Table 1.
Summary of Ca-binding patterns.
Figure 1.
Section of the sequence alignment of ANP-like domains from 12 two-domain bacterial proteins.
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 figure S1. 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 Fulvimarina pelagi HTCC2506 (Q0G341), Manganese-oxidizing bacterium (strain SI85-9A1) (Q1YMS2), Methylobacterium chloromethanicum CM4 / NCIMB 13688 (B7KW13), M. extorquens DSM 5838 / DM4 (A9W3A5), M. extorquens PA1 (C7CGY0), Pseudomonas putida F1 (A5W572), P. putida GB1 (B0KJL7), P. putida KT2440 (Q88JT6), Rhodopseudomonas palustris BisA53 (Q07SX1), Rhodopseudomonas palustris BisB5 (Q13AU2), Roseobacter sp. MED193 (A3XF15) and Roseovarius sp. TM1035 (A6E280). Letters -A and -B refer to N- and C-terminal ANP-like domains, respectively.
Figure 2.
Two animal peroxidase-like domains (according to PROSITE profile PS50292) are shown in red, with an internal region of low homology marked in fair red; sequences with the consensus G-X-D-G-X(5)-D-D interrupting the domains are shown in brown bars and hemolysin calcium binding motifs (according to PS0033) are in blue bars. Numbers indicate amino acid residues.
Figure 3.
Sequence logo of insertions interrupting the ANP-like domains of bacterial proteins.
The figure was generated at http://weblogo.berkeley.edu 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 Table S1 and Text S2 for more details.
Table 2.
Sequence variation of the eleven residues inserts found in the animal heme dependent peroxidases of bacteria.
Table 3.
Bacterial heme peroxidases containing G-x-D-G-x(5)-D-D insertions within the ANP-like domain as defined by the PS50292 profile.
Figure 4.
Neighbor-joining phylogenetic tree of bacterial ANP-like domains contained in animal heme peroxidases with G-x-D-G-x(5)-D-D.
The domain of human myeloperoxidase, which does not contain any hit, is included as an out-group member. The alignment shown in figure S2 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) (Table S1). The column on the right indicates the number of insertions in each of the domains. Bar: 0.2 substitutions per amino acid position.
Figure 5.
Zoom at the calcium-binding region of the pseudopilin structure.
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 (http://www.marcsaric.de/index.php/WebLab_Viewer_Lite).
Figure 6.
Structural basis for the design of the BACHEMP-Cons peptide.
(A) Three dimensional structure of E. coli 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 (http://www.marcsaric.de/index.php/WebLab_Viewer_Lite).
Figure 7.
Isothermal titration calorimetry studies of the interaction of the BACHEMP-Cons peptide with CaCl2.
(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 Table 4. (B) Dependence of KD 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.
Table 4.
Thermodynamic parameters derived from the microcalorimetric titrations of peptide BACHEMP-Cons in different buffer systems.
Figure 8.
Isothermal titration calorimetry studies to evaluate the specificity of molecular recognition between the peptide BACHEMP-Cons and cations.
Upper panel: Raw data for the titration of 50 µM peptide with 3.2 µl aliquots of CaCl2 (A) and MgCl2 (B) Experiments were conducted in polybuffer pH 6.0 supplemented with 100 mM NaCl. Lower panel: Integrated, dilution-corrected and concentration normalized titration data of the peptide with CaCl2. Data were fitted with the “One binding site model” of the MicroCal (Northampton, MA) version of ORIGIN. The derived thermodynamic parameters are given in Table 4.
Figure 9.
ITC analysis of Ca binding to the N-terminal ANP-like domain of PepA.
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.
Table 5.
Presence of the different versions of the PERCAL calcium binding motif in bacteria and eukaryotes.