Figure 5: Characteristics of the CaM–IQCG interfaces and ability of IQCG and CaMKIV to interact with HSP70.

(a) IQCG uses a collection of both hydrophobic and hydrophilic residues (well-defined side chains as sticks in electron density map as grey mesh) to bind CaM in the Ca²⁺-free setting. The IQ motif-defining residues (coloured red in the lower panel) undertake almost identical conformations when compared with the CaM–myosin V IQ2 complex (middle panel). Dashed lines indicate salt bridges or hydrogen bonds. (b) A compromise in charge complementarity is evident at the interface between IQCG and Ca²⁺-bound CaM when compared with either the Ca²⁺-free complex (a) or a CaMKIIδ–CaM complex (middle panel). The colouring scheme is kept consistent with Fig. 4 and the superpositions were carried out based on the boxed sequences shown in the lower panels and so were those for Fig. 4. The electron density is shown as σ_A-weighted 2F_o−F_c map contoured at either 2σ (a) or 1.5σ (b). (c) A single-point mutated forms of IQCG, Hs_IQCG (Q401A) or Dr_Iqcg (Q312A) lost the ability to interact with CaM. (d) Ability of IQCG and CaMKIV to interact with HSP70, as determined by Co-IP experiments. Two irrelevant proteins, MRG15 and MEK1, are shown as negative controls. Anti-Flag M2 affinity gel was used for IP; an antibody to HSP70 or Flag was used for western blot (WB). (e) Ability of endogenic Iqcg and CaMKIV to interact with Hsp70. Co-IP experiments were performed using mouse testis lysate. Antibodies to CaMKIV, Iqcg and normal rabbit IgG were used for IP; antibodies to HSP70, Iqcg and CaMKIV were used for WB.