In vitro analysis of RapF and RapF mutants interaction with peptides and ComA.

<p>(A) Native gel assays. The interaction of native RapF and the RapF mutants Asn227Ala (RapF^N227A), Asp194Ala (RapF^D194A), Glu303Lys (RapF^K303A), and Glu303Ala (RapF^E303A) with the inhibitory peptides PhrF and PhrC and the RR ComA were analyzed by Native-PAGE and Coomassie-stained. The positions of the individual proteins and the peptide-Rap or ComA-Rap complexes are indicated by black arrowheads and labeled. (B) Thermal-shift assays. Representative thermal denaturation curve profiles of wild type and mutant variants in the absence (―) or the presence of PhrF (—) and PhrC (····) as monitored by Sypro orange fluorescence. The Tm values from at least three independent experiments performed in duplicated are indicated.</p

PhrF induced conformational changes disrupt response regulator binding sites.

<p>(A) Side (left) and top view (right) of the superimposition of RapF 3-helix bundle from the PhrF (blue hues) and the ComA (yellow hues) complexes shows that this domain rotates ∼155° as a rigid body using the linker region (dark blue and orange for PhrF and ComA complexes, respectively) as a hinge. As a result of the movement α1 helices of TPR1 motif (cyan and dark red for PhrF and ComA complexes, respectively) superimpose but with inverted orientation, extending from three to five helices the helix bundle in the case of RapF-PhrF complex. (B) RR binding to Rap is impaired in the Phr-induced conformation. Spo0F and DNA-binding domain of ComA (green and yellow surfaces, respectively) were placed in the RapF-PhrF structure by aligning the 3-helix bundle of RapF-Spo0F (3Q15) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001511#pbio.1001511-Baker1" target="_blank">[6]</a> and RapF-ComA (3ULQ) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001511#pbio.1001511-Parashar1" target="_blank">[7]</a> with RapF-PhrF structure. RapF-PhrF is represented in ribbon and colored the N-terminal portion in magenta and the TPR domain in blue, view from the side (left) and the top (right).</p

Quantification of Phr peptide binding to wild-type and mutational RapF variants and limited proteolysis analysis.

<p>(A) RapF or its mutational variants were incubated with increasing concentrations of PhrF and/or PhrC peptides (0–1.2 mM) and separated by native PAGE. The band corresponding to the Rap-Phr complex was quantified and represented versus peptide concentration to calculate the apparent constant affinities (K_d). Representative PhrF titration experiments for Rap proteins with (RapF) or without (RapF^N227A) capacity to bind the peptide are shown in the upper part of the figure. (B–D) Limited proteolysis of wild-type RapF and mutant forms. (B) Wild-type RapF or mutants were incubated with trypsin in the presence (1 mM) or absence of inhibitory peptides. The reaction were stopped at the indicated time points and analyzed by SDS-PAGE. The proteolytic fragment analyzed by Edman sequencing is indicated by a white arrowhead. (C) Native gel analysis of RapF trypsin digestion. RapF was digested for 60 min with trypsin in the presence or the absence of PhrF (1 mM) and analyzed by native gel electrophoresis. Notice that RapF-PhrF is selectively protected against the trypsin digestion. (D) Trypsin protection is peptide-concentration dependent. RapF was incubated with increasing concentrations of PhrF and subjected to limited proteolysis with trypsin for 60 min and analyzed by SDS-PAGE. Line labeled with (-) corresponds to a control without trypsin.</p

Peptide specificity.

<p>Rap proteins shown an exquisite specificity for their inhibitory peptides as is exemplified by the closely related PhrF (QRGMI) and PhrC (ERGMT) peptides and their targets RapF and RapC/RapB, respectively. The RapF peptide-binding site is represented in semi-transparent surface colored in green and red for conserved and variable residues, respectively, among RapF, RapB, and RapC. PhrF is shown in sticks rendering with carbon atoms colored in cyan for identical positions with PhrC and purple for variable. As in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001511#pbio-1001511-g002" target="_blank">Figure 2</a>, the active center is cut along the peptide axis and presented in two halves for an easier visualization.</p

PhrF recognition by RapF.

<p>(A) Close view of the PhrF binding site that is presented in two halves dissected along the peptide axis for easier visualization. Colors are as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001511#pbio-1001511-g001" target="_blank">Figure 1B</a>. Peptide interacting residues are shown in stick, labeled and colored with the carbon atoms as the corresponding structural element. PhrF is shown in sticks, labeled and colored with carbon atoms in pink. RapF-PhrF polar interactions are drawn as dashed black lines. (B) Peptide interacting residues in Rap proteins from <i>B. subtilis</i>. Residues for the 11 Rap proteins from <i>B. subtilis</i> corresponding to the RapF positions interacting with PhrF are aligned. Anchor and specificity residues are highlighted by magenta and light blue boxes, respectively. The numbers indicate amino acid positions of RapF. (Right) For each Rap protein the corresponding Phr Inhibitory peptide is shown. The conserved positive charged residue at position 2 is highlighted in mustard.</p

Conformational changes in RapF upon PhrF binding.

<p>(A) Superimposition of RapF structures from the PhrF (blue hues) and ComA (orange hues) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001511#pbio.1001511-Baker1" target="_blank">[6]</a> complexes. The view shows two cartoon diagrams (90° rotation along the vertical axis). Superimposition reveals a huge (155°) angular movement of approximation to the TPR domain of the N-terminal portion (darker hues) induced by PhrF (in sticks rendering with carbon atoms colored in pink) binding. TPR1–TPR3 motifs (bright hues) are displaced towards the TPR4–TPR7 motifs (tint hues) that present an almost fixed disposition in both structures. Helices are shown as cylinders and labeled for the N-terminal domain. TPR motifs are labeled in the TPR domain. (B) Detailed view of the conformational changes induced in TPR1–TPR3 (left) and TPR4–TPR7 (right) motifs of RapF upon PhrF binding. TPR motifs are represented in ribbon, labeled, and colored in blue and orange hues for RapF in complex with PhrF and ComA, respectively. Interacting amino acids are shown in sticks, labeled, and colored with carbons in the same color that the corresponding TPR motif. Dashed lines indicate the displacements. PhrF is shown as a stick model with carbons colored in pink.</p

Schematic domain organization of human endoglin and ALK1 and western blots of endoglin domains.

<p>Bar diagram of (A) endoglin and (B) ALK1 with the domains indicated and highlighted in different styles. TM, transmembrane region, ZP, zona pellucida. The putative Asn glycosylation sites Asn88, Asn102, Asn121, Asn134 and Asn306 of endoglin and Asn98 of ALK1 are labelled within green ovals. The constructs used in this study and the domains encompassed by these are shown below the bar diagram of the respective full length protein. (C) Western blots of endoglin constructs. Endo₃₃₈, Endo₃₆₂ and LG-Endo_EC were analyzed by 10% SDS polyacrylamide electrophoresis gel followed by western blotting with an anti-His₆ antibody. Samples reduced with dithithreitol (DTT) were incubated for 1 h with 10 mM of this reagent at 65°C. All samples (0.5 µg of protein) were then denaturated by boiling at 95°C for 5 minutes prior to charging onto the gel. The molecular weight markers (M) are indicated at the left of the samples. For both Endo₃₆₂ and LG-Endo_EC, dimeric species are visible, while Endo₃₃₈ was only observed in monomeric form.</p

Experimental and modelling SAXS parameters.

a<p>Values for I(0) have been extrapolated by the Guinier approximation from the experimental scattering profiles.</p>b<p>Concentration of the protein used for the calculation of the estimated Mw.</p>c<p>Relative molecular mass estimated from I(0) and the concentration of the protein through BSA calibration.</p>d<p>The Porod volume was calculated using PRIMUS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029948#pone.0029948-Mertens1" target="_blank">[41]</a>.</p>e<p>Expected molecular mass predicted from the sequence and assuming full occupation of the glycosylation sites.</p>f<p>Rg (Guinier), Rg (GNOM), radius of gyration given by the Guinier approximation, and calculated by the program GNOM, respectively, given in nm.</p>g<p>Maximum dimension of the macromolecules. χ²^(over) Discrepancy between the SAXS profile and its fit by the overall shapes-models calculated by DAMMIF, and χ^{2 (sasref)} the average discrepancy of the best atomic models estimated with the program CRYSOL. ND, not determined.</p

Functional interactions between endoglin, ALK1 and BMP-9.

<p>The interactions of (A) Endo_EC, (B) ALK1_EC, (E) Endo₃₃₈ and (F) Endo₃₆₂ with BMP-9 were investigated by SPR. While HG-Endo_EC and Endo₃₆₂ dissociated slowly, Endo₃₃₈ dissociated much faster, indicating a rigid body type of binding as opposed to an induced fit mechanism. For affinity measurements, the indicated recombinant proteins were injected at six concentrations ranging from 12.5 to 400 nM over BMP-9 (which was immobilized on a CM5 sensor chip by amine coupling) to generate sensorgrams (colored curves). When testing competition between HG-Endo_EC and HG-ALK1_EC (C and D) the chip was first pre-equilibrated with 750 mM of either HG-Endo_EC (C, left) or HG-ALK1_EC (D, left) before injecting the various concentration of the second ligand, showing the curve for the highest concentrations of the 2^nd ligand. Both HG-ALK1_EC (C, right) and HG-Endo_EC (D, right) yielded, after subtracting the background, similar results to those in runs in which no first ligand was preequilibrated before injecting the second ligand (D right vs. E; C right vs. B). This leads to the conclusion that endoglin and ALK1 bind independently to different sites on BMP-9. The kinetic parameters for the interaction were determined by global fitting (curves in black) of the 1∶1 Langmuir binding model to these data, providing values for the association (k_a) and dissociation (k_d) rate constants and the dissociation affinity constant (K_D).</p

Analysis of ligand binding to BMP-9 assessed by Surface Plamson Resonance.

<p>Kinetic analysis of endoglin and ALK1 binding to BMP-9 was performed in triplicates on a Biacore T100 at 25°C as described in the experimental procedures. Data were globally fit to a 1∶1 binding model using the Biacore T100 evaluation software.</p