A mutually induced conformational fit underlies Ca 2 -directed interactions between calmodulin and the proximal C terminus of KCNQ4 K channels

Crystal R. Archer, Benjamin T. Enslow, Alexander B. Taylor, Victor De la Rosa, Akash Bhattacharya, Mark S Shapiro

Research output: Contribution to journalArticle

Abstract

Calmodulin (CaM) conveys intracellular Ca 2 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 [Ca 2 ] 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 Ca 2 /CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca 2 /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 Ca 2 /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 Ca 2 -free CaM to interact with the KCNQ4 B domain (K d 10 –20 M), with increasing Ca 2 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 Ca 2 , 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 Ca 2 -dependent regulation of KCNQ gating.

Original languageEnglish (US)
Pages (from-to)6094-6112
Number of pages19
JournalJournal of Biological Chemistry
Volume294
Issue number15
DOIs
StatePublished - Jan 1 2019

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Calmodulin
Peptides
Thermophoresis
Calorimetry
X ray crystallography
Structural Models
X Ray Crystallography
Titration
Ion Channels
Nuclear magnetic resonance spectroscopy
Magnetic Resonance Spectroscopy

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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A mutually induced conformational fit underlies Ca 2 -directed interactions between calmodulin and the proximal C terminus of KCNQ4 K channels . / Archer, Crystal R.; Enslow, Benjamin T.; Taylor, Alexander B.; De la Rosa, Victor; Bhattacharya, Akash; Shapiro, Mark S.

In: Journal of Biological Chemistry, Vol. 294, No. 15, 01.01.2019, p. 6094-6112.

Research output: Contribution to journalArticle

Archer, Crystal R. ; Enslow, Benjamin T. ; Taylor, Alexander B. ; De la Rosa, Victor ; Bhattacharya, Akash ; Shapiro, Mark S. / A mutually induced conformational fit underlies Ca 2 -directed interactions between calmodulin and the proximal C terminus of KCNQ4 K channels In: Journal of Biological Chemistry. 2019 ; Vol. 294, No. 15. pp. 6094-6112.
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abstract = "Calmodulin (CaM) conveys intracellular Ca 2 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 [Ca 2 ] 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 Ca 2 /CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca 2 /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 Ca 2 /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 Ca 2 -free CaM to interact with the KCNQ4 B domain (K d 10 –20 M), with increasing Ca 2 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 Ca 2 , 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 Ca 2 -dependent regulation of KCNQ gating.",
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AU - Archer, Crystal R.

AU - Enslow, Benjamin T.

AU - Taylor, Alexander B.

AU - De la Rosa, Victor

AU - Bhattacharya, Akash

AU - Shapiro, Mark S

PY - 2019/1/1

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N2 - Calmodulin (CaM) conveys intracellular Ca 2 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 [Ca 2 ] 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 Ca 2 /CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca 2 /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 Ca 2 /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 Ca 2 -free CaM to interact with the KCNQ4 B domain (K d 10 –20 M), with increasing Ca 2 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 Ca 2 , 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 Ca 2 -dependent regulation of KCNQ gating.

AB - Calmodulin (CaM) conveys intracellular Ca 2 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 [Ca 2 ] 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 Ca 2 /CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca 2 /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 Ca 2 /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 Ca 2 -free CaM to interact with the KCNQ4 B domain (K d 10 –20 M), with increasing Ca 2 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 Ca 2 , 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 Ca 2 -dependent regulation of KCNQ gating.

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