Identification of a Novel Calcium Binding Motif Based on the Detection of Sequence Insertions in the Animal Peroxidase Domain of Bacterial Proteins

<div><p>Proteins of the animal heme peroxidase (ANP) superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of <em>Pseudomonas putida</em> KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20), where it was found to be involved in Ca²⁺ coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33–79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca²⁺ binding with a <em>K_D</em> of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821) is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of life.</p> </div

Presence of the different versions of the PERCAL calcium binding motif in bacteria and eukaryotes.

<p>An expanded table with more motifs and the details required for the generation of this table are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040698#pone.0040698.s006" target="_blank">Table S2</a>.</p>a<p>Percentage of insertions (of a total of 74) within ANP-like domains recognized by the corresponding motif.</p>b<p>Number of total entries in the database 18215214</p>c<p>Entries from Bacteria (63%)</p>d<p>Entries from Eukaryota (28%)</p

Zoom at the calcium-binding region of the pseudopilin structure.

<p>The amino acids which establish contacts with the calcium ion are shown in ball-and-stick mode and the corresponding distances are indicated (in Å). The structure is deposited at the protein data bank under the code 3G20. The figure was produced using the program WebLabViewer (<a href="http://www.marcsaric.de/index.php/WebLab_Viewer_Lite" target="_blank">http://www.marcsaric.de/index.php/WebLab_Viewer_Lite</a>).</p

Section of the sequence alignment of ANP-like domains from 12 two-domain bacterial proteins.

<p>ANP-like domains as defined by PS50292 were aligned with MEGA 4.0. The numbers indicate the protein residues. The complete alignment is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040698#pone.0040698.s001" target="_blank">figure S1</a>. A region with two insertions (in boxes) matching the G-x-D-G-x(5)-D-D/E consensus and interrupting the major alignment are shown. The individual ANP-like domains are of <i>Fulvimarina pelagi</i> HTCC2506 (Q0G341), Manganese-oxidizing bacterium (strain SI85-9A1) (Q1YMS2), <i>Methylobacterium chloromethanicum</i> CM4 / NCIMB 13688 (B7KW13), <i>M. extorquens</i> DSM 5838 / DM4 (A9W3A5), <i>M. extorquens</i> PA1 (C7CGY0), <i>Pseudomonas putida</i> F1 (A5W572), <i>P. putida</i> GB1 (B0KJL7), <i>P. putida</i> KT2440 (Q88JT6), <i>Rhodopseudomonas palustris</i> BisA53 (Q07SX1), <i>Rhodopseudomonas palustris</i> BisB5 (Q13AU2), <i>Roseobacter</i> sp. MED193 (A3XF15) and <i>Roseovarius</i> sp. TM1035 (A6E280). Letters -A and -B refer to N- and C-terminal ANP-like domains, respectively.</p

Neighbor-joining phylogenetic tree of bacterial ANP-like domains contained in animal heme peroxidases with G-x-D-G-x(5)-D-D.

<p>The domain of human myeloperoxidase, which does not contain any hit, is included as an out-group member. The alignment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040698#pone.0040698.s002" target="_blank">figure S2</a> was cut at the position which corresponds to the sequence boundaries of the human myeloperoxidase domain. The tree is based on this reduced alignment. The bootstrap consensus tree inferred from 500 replicates is shown. Letters –A and -B refer to N- and C-terminal ANP-like domains, respectively. All the 23 sequences here considered presented at least one insert with the consensus of PERCAL (G-x-D-G-x(2)-G/N-T/N-x-D-D) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040698#pone.0040698.s005" target="_blank">Table S1</a>). The column on the right indicates the number of insertions in each of the domains. Bar: 0.2 substitutions per amino acid position.</p

Isothermal titration calorimetry studies of the interaction of the BACHEMP-Cons peptide with CaCl₂.

<p>(A) Upper panel: raw data for the titration of 50 µM peptide with 3.2 µl aliquots of 5 mM CaCl₂. Experiments were conducted in polybuffer at the pH values indicated. Lower panel: Integrated, dilution-corrected and concentration-normalized raw data. Data were fitted with the “One binding site model” of the MicroCal (Northampton, MA) version of ORIGIN. pH 6.0 (○), pH 7.0 (□), pH 8.0 (Δ). The derived thermodynamic parameters are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040698#pone-0040698-t004" target="_blank">Table 4</a>. (B) Dependence of <i>K</i>_D on the pH. Experiments were conducted in polybuffer which was adjusted to the pH indicated by the addition of concentrated HCl or NaOH. Shown are means and standard errors derived from three individual experiments.</p

ITC analysis of Ca binding to the N-terminal ANP-like domain of PepA.

<p>Upper panel: Raw titration data for the injection of 6.4 µl aliquots of 1 mM CaCl₂ into 11.2 µl of recombinant protein. Ligand and protein were in buffer Tris-HCl 10 mM, NaCl 50 mM, Glycerol 10%, pH 7.5. Experiments were carried out at 25°C. Lower panel: Integrated, dilution-corrected and concentration-normalized peak areas of titration raw data. Shown is the fit with the “one binding site model” of the MicroCal version of ORIGIN.</p

Bacterial heme peroxidases containing G-x-D-G-x(5)-D-D insertions within the ANP-like domain as defined by the PS50292 profile.

a<p>The number of insertions per domain is shown.</p>b<p>The second domain is incomplete.</p

Sequence logo of insertions interrupting the ANP-like domains of bacterial proteins.

<p>The figure was generated at <a href="http://weblogo.berkeley.edu" target="_blank">http://weblogo.berkeley.edu</a> based on an alignment of 74 domain insertions matching the motif x-x-x-G-x-D-x(6)-D/E-D/E-x-x-x from 24 bacterial proteins. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040698#pone.0040698.s005" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040698#pone.0040698.s008" target="_blank">Text S2</a> for more details.</p

Structural basis for the design of the BACHEMP-Cons peptide.

<p>(A) Three dimensional structure of <i>E. coli</i> pseudopilin (pdb 3G20). Bound calcium is shown in green. The fragment which forms the basis for the design of peptide BACHEMP-Cons is shown in blue and gold. The BACHEMP-Cons peptide is composed of amino acids from this structure (shown in blue) and amino acids that correspond to the consensus defined (shown in gold). (B) The fragment of the structure that corresponds to the BACHEMP-Cons peptide. The sequence of the peptide is colored according to the color coding explained above. The figure was produced using the program WebLabViewer (<a href="http://www.marcsaric.de/index.php/WebLab_Viewer_Lite" target="_blank">http://www.marcsaric.de/index.php/WebLab_Viewer_Lite</a>).</p