TY - JOUR
T1 - A mutually induced conformational fit underlies Ca2-directed interactions between calmodulin and the proximal C terminus of KCNQ4 K channels
AU - Archer, Crystal R.
AU - Enslow, Benjamin T.
AU - Taylor, Alexander B.
AU - De la Rosa, Victor
AU - Bhattacharya, Akash
AU - Shapiro, Mark S.
N1 - Funding Information:
Acknowledgments—We thank Yinghua Chen (Case Western Reserve University, Cleveland, OH) for preparing the Alexa Fluor 594 proteins and providing expertise in MST analysis and Pamela Reed, MaryAnn Hobbs, and Isamar Sanchez for expert technical support. We are indebted to the following facilities of the Institutional Research Cores at UT Health San Antonio in the Department of Biochemistry: the Center for Macromolecular Interactions, directed by Drs. Eileen Lafer and Bo Demeler; the X-ray Crystallography Core Laboratory, directed by Drs. P. John Hart and Alex Taylor; and the NMR spectroscopy core, directed by Dr. Dmitri Ivanov. Institutional Research Cores at UT Health San Antonio are supported by the Office of the Vice President for Research and the Mays Cancer Center, the center home to the UT Health San Antonio MD Anderson Cancer Center (National Institutes of Health Grant P30 CA054174). We also gratefully thank Drs. Eileen Lafer, Bo Demeler, and Dmitri Ivanov (Department of Biochemistry and Structural Biology), Robert Brenner (Department of Cell and Integrative Physiology), Brad Rothberg (Temple University), Chad Brautigam (UT Southwestern), Sudha Chakrapani (Case Western Reserve University), and William N. Zagotta (University of Washington) for many helpful discussions regarding this project. We are further indebted to James D. Stockand for providing the necessary time for C. R. A. to finish the manuscript.
Funding Information:
This work was supported by National Institutes of Health Grants R01 NS 043394 and NS 094461 (to M. S. S.), a Faculty Scholar Award (to M. S. S.), a grant from the Morrison Trust Foundation (to M. S. S.), NINDS/National Institutes of Health NRSA predoctoral training fellowship F31 NS090887 (to C. R. A.), National Institutes of Health/NHLBI postdoctoral training grant T32 HL007446 (to C. R. A.) (directed by James D. Stockand), NIH Grant R01 AI104476 (to Dmitri Ivanov supporting A. B.) and a core usage support grant from the IIMS CTSA at UT Health San Antonio (Department of Medi-cine). Part of this work was performed at the Northeastern Collaborative Access Team beamlines, which are funded by a program project grant from the NIGMS/NIH (P30 GM124165). The Eiger 16M detector on the 24-ID-E beam line is funded by NIH-ORIP HEI Grant S10-OD021527. This research used resources of the Advanced Photon Source, a user facility operated for the DOE, Argonne National Laboratory under Contract DE-AC02–06CH11357. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the respon-sibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
This work was supported by National Institutes of Health Grants R01 NS 043394 and NS 094461 (to M. S. S.), a Faculty Scholar Award (to M. S. S.), a grant from the Morrison Trust Foundation (to M. S. S.), NINDS/National Institutes of Health NRSA predoctoral training fellowship F31 NS090887 (to C. R. A.), National Institutes of Health/NHLBI postdoctoral training grant T32 HL007446 (to C. R. A.) (directed by James D. Stockand), NIH Grant R01 AI104476 (to Dmitri Ivanov supporting A. B.) and a core usage support grant from the IIMS CTSA at UT Health San Antonio (Department of Medicine). Part of this work was performed at the Northeastern Collaborative Access Team beamlines, which are funded by a program project grant from the NIGMS/NIH (P30 GM124165). The Eiger 16M detector on the 24-ID-E beam line is funded by NIH-ORIP HEI Grant S10-OD021527. This research used resources of the Advanced Photon Source, a user facility operated for the DOE, Argonne National Laboratory under Contract DE-AC02– 06CH11357. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
PY - 2019/4/12
Y1 - 2019/4/12
N2 - 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 (Kd 10 –20 M), 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.
AB - 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 (Kd 10 –20 M), 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.
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U2 - 10.1074/jbc.RA118.006857
DO - 10.1074/jbc.RA118.006857
M3 - Article
C2 - 30808708
AN - SCOPUS:85064336838
VL - 294
SP - 6094
EP - 6112
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 15
ER -