Extended Data Fig. 3: Biochemical property and structural mechanism of CaM interaction with OspC3.

a, ITC profiles of CaM binding to OspC3 or OspC3^ARD. Calculated dissociation constant (K_D) and binding stoichiometry (N) are expressed as means ± SD from three determinations. ND, not detectable. b, c, Activation of OspC3 by Ca²⁺-loaded CaM. b, Gel-filtration chromatography combined with SDS-PAGE analyses of the complex between OspC3 and Ca²⁺-loaded CaM. c, Caspase-4-p30-C/A was reacted with NAD⁺ and OspC3 that had been complexed with Ca²⁺-free or Ca²⁺-loaded CaM. The reactions were subjected to native/SDS-PAGE analyses. Control, fully ADP-riboxanated caspase-4 obtained by co-expression with OspC3 in E. coli. d, Measurements of Ca²⁺ contents in OspC3-bound CaM by ICP-MS. Purified Ca²⁺-free and Ca²⁺-loaded CaM were included as controls. Data are means ± SD from three determinations. e, f, Complex formation and activation of MBP-OspC3 by Ca²⁺-free CaM. e, Gel-filtration chromatography combined with SDS-PAGE analyses of the complex between MBP-OspC3 and Ca²⁺-free CaM. f, Caspase-4/11-p30-C/A was reacted with NAD⁺ and the OspC3–CaM or MBP-OspC3–CaM complex. The reactions were subjected to native/SDS-PAGE analyses. Control, fully ADP-riboxanated caspase-4/11 obtained by co-expression with OspC3 in E. coli. g, Structural comparison of OspC3^ARD in the CaM complex with its apo-state. h, Cartoon model of Ca²⁺-free CaM binding to the IQ motif of myosin V (PDB code: 2IX7). On the right are close-up views of structural comparisons of the two lobes in OspC3-bound CaM and those in myosin V-bound CaM. i, Gel-filtration chromatography analyses of complex formation between CaM and OspC3 (WT or an indicated mutant). Shown are elution profiles along with SDS-PAGE analyses of the elution fractions. Data in representative of three (a–c, e, f, i) or two (d) independent experiments.

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