Fig 1.
Protein sequence, secondary structure and a cartoon representation of the three-dimensional structure of FasDD (PDB ID: 1DDF). The CaM-binding regions (Fas-Pep1 and Fas-Pep2) are highlighted in red and green, respectively.
Fig 2.
Proteolysis assay and gel filtration of Ca2+/CaM–FasDD digests.
(A) Gel filtration and SDS-PAGE of the proteolysis product of a 2:1 Ca2+/CaM:FasDD sample left at 4°C for one week. The SDS-PAGE data show that the degraded FasDD protein gave rise to ~5 kDa fragment(s). The Ca2+/CaM protein, however, remained stable. Analysis of the digestion products by mass spectrometry confirmed the identity of the FasDD peptides that are resistant to proteolysis. The most abundant peptides are located in the N-terminus (205–238, 205–239, and 205–240) and in the C-terminus (251–288, 259–288 and 262–288). (B) SDS-PAGE of FasDD, Ca2+/CaM and Ca2+/CaM:FasDD complex without or with added subtilisin at 1:500 (enzyme:protein) 24 hours after initiation of the reaction. Unbound FasDD and Ca2+/CaM were stable in the presence of subtilisin. (C) Gel filtration and SDS-PAGE data of the proteolysis product of a 2:1 Ca2+/CaM:FasDD sample treated with subtilisin for 24 hours. Similar to our observation in A, the SDS-PAGE data show a band at ~5 kDa eluting with Ca2+/CaM. FasDD fragments were identified by mass spectrometry (S1 Table).
Fig 3.
Gel filtration data of Ca2+/CaM complexes with recombinant FasDD peptides.
(A) Gel filtration chromatograms of Ca2+/CaM complexes with FasDD peptides using a HiLoad Superdex 75 (10/300 GL) column. (B) Complex formation has been confirmed by SDS-PAGE. Two bands of CaM and peptides are clearly observed. Elution of the peptides with CaM indicates direct binding. (C) Gel filtration calibration curve with indicated mobility of Ca2+/CaM, FasDD peptides and their complexes. The approximate molecular weights of complexes suggest a 1:1 stoichiometry.
Fig 4.
ITC data of Ca2+/CaM binding to FasDD peptides.
ITC data obtained for titration of (A) Ca2+/CaM at 450 μM into Fas-Pep1 at 25 μM, and (B) Ca2+/CaM (195 μM) into Fas-Pep2 at 17 μM. Data fitting afforded Kd values of 0.3 and 1.1 μM for Fas-Pep1 and Fas-Pep2, respectively.
Fig 5.
2D HSQC NMR data of Ca2+/CaM:Fas-Pep1 complex.
Overlay of 2D 1H-15N HSQC spectra obtained for 15N-labeled Ca2+/CaM in the free state (black) and in complex with Fas-Pep1 (red) at 1.5:1 peptide: Ca2+/CaM. No chemical shift changes were observed in the HSQC spectra with further addition of Fas-Pep1, indicating saturation at this ratio. Signal labels correspond to residues of CaM in the bound form. Signals labeled in blue are folded in the spectrum by 20 ppm.
Fig 6.
2D HSQC NMR data for Ca2+/CaM:Fas-Pep2 complex.
Overlay of 2D 1H-15N HSQC spectra obtained for 15N-labeled Ca2+/CaM in the free state (black) and in complex with Fas-Pep2 (green) at 1.5:1 peptide:CaM ratio. No chemical shift changes were observed in the HSQC spectra with further addition of Fas-Pep2, indicating saturation at this ratio. Signal labels correspond to residues of CaM in the bound form. Signals labeled in blue are folded in the spectrum by 20 ppm.
Fig 7.
Histograms of the chemical shift changes of Ca2+/CaM upon binding to FasDD peptides.
A schematic representation of the secondary structure of Ca2+/CaM is shown. Calcium ions are indicated by green dots. Histograms of normalized chemical shift changes vs. residue number for Ca2+/CaM bound to Fas-Pep1 and Fas-Pep2. Notice the differences in the chemical shift changes especially in the central linker (H4/H5) of Ca2+/CaM, which may suggest significant differences in the binding mode of FasDD peptides.
Fig 8.
Role of Ca2+/CaM Met residues in binding of FasDD peptides.
(A) Surface representation of the Ca2+/CaM structure (PDB ID: 3CLN) showing the nine Met residues localized in the N- and C-terminal lobes (red sticks). Ca2+ ions are colored in green. (B) Overlay of a selected region of 2D 1H-13C HMQC spectra obtained for 13C-labeled Ca2+/CaM as a function of added Fas-Pep1 (top) or Fas-Pep2 (bottom). [peptide:Ca2+/CaM = 0:1 (black), 0.5:1 (red), 1:1 (blue), 1.5:1 (green)]. Only the 1H-13C signals for the Met methyl groups are shown.