Fig. 5: NMR mapping of the residues contributing to the CaM–PYK2 KFL association.

CSP analyses of 150 µM [¹³C,¹⁵N] PYK2 KFL_728-839 titrated with CaM in the a absence and c presence of Ca²⁺. Orange and green horizontal lines indicate the threshold for major shifts (∆ppm + 2 std) and minor shifts (∆ppm + 1 std), respectively. Resonances that disappeared are indicated in black. CSPs from a and c are mapped onto a structural representative of PYK2 KFL_728-839 in b and d, respectively. Dark blue: residues for which peaks disappeared; slate blue: major shifts; cyan: minor shifts; black: prolines and unassigned residues. N and C indicate the N-terminal and C-terminal of the molecule. e CSP analysis of 150 µM [¹³C,¹⁵N] apo-CaM titrated with PYK2 KFL_728–839. Colouring according to a. f Mapping of the data from e onto the 3D structure of apo-CaM (PDB ID 4e53). Colouring according to b. g Plot of peak intensities recorded for 150 µM [¹³C,¹⁵N] Ca²⁺/CaM titrated with PYK2 KFL_728–839 at 10 °C. The intensity value without the binding partner (corresponding to a ratio of 1:0) is taken as a reference and used to normalise the intensity values of subsequent titrations (1:0.5, 1:1, 1:2) to compensate for an overall loss of intensity as the binding partner is added to the solution at increasing concentrations. Intensities per residue are colour-coded by titration ratio as indicated. h Intensity changes from g are mapped onto the 3D structure of CaM (PDB ID 1 × 02) according to dark blue: residues that disappeared at ratio 1:0.5; slate blue: residues with an intensity less than 0.045 at a ratio of 1:0.5; cyan: residues that have an intensity more than 0.045 at 1:0.5 but disappear at 1:1; black: prolines and unassigned residues. Red spheres represent Ca²⁺. i Sequence of the PYK2 KFL_728–839 construct used. The non-natural 6xHis-tag is boxed in blue. Pink residues indicate residues identified as contributing to both dimerisation and CaM binding. Blue residues are those assumed contributing only to dimerisation (based on CSPs), and red residues are those that only contribute to CaM binding (in the absence or presence of Ca²⁺). Prolines and unassigned residues are marked in bold black. Note that the confidence of the residue mapping is low because of the fuzzy nature of the binding events.