of CTP1L on <i>C. tyrobutyricum</i> cells showing the effect of mutants that reduce autocleavage (V195P, T221C and T221R).</p

Sequence alignment of the C-terminal domains of CD27L and CTP1L showing this fold is prevalent among lysins that target <i>Clostridia</i>.

<p>A. Sequence alignment of the C-terminal domain of CD27L and other sequences with a significant BLAST score (E<0.01) produced with ESPRIPT <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004228#ppat.1004228-Gouet1" target="_blank">[49]</a>. Conserved residues are colored red. The secondary structure of the C-terminal domain of CD27L is depicted with arrows for beta strands and curls for alpha helices. Hydrophobic residues that contribute to the head-on dimer interface are colored blue, and the cysteine residue involved in the side-by-side dimer formation is colored green. B. Structure-based sequence alignment of the C-terminal domain of CD27L with the C-terminal domain of CTP1L. Residues where the cross-linking amino acid pBpa is inserted are marked by a blitz, and the area involved in the side-by-side dimer is marked SD. C. Sequence alignment of the C-terminal domain of CTP1L with other sequences that give a significant BLAST score. All sequences listed from the BLAST analysis are annotated as Clostridial lysins or endolysins.</p

Equilibrium analysis of the SAXS data using the program OLIGOMER.

a<p>Values reported for merged data sets (wild-type: 0.9 & 4.0 mg/mL, C238R: 1 & 8.4 mg/mL).</p