A mutually induced conformational fit underlies Ca2+-directed interactions between calmodulin and the proximal C terminus of KCNQ4 K+ channels

Abstract

Calmodulin (CaM) conveys intracellular Ca²⁺ signals to KCNQ (Kv7, "M-type") K⁺ channels and many other ion channels. Whether this "calmodulation" involves a dramatic structural rearrangement or only slight perturbations of the CaM/KCNQ complex is as yet unclear. A consensus structural model of conformational shifts occurring between low nanomolar and physiologically high intracellular [Ca²⁺] is still under debate. Here, we used various techniques of biophysical chemical analyses to investigate the interactions between CaM and synthetic peptides corresponding to the A and B domains of the KCNQ4 subtype. We found that in the absence of CaM, the peptides are disordered, whereas Ca²⁺/CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca²⁺/CaM has higher affinity for the B domain than for the A domain of KCNQ2-4 and much higher affinity for the B domain when prebound with the A domain. X-ray crystallography confirmed that these discrete peptides spontaneously form a complex with Ca²⁺/CaM, similar to previous reports of CaM binding KCNQ-AB domains that are linked together. Microscale thermophoresis and heteronuclear single-quantum coherence NMR spectroscopy indicated the C-lobe of Ca²⁺-free CaM to interact with the KCNQ4 B domain (K_d ∼10-20 μm), with increasing Ca²⁺ molar ratios shifting the CaM-B domain interactions via only the CaM C-lobe to also include the N-lobe. Our findings suggest that in response to increased Ca²⁺, CaM undergoes lobe switching that imposes a dramatic mutually induced conformational fit to both the proximal C terminus of KCNQ4 channels and CaM, likely underlying Ca²⁺-dependent regulation of KCNQ gating.

Keywords: M current; X-ray crystallography; biophysical chemical analysis; calcium signaling; calcium-binding protein; calmodulin (CaM); conformational change; isothermal titration calorimetry (ITC); microscale thermophoresis (MST); nuclear magnetic resonance (NMR); potassium channel; potassium voltage-gated channel subfamily Q member (KCNQ).