Arrhythmia mutations in calmodulin cause conformational changes that affect interactions with the cardiac voltage-gated calcium channel Kaiqian Wanga,1, Christian Holtb,1, Jocelyn Lua, Malene Brohusb, Kamilla Taunsig Larsenb, Michael Toft Overgaardb, Reinhard Wimmerb,2, and Filip Van Petegema,2 aDepartment of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; and bDepartment of Chemistry and Bioscience, Aalborg University, 9220 Aalborg, Denmark Edited by Richard W. Aldrich, The University of Texas at Austin, Austin, TX, and approved September 26, 2018 (received for review May 21, 2018) Calmodulin (CaM) represents one of the most conserved proteins found in patients with CPVT (6) and were subsequently found to among eukaryotes and is known to bind and modulate more than affect the function of RyR2 (13, 14). a 100 targets. Recently, several disease-associated mutations have In addition, several CaM mutations have been found to affect been identified in the CALM genes that are causative of severe an L-type voltage-gated calcium channel (CaV1.2). This channel 2+ cardiac arrhythmia syndromes. Although several mutations have has the intriguing property to undergo Ca -dependent in- 2+ been shown to affect the function of various cardiac ion channels, activation (CDI), a process whereby Ca accelerates the kinetics direct structural insights into any CaM disease mutation have been of inactivation (19, 20). This process plays a role in shaping the lacking. Here we report a crystallographic and NMR investigation action potential in cardiac myocytes and slowing of inactivation of several disease mutant CaMs, linked to long-QT syndrome, in can lead to LQT (12, 21). CDI requires the preassociation of 2+ complex with the IQ domain of the cardiac voltage-gated calcium CaM to the channel in low Ca conditions, and upon binding 2+ channel (CaV1.2). Surprisingly, two mutants (D95V, N97I) cause a Ca , a conformational change is transmitted to the channel to major distortion of the C-terminal lobe, resulting in a pathological impose inactivation. A region of prime importance is the IQ conformation not reported before. These structural changes result domain, located within the channel’s proximal C-terminal tail. 2+ 2+ BIOCHEMISTRY in altered interactions with the CaV1.2 IQ domain. Another muta- CaM can bind this region in both Ca -free and Ca -loaded + tion (N97S) reduces the affinity for Ca2 by introducing strain in EF states (22–24), and mutations in the IQ domain can diminish and hand 3. A fourth mutant (F141L) shows structural changes in the even abolish CDI (19, 24). Accordingly, LQT-associated muta- Ca2+-free state that increase the affinity for the IQ domain. These tions in the C-lobe of CaM (D95V, N97S, F141L) have been results thus show that different mechanisms underlie the ability of shown to either obliterate or decrease the extent of CDI (12). + CaM disease mutations to affect Ca2 -dependent inactivation of Despite the substantial impact of CaM disease mutations on the voltage-gated calcium channel. the functions of various channels, we still lack insights into their calcium signaling | X-ray crystallography | NMR | calcium channels | Significance inactivation Calmodulin is a ubiquitous Ca2+-sensing protein that can bind 2+ to more than a 100 different targets. In doing so, it endows almodulin (CaM) is an essential Ca sensor that plays a + Cpivotal role in many signaling pathways (1). It has a simple many of these with Ca2 -dependent modulation. As one of the architecture, consisting of two domains (N-lobe and C-lobe) that most conserved proteins throughout evolutionary history, all + can each bind two Ca2 ions in a highly cooperative manner. The calmodulin genes in vertebrates are identical. However, several + binding of Ca2 results in the exposure of hydrophobic residues, disease-associated mutations have been uncovered in human which affects the ability of CaM to bind other target proteins (2, calmodulin genes, all of which are linked to inherited cardiac + 3). CaM thus confers Ca2 sensitivity to a large list of both cy- arrhythmia syndromes. This report shows high-resolution tosolic and membrane-embedded proteins (2–5). glimpses into calmodulin disease mutations and their effect on The human genome contains three individual CALM genes binding the L-type voltage-gated calcium channel, a channel that encode identical proteins. The sequence of CaM has been involved in the cardiac action potential that receives calcium- highly conserved throughout evolution, with no variation found dependent feedback. Although each mutant is able to affect among vertebrates (1). Given this degree of sequence conser- calcium-dependent inactivation, the structures show that they vation, it was long thought that any mutation in the CALM genes adopt different mechanisms. would be fatal, because CaM is known to bind hundreds of target proteins and any mutation would thus likely affect multiple Author contributions: M.T.O., R.W., and F.V.P. designed research; K.W., C.H., J.L., M.B., pathways simultaneously. However, several studies have recently and K.T.L. performed research; K.W., C.H., J.L., M.B., K.T.L., M.T.O., R.W., and F.V.P. ana- reported mutations in the CALM genes of human patients (6– lyzed data; and K.W., C.H., M.B., M.T.O., R.W., and F.V.P. wrote the paper. 11). The finding that any CaM mutations are viable is already of The authors declare no conflict of interest. note, but even more surprising is that they are linked to cardiac This article is a PNAS Direct Submission. phenotypes, suggesting that other pathways involving CaM are Published under the PNAS license. less affected. The clinical phenotypes include long-QT syndrome Data deposition: The atomic coordinates and structure factors have been deposited in the (LQT), catecholaminergic polymorphic ventricular tachycardia Protein Data Bank, www.wwpdb.org (PDB ID codes 6GDL, 6GDK, 6DAE, 6DAH, 6DAF, and (CPVT), and idiopathic ventricular fibrillation (IVF). 6DAD). The NMR chemical shifts have been deposited in the BioMagResBank, www.bmrb. Subsequent studies have shown that the mutations can alter wisc.edu (accession nos. 34263 and 34262). the functional behavior of several cardiac ion channels (10–16). 1K.W. and C.H. contributed equally to this work. 2+ This includes the cardiac ryanodine receptor (RyR2), a Ca 2To whom correspondence may be addressed. Email: [email protected] or petegem@mail. release channel located in the sarcoplasmic reticulum (17). ubc.ca. Mutations in this channel are often associated with CPVT, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. + causing a gain-of-function phenotype with excessive Ca2 release 1073/pnas.1808733115/-/DCSupplemental. (18). The first two reported CaM mutations (N53I, N97S) were www.pnas.org/cgi/doi/10.1073/pnas.1808733115 PNAS Latest Articles | 1of10 Downloaded by guest on September 29, 2021 + + mechanism of action. Do they merely affect Ca2 affinity or do These results suggest substantial changes in the Ca2 -free form they alter structure and dynamics? Do they affect target recog- of CaM, due to the F141L mutation. Indeed, native PAGE ex- nition? Here we report an integrated structural biology ap- periments show a different mobility of F141L CaM, relative to + + proach, combining NMR and X-ray crystallography. Together wild-type, under Ca2 -free but not Ca2 -loaded conditions (SI with quantitative binding assays, we describe the mechanism of Appendix, Fig. S2). + action for four different CaM disease mutants. Although each At high Ca2 concentrations, the affinities remain high for mutant is able to reduce CaV1.2 CDI, their mechanisms of action each mutant. To confirm this, and to obtain further thermody- are highly divergent. We observe at least three distinct primary namic insights, we also performed isothermal titration calorim- effects, indicating that the disease mutations act in different ways etry (ITC) experiments on the individual C-lobes at saturating + to affect CDI of L-type calcium channels. (10 mM) Ca2 levels (Fig. 1 B–F). These confirm that the binding affinities remain high (KD in the low nanomolar range). Results However, the D95V mutant has a more negative ΔH(−15 vs. − Throughout this report we utilize the numbering for mature −10 kcal·mol 1). The N97I mutant binds the weakest, with an CaM in which the initial methionine (residue 0) is removed. affinity ∼3.5-fold lower compared with wild-type CaM (SI Appendix,TableS2). Effect of CaM Mutations on Binding to the Ca 1.2 IQ Domain. It has These changes in affinity for the IQ domain have implications V + been shown previously that several CaM disease mutants, linked on the inherent affinity of the CaM:IQ complexes for Ca2 .To 2+ to LQT, have a dominant effect on CDI of CaV1.2 (12). We obtain experimental estimates of the CaM affinity for Ca when therefore started by analyzing their effect on the ability to bind to bound to the IQ domain, we monitored the fluorescence an- the IQ domain of Ca 1.2, a prerequisite for normal CDI. We isotropy development of the TAMRA-labeled IQ domain as a V + + covered a range of free Ca2 concentrations and measured the function of the free Ca2 concentration at the highest CaM affinities using fluorescence anisotropy, making use of a concentration (SI Appendix, Fig. S3). We find an approximately + TAMRA-labeled IQ domain (SI Appendix, Fig. S1 G–K). The threefold reduction in Ca2 affinity for the D95V and N97I 2+ overall plot of KD vs. Ca concentration for each mutant is mutants compared with wild-type. The F141L mutation affected displayed in Fig. 1A. These data show that several mutants have affinity the most, ∼5.5-fold lower than wild-type CaM, reflecting a decreased affinity for the IQ domain in the range of 100 nM– the increase in affinity for the IQ domain in the apo-state and the + + 10 μMCa2 .