Arrhythmia mutations in calmodulin cause conformational changes that affect interactions with the cardiac voltage-gated

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 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 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 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 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 , 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 (RyR2), a Ca 2To whom correspondence may be addressed. Email: [email protected] or petegem@mail. release channel located in the (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 . In this range, the affinity is the weakest for D95V maintained high affinity for the IQ domain in the Ca2 -saturated + and N97I CaM, whereas N97S CaM has a smaller effect on the state. Thus, all three mutations display a reduced Ca2 -sensing affinity and is closer to wild-type. ability when in complex with the Cav1.2 IQ domain compared A peculiar observation is made for F141L CaM, which has with wild-type CaM. a >10-fold higher affinity for the IQ domain than wild-type un- + der low Ca2 concentrations (SI Appendix, Table S1). At con- Crystal Structure of D95V Ca2+/CaM Bound to the Ca 1.2 IQ Domain. − + V centrations near 10 6 M free Ca2 , the affinity of F141L CaM is Because the D95V mutation did not seem to abolish CaM + similar to those of D95V and N97I CaM. Although both D95V binding to the IQ domain at high Ca2 concentrations, we solved and F141L CaM have previously been shown to abolish CDI of a crystal structure of this complex at 2.0-Å resolution. The CaV1.2 (12), these results suggest that there are differences in structure contains two complexes per asymmetric unit, which are the inherent mechanisms for each mutant. To confirm the pe- very similar. The figures and analysis are shown for complex A. culiar observation for F141L CaM, we also performed affinity We found no electron density that could fit a fully occupied + + measurements on the isolated C-lobes (SI Appendix, Fig. S1 and Ca2 ion in EF hand 3 (EF3). At longer wavelengths, Ca2 has Table S1). These confirm that the F141L mutant has an in- an anomalous signal, so we collected an anomalous dataset. The + creased affinity for the IQ domain at low Ca2 concentrations. corresponding anomalous density map shows the positions of

A Full-length CaM BCTime (min) Time (min) 10-4 010203040 010203040 0.0

-1 -1 0.0 -5 10 WT -0.1 D95V

µcal s -0.1 µcal s 10-6 N97I -0.2 (M) N97S -7 -0.2 D,app 10 F141L -0.3 K 0 0 10-8 -4 -4

of injectant of injectant -8

-1 10-9 -1 -8 -12

-10 -16 10 -12 10-9 10-8 10-7 10-6 10-5 10-4 10-3 kcal mol 0.0 1.0 2.0 kcal mol 0.0 1.0 2.0 Free Ca2+ concentration (M) Molar ratio Molar ratio

D Time (min) E Time (min) F Time (min) 2+ 2+ 010203040 0 10203040 0 10203040 Fig. 1. Ca /CaM- and Ca /C-lobe-CaV1.2 IQ do- main binding affinities. (A) Binding affinity (KD,app)

-1 0.0

-1 0.0 -1 0.0 of full-length CaM variants to the IQ domain at + various free Ca2 concentrations by TAMRA fluores-

µcal s µcal s -0.1 µcal s -0.1 -0.1 cence anisotropy. Error bars represent the SD of -0.2 -0.2 -0.2 three replicates. Isothermal titrations of 75 μM(B) 2+ 2+ 0 0 0 wild-type Ca /C-lobe, (C) D95V Ca /C-lobe, (D) N97I + + + Ca2 /C-lobe, (E)N97SCa2 /C-lobe, and (F) F141L Ca2 /C- -4 -4 -4 μ

of injectant of injectant of injectant lobe into the 11- M IQ domain. Isotherms were fit to

-1

-1 -1 -8 -8 -8 a one-site binding model. Panels show injections of 2 μL of titrant into IQ domain (Upper) and binding -12 -12 -12

kcal mol kcal mol 0.0 1.0 2.0 kcal mol 0.0 1.0 2.0 0.0 1.0 2.0 isotherms (Lower). Experiments were conducted in triplicates; representative curves are shown.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1808733115 Wang et al. Downloaded by guest on September 29, 2021 + + Ca2 in the structure. We show this map around EF3 and EF4 in the NMR spectra of wild-type apoCaM and D95V Ca2 /CaM (SI + the C-lobe (SI Appendix, Fig. S4A). Whereas the density for Ca2 Appendix, Fig. S6) shows that all C-lobe resonances are altered, in EF4 is clearly visible, no density is found for EF3. This shows also at positions remote from the mutation site. Thus, we con- + + that even under saturating Ca2 conditions (10 mM in the clude that the structure of the C-lobe of D95V Ca2 /CaM nei- + crystallization condition), the D95V mutation completely dis- ther resembles the wild-type apo nor the wild-type Ca2 -loaded + rupts Ca2 binding to EF3. form, and that large changes also occur in the absence of the + This loss of Ca2 results in major allosteric changes, most IQ domain. readily observed in animations (Movies S1 and S2). Within EF3, Despite the large conformational changes in the C-lobe, the + Glu104, which normally coordinates the Ca2 ion, has now re- D95V mutant is still able to bind to the IQ domain with nano- + laxed to a different position, with its side chain shifting up to molar affinity at saturating Ca2 concentrations (Fig. 1). How- 6.6 Å (Fig. 2 A and B). This has long-range allosteric consequences, ever, because the surface of the D95V C-lobe has altered as it completely rearranges the α-helical packing in the C-lobe dramatically, the mode of the interaction has changed, as shown (Fig. 2C). Methionine residues in the C-lobe form major con- by a direct comparison with the wild-type complex structure (22, tributions to the binding to the IQ domain (22, 23). The effect of 23) (Fig. 2 E and F). In one such structure (PDB ID code 2BE6), the structural changes on these methionine residues is most three complexes are present in the asymmetric unit. In each of easily observed for Met109 and Met144: their side chains are these, the N-lobe adopts different positions, supporting the no- + ∼15 Å apart in the wild-type Ca2 /CaM:IQ complex structure tion that the N-lobe is bound weakly to the IQ domain and may but are in Van der Waals contact (3.8 Å) in the D95V mutant. have another binding site in full-length channels (25, 26). How- The wild-type CaM lobes are known to undergo large con- ever, the C-lobe is bound in identical ways in all published + formational changes upon binding or unbinding Ca2 ,sowe structures, with three aromatic IQ domain residues binding to + wondered whether the conformation of D95V Ca2 /CaM is hydrophobic pockets within the C-lobe. Ile1624, which defines the + similar to that of a Ca2 -free lobe. We superposed the D95V C- “I” in the canonical IQ motif, is at the periphery, partially exposed + + lobe with Ca2 -free and Ca2 -occupied wild-type C-lobes and to solvent. In the D95V CaM, however, the structural distortions with CaM1234 (SI Appendix, Fig. S4). This shows that the helical lead to a new hydrophobic pocket that fully sequesters Ile1624. The packing in D95V CaM is different from either, and thus repre- three aromatic anchors in the IQ domain (Y1627, F1628, F1631), sents a conformation that has not been reported before. which contribute most to the binding in the wild-type CaM struc- To check whether the altered conformation also occurs in the ture, have altered environments. absence of the IQ domain, we compared HSQC spectra of WT These altered interactions result in a rotation of the C-lobe + and D95V Ca2 /CaM in the absence of IQ peptide (SI Appendix, relative to the IQ domain (Fig. 2D), with positional shifts in the BIOCHEMISTRY Fig. S5). These show that the C-lobe resonances display great main chain up to 11 Å. There are smaller shifts in the relative shifts, whereas the N-lobe resonances superpose well. Overlaying position of the N-lobe, but because this lobe can adopt multiple

A D95 B V95 N97 N97

D93 D93 E104 E104

C D95V Ca2+/C-lobe D WT Ca2+/C-lobe + Fig. 2. Comparisons of the wild-type Ca2 /CaM- and 2+ M145 D95V Ca /CaM:CaV1.2 IQ domain structures. Stick representation of EF3 of (A) wild-type Ca2+/CaM (PDB + ID code 2BE6) and (B) D95V Ca2 /CaM. Residue 95 is M109 M144 shown in black, calcium ion as a green sphere, and M124 water molecule as a red sphere. (C) Superpositions of WT Ca2+/CaM:IQ C-lobes of wild-type (PDB ID code 2BE6, gray) and D95V Ca2+/CaM:IQ D95V Ca2+/CaM (blue). The superposition is based on the first helix of the lobes, highlighting the different E F conformation. Methionine residues are labeled, showing how M109 and M144, far apart in wild-type, form Van der Waals interactions in the mutant. (D) + Superposition of WT Ca2 /CaM (PDB ID code 2BE6, Y1627 Y1627 2+ gray) and D95V Ca /CaM (N-lobe in green, C-lobe in blue, Ca2+ ions in black) based on the IQ domain. Interactions of the C-lobe of (E) wild-type Ca2+/CaM + (PDB ID code 2BE6) or (F) D95V Ca2 /CaM with the

F1631 F1631 CaV1.2 IQ domain. The IQ domain is shown in stick representation with I1624 colored in green; CaM C- I1624 I1624 lobe is shown in surface representation with the F1628 F1628 colors indicating the electrostatic potential (red: negative; blue: positive).

Wang et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 29, 2021 + conformations around the IQ domain (22, 23), this change in therefore solved a structure of the N97I Ca2 /CaM:IQ domain the N-lobe position is not necessarily the result of the disease complex at 1.65-Å resolution (Fig. 4). In contrast to the N97S mutation, N97I results in large con- mutation. + The Ile1624 residue has previously been shown to be crucial formational changes reminiscent of the D95V mutant. Ca2 for CDI (24). Its altered interactions with the D95V C-lobe, binding to EF3 is abolished, resulting in relaxation of Glu104 to + along with the complete loss of Ca2 binding to EF3, may both a position different from the wild-type. This causes a distortion contribute to the loss of CDI observed for D95V CaM (12). of the C-lobe and a rotation of the C-lobe relative to the IQ domain. Fig. 4A shows a superposition of the D95V and N97I 2+ Structure of N97S Ca2+/CaM. CaM Asn97 is another residue in Ca /CaM:IQ domain complexes, showing that these two CaM EF3. The N97S mutation has been linked to both CPVT and mutants share similar overall conformations, thus both very + LQT (6, 7). Because it coordinates the same Ca2 ion as CaM different from the wild-type complex. Although both the N97I and D95V mutations decrease the Asp95, we wondered whether it also results in large conforma- + affinity of the C-lobe for Ca2 , N97I CaM, in the absence of the tional changes, similar to the D95V mutation. We were unable to 2+ obtain a crystal structure of a complex with the IQ domain, but IQ domain, still binds Ca with a twofold higher affinity com- + were able to solve a 2.5-Å crystal structure of Ca2 -saturated pared with D95V CaM (7, 8, 27). We therefore compared the B N97S CaM, in the absence of any target peptide. Fig. 3A shows a area around the mutation site for possible clues. Fig. 4 shows a superposition of the region in EF3 with a wild-type CaM struc- local superposition in EF3. In addition to small changes in main- ture obtained in the absence of a target peptide (PDB ID code chain conformation, Tyr99 is seen to adopt a different confor- + 4BW8). In this case, N97S CaM is still able to bind Ca2 , and the mation, with its side chain stacking with Ile97 and Gln135. This conformational changes are highly localized and much smaller conformation is present in both complexes of the asymmetric unit, whereas it is not observed for any of the D95V or wild-type than for D95V CaM (Fig. 3B and Movie S3). Interestingly, the + CaM complexes. We postulate that this additional stabilizing Ser97 side-chain hydroxyl oxygen is able to compensate for Ca2 interaction, not observed for D95V CaM, results in a more stable + + coordination. However, because this oxygen is one position Ca2 -bound state, and hence a higher Ca2 affinity. closer to the main chain compared with the Asn97 side-chain 2+ Overall, these results show that not only the exact location, but oxygen, the residue needs to move closer to the Ca ion, with also the identity of the substitution determines the effect on the changes in the conformation of the main chain. We hypothesize structure. Given the similar impact of the N97I and D95V mu- that this results in a less-favorable conformation, which would 2+ + tations on the conformation of Ca /CaM, we predict that N97I impact the affinity for Ca2 . Indeed, it has previously been found CaM would also abolish CDI of CaV1.2. that the N97S mutation leads to a reported 1.5- to 4-fold re- + 2 2+ duction in the affinity for Ca (6, 13, 14, 27). Crystal Structure of F141L Ca /CaM Bound to the CaV1.2 IQ Domain. Another feature of the N97S structure is an altered confor- The three mutations described above affect residues directly + mation of Gln135, located in EF4. In wild-type CaM, this residue involved in Ca2 binding. F141L is a mutation originally linked to makes water-mediated hydrogen bonds with Asn97, but not with LQT (8) and has been shown to abolish CDI of Ca 1.2 (12). + V Ser97 in the N97S structure. As a result, the side-chain confor- Phe141 is not directly involved in Ca2 coordination, but nev- mation is different, with small shifts in the main chain, showing ertheless, its mutation to Leu was shown to decrease the affinity + that changes in EF3 can be transmitted to EF4. of the C-lobe for Ca2 approximately fivefold in the absence of A full superposition of the wild-type and N97S C-lobes shows any binding partner (8). We crystallized the F141L mutant in the that the two structures superpose well, with an RMSD of 0.38 Å presence of the CaV1.2 IQ domain. for 55 Cα atoms (Fig. 3A). The main effect of the mutation is Fig. 5A shows a superposition of wild-type (PDB ID code + thus a local change in the energetics. 2BE6) and F141L Ca2 /CaM bound to the IQ domain around the site of the mutation. In wild-type CaM, Phe141 is involved in 2+ Crystal Structure of N97I Ca /CaM Bound to the CaV1.2 IQ Domain. interactions with the IQ domain, but also in forming hydropho- + Residue 97 in CaM is the target for another mutation (N97I), bic interactions with other Ca2 /C-lobe residues. It interacts with linked to LQT (7). Because the D95V and N97S mutations have Tyr1627 of the IQ domain, a residue that forms one of the main + such different effects on the structure of CaM, despite co- anchor points for the Ca2 /C-lobe (22). In the mutant, Leu141 is + ordinating the same Ca2 ion, we wondered whether this is due more than 4 Å away from the Tyr1627 side chain, and so con- to the exact position, or due to the nature of the substitution. We tributes minimally to the binding. In addition, there is an overall

A B S97 N97 WT Ca2+/C-lobe D95 N97S Ca2+/C-lobe Q135

S97 N97

E104

+ + + Fig. 3. Comparisons of the wild-type Ca2 /CaM and N97S Ca2 /CaM structures. (A) Overall superposition of the C-lobes of wild-type Ca2 /CaM (PDB ID code + 4BW8, gray) and N97S Ca2 /CaM (blue). C-lobes are shown in ribbon representation and calcium ions as green spheres. S97 is colored black. (B) Superposition + + of wild-type Ca2 /CaM (gray) and N97S Ca2 /CaM (blue) based on residues 92–102; shown are details around EF3. CaM is in stick representation, with the + mutated S97 residue in black, and calcium ion as a green sphere. The dashed lines indicate hydrogen bonds. To maintain a similar Ca2 -coordination, the main chain and side chain of Ser97 move closer, likely resulting in strain that reduces the Ca2+ affinity. Due to the absence of hydrogen bonds with Gln135 in the mutant, the Gln135 conformation is altered, resulting in small changes in EF4.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1808733115 Wang et al. Downloaded by guest on September 29, 2021 I97 A B Y99 V95 Q135 D95

D93

E104 D95V Ca2+/CaM:IQ N97I Ca2+/CaM:IQ

2+ 2+ 2+ 2+ Fig. 4. Comparison of the D95V Ca /CaM - and N97I Ca /CaM:CaV1.2 IQ domain structures. (A) Superposition of D95V Ca /CaM:IQ (gray) and N97I Ca /C- lobe:IQ (N-lobe in green, C-lobe in blue, IQ in orange) based the IQ domain. CaM is shown in cartoon representation, IQ domain as sticks, and calcium ionsas spheres. The overall conformation of N97I is similar to D95V, and thus very different from wild-type Ca2+/CaM. (B) Superposition of D95V Ca2+/CaM (gray) and N97I Ca2+/CaM (blue) based on residues 92–102; shown are details around EF3. CaM is in stick representation, with the mutated residues in black, and calcium + ion as a green sphere. In both mutants, no Ca2 is visible in EF3, leading to a conformational switch, mediated by Glu104. A noticeable difference is the + stacking of Tyr99 against the hydrophobic Ile97 residue. This conformation of Tyr99 is unique, and not found in wild-type Ca2 /CaM or any of the other disease mutant structures. Because Tyr99 now stacks against EF4, small changes in the positions of EF4 residues can be observed.

+ reduction in hydrophobic interactions with other Ca2 /C-lobe spectrum. However, for the peaks that are present, the chemical + residues for the F141L mutant, suggesting a less-stable Ca2 -bound shifts are very similar to wild-type. These belong to the N-lobe, conformation. So although Phe141 is not directly involved in suggesting this lobe is minimally impacted by the mutation. A + Ca2 coordination, and the Leu141 mutation does not lead to a peak intensity histogram (Fig. 6C) shows an almost identical + visible change in coordination of the Ca2 ions in EF3 and EF4, intensity profile of the first 77 peaks (corresponding roughly to + a decreased stability of the F141L Ca2 /C-lobe may explain the the N-lobe), while the remaining amino acids display significantly + reduction in Ca2 affinity. weaker NMR peaks and the overall number of peaks is reduced Despite the less-favorable packing, the overall conformation in the mutant. The direct carbon-detected CACO also show BIOCHEMISTRY + of the F141L Ca2 /C-lobe and its relative position to the IQ fewer than the 148 expected peaks. While signal intensities in the domain are very similar to wild-type (Fig. 5B). As discussed N-lobe allowed for a full assignment, the weak signals almost + earlier, relative positional changes of the Ca2 /N-lobe are less exclusively come from C-lobe residues, making resonance as- likely to be relevant due to the relative mobility of this lobe in the signment of large stretches of the C-lobe residues impossible. wild-type structure (22). Overall, the minor structural changes Those that could be assigned are all located in positions where + observed for the F141L Ca2 /CaM:IQ complex seem insufficient the wild-type apo C-lobe is flexible (Fig. 6D). Those chemical to explain its ability to obliterate CDI, suggesting that there are shifts that could be assigned for F141L apoCaM display very changes in dynamics, not captured with the crystal structure, or little difference to the wild-type apoCaM (SI Appendix, Fig. S7). that there are changes in the apo-form of this mutant. Correspondingly, the 3D structure of the N-lobe of F141L apoCaM is indistinguishable from the structure of the wild-type NMR Analysis of F141L apoCaM. The F141L mutation shows the (Fig. 6E), whereas no structure could be calculated for the C- puzzling effect of increasing the affinity for the IQ domain, rel- lobe. + ative to wild-type CaM, in the absence of Ca2 . We therefore An important observation is that the F141L apo C-lobe is not + expect substantial structural changes in the Ca2 -free state. Be- fully disordered, because in such a case we would easily have cause our efforts to crystalize a F141L apoCaM were un- been able to detect all resonances. Instead, these observations successful, we resorted to NMR (Fig. 6). The NMR spectra suggest it is likely in an equilibrium between multiple states ex- reveal major changes in F141L vs. wild-type apoCaM, because changing at rates that correspond roughly to the chemical-shift F141L apoCaM displays fewer peaks than wild-type apoCaM. differences, making it difficult to observe NMR resonances. This 1 15 Fig. 6 A and B show an overlay of [ H- N]-HSQC spectra, is corroborated by T2 relaxation dispersion data showing only demonstrating that there are several peaks missing in the mutant few residues with relaxation dispersion in the N-lobe, but

A B

Y1627 M145

F141 E87 E84 L141

I85

WT Ca2+/CaM:IQ F141L Ca2+/CaM:IQ

Fig. 5. Comparisons of the wild-type and F141L Ca2+/CaM:IQ domain structures. (A) Superposition of WT Ca2+/CaM (PDB ID code 2BE6) (gray) and F141L Ca2+/CaM + (N-lobe in green, C-lobe in blue) based on the IQ domain (orange IQ corresponds to the F141L Ca2 /CaM structure). CaM is shown in cartoon representation, IQ domain as sticks, and calcium ions as spheres. (B) Details around the F141L mutation site. IQ domain is shown in stick representation, and CaM in cartoon and stick representation. Residue L141 is colored black. Because Phe141 is part of a hydrophobic stacking interaction, multiple side chains have different conformations in the F141L mutant. This leads to altered interactions that result in the shift of C-lobe residues up to 2.3 Å.

Wang et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 29, 2021 Fig. 6. NMR analysis of F141L apoCaM. (A and B) [1H-15N]-HSQC spectra of wild-type (black) and F141L apoCaM (red) overlaid. (A) Whole spectrum; (B)re- gion of interest. (C) Comparison of the peak volumes (normalized to the strongest peak) found in the [1H-15N]-HSQC spectra of both wild-type (red bars) and F141L (blue bars) apoCaM along the positive y axis. In each spectrum, each visible backbone amide peak was picked, integrated, and then normalized. The negative y axis shows the corresponding values (multiplied by −1) for the directly C-detected CACO spectrum of wild-type (green) and F141L apoCaM (light blue). The values are sorted by descending volume (i.e., their x coordinate does not correspond to any specific residue number). It is seen that the peak volume profile of both HSQC and CACO spectra is very similar for the most intense ∼80 peaks, slightly more than half the number of amino acid residues. However, the remaining peaks are much less intense in F141L apoCaM and there is an overall smaller numer of peaks in the mutant. Also the C-detected CACO yields a significantly smaller number of peaks for F141L CaM than for the wild-type. (D) Structure of WT apoCaM (first model of 1DMO) color-coded according to the assignment status of F141L apoCaM: amino acids shown in purple could be assigned in the mutant, amino acids shown in yellow could not. (E) Bundle of 20 NMR structures of F141L apoCaM (PDB ID code 6GDL, backbone only, carbon atoms in cyan, nitrogen atoms in blue) overlaid with a ribbon drawing (magenta) of the first model of the NMR structure of wild-type apoCaM (PDB ID code 1DMO) (28). Numbers indicate residue numbers.

+ significant relaxation dispersion throughout the C-lobe (SI Ap- We therefore analyzed the F141L Ca2 /CaM, in the absence of pendix, Fig. S8). We also tried to alter both exchange rates (by IQ domain, with NMR. In contrast to the apoCaM data, the NMR + changing temperature) and resonance frequency (by recording spectra of F141L Ca2 /CaM are more similar to wild-type (Fig. 7 A NMR spectra at higher field strength) to see whether this would and B). A similar number of signals is seen in the [1H-15N]-HSQC improve the spectra. Only minute changes could be observed spectrum and structure determination shows that the N-lobe within the permissible temperature range (277–310 K) and at structure resembles the wild-type closely (Fig. 7C). The C-lobe accessible field strengths (14.1 and 22.3 T) (SI Appendix, Fig. S9). displays the same overall fold as the wild-type, but there are NMR structures of wild-type apoCaM show that F141 is fully distinct differences around the site of mutation and notably in buried (28), forming part of a hydrophobic core involving EF4 (Fig. 7D). The final α-helix from residues 138–146 has a Leu105, Met124, Met144, and Met145. Destabilization of these slightly changed orientation, with the helical axis tilted 18 ± 3.6° interactions by introducing of a shorter Leu side chain most closer toward residue 128. As has been shown for wild-type + likely underlies the conformational changes. Ca2 /CaM (29), the relative orientations of N-lobe and C-lobe + In conclusion, the F141L mutation imparts substantial for F141L Ca2 /CaM are entirely dynamic in solution and thus conformational changes in the apo C-lobe, resulting in a undefined. Residues 75–87, linking the two lobes, are undefined mixture of states. This most likely underlies the ability of this and adopt a variety of conformations. In the wild-type, F141 is mutant to bind the IQ domain with 10-fold higher affinity at involved in several π–π interactions to both F92 and F89. In + low Ca2 concentrations. addition, strong hydrophobic interactions exist with M145 and I100, as well as weaker interactions with Y138, V142, and V136. NMR Analysis of F141L Ca2+/CaM. F141L imparts minimal changes Leucine cannot form π–π interactions and its side chain pro- + to the Ca2 /CaM:IQ complex, but this may be due to the pres- trudes less far from the backbone, thus leaving an unfavorable ence of the IQ domain, which might stabilize the folding of CaM. void in the hydrophobic packing. This could be the reason why

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1808733115 Wang et al. Downloaded by guest on September 29, 2021 + Fig. 7. NMR analysis of F141L Ca2 /CaM. (A and B) [1H-15N]-HSQC spectra of WT (black) and F141L (red) Ca2+/CaM overlaid. (A) Whole spectrum; (B) region of interest with selected peaks labeled with the corresponding amino acid number. (C and D) Struc- tures of the N-lobe and C-lobe, respectively, of F141L + BIOCHEMISTRY Ca2 /CaM (PDB ID code 6GDK, backbone only, carbon atoms in cyan, nitrogen atoms in blue, calcium in yellow) overlaid with a ribbon drawing (magenta) of wild-type Ca2+/CaM lobes (PDB ID code 1CLL) (44). Numbers refer to the position of selected amino acids. (E) Close-up of residue F141 (shown in red) in wild- + type Ca2 /CaM. YASARA was used to identify possible π–π interactions of F141 with other amino acids (or- ange) as well as its hydrophobic interactions with other amino acids (purple). (F) Same information as E, but for the F141L Ca2+/CaM structure: two represen- tative structures of the the F141L Ca2+/CaM bundle are shown with each their orientation of F89. YASARA was used to identify possible hydrophobic interactions of L141 with other amino acids (purple). (G)Super- position of the wild-type structure (magenta) overlaid withabundleof10structuresofF141LCa2+/CaM (PDB ID code 6GDK, blue and gray ribbon). The side chain of residue 141 is shown in red. Side chains of other residues no more than 4.5 Å away from any atom in thesidechainofresidue141areshown(carbonatoms in cyan, sulfur in green).

the terminal α-helix is bent toward the hydrophobic core, thus the IQ domain. However, because differences may also arise allowing the shorter leucine side chain to form hydrophobic from a change in dynamics, we performed {1H}-15N-NOE interactions and avoiding the formation of a void volume. Fig. 7 E– measurements. In rigid parts of a folded protein, the NOE is on G show the environment of residue 141 in both wild-type and the order of 0.7–0.8, while smaller values indicate intramolecular mutant. Interestingly, F89 adopts two different orientations in the mobility, typically found in loops, termini, or disordered regions. bundle of NMR structures, both satisfying the NOE data available For F141L apoCaM, NOE data show a well-ordered N-lobe with and both allowing for hydrophobic interactions between F89 and some disordered loops (SI Appendix,Fig.S10). Only few NOEs can L141, as well as π–π interactions between F89 and Y138. Hydro- be observed in the C-lobe, but all of them display a higher degree of phobic and π–π interactions between residue 141 and its sur- intramolecular mobility than whatcanbeexpectedfromastable roundings are highlighted in Fig. 7E (wild-type) and Fig. 7F folded protein. Although only 50% of the C-lobe resonances could (F141L). F92, which is close enough to F141 in the wild-type to be detected, and only one-third of these assigned, this further form both hydrophobic and π–π interactions, is too far away for supports the observation that F141L imparts significant changes to hydrophobic interactions with L141 in the mutant. L141 still inter- the apoCaM structure. + acts with I100, V136, and M145. In addition, the side chain of For Ca2 /CaM, NOE data show no significant differences L141 interacts with the side chain of M144, which is not the case in between wild-type and F141L (SI Appendix, Fig. S11). In both the wild-type. The final turn of the terminal α-helix is distorted to cases, the region around amino acid 43 in the N-lobe displays allow for this interaction to happen. higher mobility, and lower NOE values are also observed for the + Overall, the changes in Ca2 /CaM, induced by F141L, are linker regions (approximately residues 75–86), and the loop from 15 much smaller compared with apoCaM, even in the absence of residues 111–118 in the C-lobe. Similarly, N-T1 and T2 data

Wang et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 29, 2021 + (SI Appendix, Figs. S12 and S13) do not show any significant crystallographic analysis shows that N97S indeed still binds Ca2 , differences in molecular mobility between wild-type and F141L but because the Ser97 hydroxyl group is closer to the main chain + Ca2 /CaM. Although these relaxation experiments only probe than the Asn97 amide group, the side chain needs to move in + the nanosecond to picosecond mobility range and cannot detect closer to the Ca2 , thus introducing strain. This is in stark con- changes in processes occurring on the microsecond timescale, trast to two other mutants, D95V and N97I, which completely + our inability to find significant differences between wild-type and obliterate Ca2 binding to EF3. Surprisingly, these two mutants + F141L Ca2 /CaM further highlight that the cause for loss of CDI display a highly distorted conformation of the C-lobe that resem- + + + for this mutant lies in the Ca2 -free state. bles neither a Ca2 -occupied nor a Ca2 -free wild-type C-lobe. This results in a complete reorganization of the hydrophobic surface that Discussion binds target peptides, but despite this both mutants retain the ability + CaM is one of the most conserved proteins throughout evolu- to bind the IQ domain with high affinity under saturating Ca2 tion, but genetic linkage in a large family (6), followed by se- concentrations. However, the interactions between the C-lobe and quence analysis of several patients, have revealed mutations in IQ domain are altered, a direct effect of the distorted C-lobe the CALM genes that are linked to cardiac arrhythmias, in- conformation. A new hydrophobic pocket is formed, which binds cluding CPVT, LQT, and IVF (6–11). The disease-associated the canonical Ile1624 of the IQ motif. In the wild-type structure, mutations have a dominant effect, and with three different Ile1624 is partially exposed to solvent. Because this residue is crucial CALM genes encoding identical CaM proteins, only one of six for CDI (24), its sequestration by the mutant C-lobe may contribute to loss of CDI observed for D95V (12). Although both mutants alleles is found to be mutated in each of the patients, enough to + create a disease phenotype. have a complete loss of Ca2 binding in EF3, they have a different 2+ CaV1.2isamajorvoltage-gatedcalcium channel isoform expressed effect on the affinity for Ca in EF4, with N97I binding twofold in cardiac myocytes. Because CaV1.2 contributes to depolarization, it stronger compared with D95V (7). Our structures suggest that this is imperative that it inactivates to allow a timely repolarization. In- is due to an additional stabilizing interaction unique to N97I, deed, a delayed inactivation will result in a prolonged plateau phase whereby the Tyr99 side chain stacks against the hydrophobic of the action potential in ventricular myocytes, and may lead to LQT Ile97 side chain. This is not possible with Asn97 in D95V. 2+ (30). Inactivation of CaV1.2 carries both a voltage- and a Ca - A fourth mutant, F141L, seems to adopt a completely differ- dependent component. CaM, an essential component for CDI, is ent mechanism. Although it has also been found to abolish CDI + + knowntopreassociatewiththechannelunderrestingCa2 concen- (12), this mutation does not affect a Ca2 -coordinating residue, trations and undergoes conformational changes upon saturation with but instead interferes with hydrophobic packing within the C- + Ca2 . Importantly, the C-lobe has previously been shown to be crucial lobe, as shown by both X-ray and NMR structures. Although 2+ for CDI of CaV1.2, as site-directed mutations that knock out Ca the mutation directly affects the interaction with Y1627 in the IQ domain, F141L CaM retains high-affinity binding to the IQ do- binding in both EF3 and EF4 can obliterate CDI (31). It is therefore + interesting to note that all CaM variants, known to affect CDI of main in Ca2 -saturating conditions. However, there appears a 2+ CaV1.2, are located in the C-lobe. significant effect of this mutation in Ca -free conditions, be- The major interaction site for CaM in CaV1.2 is the IQ domain, cause this mutant binds 10-fold stronger to the IQ domain than located in the cytoplasmic C-terminal tail of the channel. The Ca 1.2 wild-type CaM. This is corroborated by NMR experiments on + V IQ domain can associate with CaM under both Ca2 -occupied this mutant. Although the N-lobe of F141L apoCaM is well + and Ca2 -free conditions (24, 31). In addition, it has been found defined, very few peaks are visible for the C-lobe. Importantly, that the N-lobe can associate with an N-terminal fragment, the C-lobe is not a completely random coil, because this would + previously termed NSCaTE, under Ca2 -saturating conditions have resulted in clearly visible peaks. Instead, the data suggest (25, 26). Although the NSCaTE segment can modulate the that the F141L apo C-lobe adopts multiple conformations. Most likely, one or more of these result in increased affinity for the IQ degree of CDI, it is not essential for robust CDI to occur. + domain. The affinity of Ca2 for the CaM:IQ complex is deter- Mutations in the IQ domain, however, can completely knock + out CDI (24). We therefore utilized the CaM:IQ domain in- mined by the relative affinities of apoCaM or Ca2 /CaM for the IQ domain, and a stronger apoCaM interaction is predicted to teraction as a probe to test the impact of several disease vari- + decrease the Ca2 affinity. Indeed, the fluorescence experiments ants on the structure, dynamics, and binding properties using a + combination of biochemical, X-ray crystallography, and NMR suggest at an ∼5.5-fold decrease in Ca2 affinity for the F141L approaches. CaM:IQ complex. We investigated four different CaM variants, all of which af- Our data also explain the ability of these mutants to have a fect CDI. N97S was first identified as a causative variant in dominant effect on CDI in the presence of wild-type CaM. Be- cause they have very minimal effects on structure and binding to CPVT (6), where an affected individual presented with cardiac + arrest at age 4 y. A later study also linked it to a case of pro- the IQ domain in low Ca2 conditions, or even enhance affinity longed QT interval (∼480 ms), with the first report at age 5 y (7). (F141L), all four mutants can preassociate with CaV1.2, thus The exact phenotype may thus depend on the genetic makeup of occupying a significant fraction of the binding sites. However, + the patient or environmental factors. In contrast, the D95V, because of large structural changes in the Ca2 -bound state, al- + F141L, and N97I mutations were found in cases of a more severe tered Ca2 affinities, different interactions with the IQ domain, LQT (QT interval > 600 ms) (7, 8, 10), where the first symptoms or a combination thereof, these mutants are unable to adopt the were found as early as 17 mo (N97I) (7), right after birth (F141L) proper interactions with CaV1.2 to mediate CDI. Although the (10), or even prenatally (D95V) (10). F141L, D95V, and N97S channels occupied with wild-type CaM can still undergo CDI, all prolonged the action potential in transfected ventricular having a significant fraction of channels (e.g., one-sixth) in- myocytes, with N97S showing a milder effect (12). capable of CDI is sufficient to affect the overall inactivation The variants linked to LQT are scattered throughout the C- profile of the CaV1.2 population in a cell (12). In this regard, the lobe, and we therefore wondered whether they affect CDI via higher affinity of F141L apoCaM for the IQ domain would result different mechanisms. Because CaM binds Ca 1.2 at different in a better competition compared with wild-type CaM, and thus a + V Ca2 concentrations, the mutations could affect binding of either higher occupancy of mutant CaM bound to Ca 1.2 than expec- + V Ca2 /CaM or apoCaM to the IQ domain. They could interfere ted from its expression relative to wild-type CaM. with binding directly through altering the interaction interface, Importantly, both F141L and N97S CaM display only minor + or indirectly by causing conformational changes or by altering structural differences in the Ca2 -bound state, implying that the + the Ca2 affinity. The main observations for each mutant are reduction in CDI is a result from the altered interactions in the + + summarized in SI Appendix, Table S5. Ca2 -free state (F141L CaM) or the reduction in Ca2 affinity. The mutant with the subtlest effect on CDI of Ca 1.2, N97S, The latter implies a reduction in the on-rate or increase in off- + V + decreases the affinity for Ca2 1.5- to 4-fold (6, 13, 14, 27). Our rate for Ca2 binding, resulting in a delayed or reduced CDI,

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1808733115 Wang et al. Downloaded by guest on September 29, 2021 thus prolonging the plateau phase of the cardiac myocyte action TAMRA Fluorescence Anisotropy Experiments. ACaV1.2 IQ peptide with an N- terminal 5-TAMRA label was obtained from Proteogenix (1659- potential and contributing to LQT. Interestingly, our estimated + 2+ DEVTVGKFYATFLIQEYFRKFKKRKEQGLVGKPS-1692). Ca2 -buffered solu- Ca affinities for the CaM:IQ complexes suggest a KD of ∼130 nM for wild-type CaM. If this affinity holds true for CaM tions contained 50 mM Hepes, 100 mM KCl, 0.5 mM EGTA, and 2 mM NTA at pH 7.2, 25 °C. The total amount of CaCl2 and MgCl2 needed to attain a de- bound to full-length CaV1.2, which remains to be confirmed, this 2+ 2+ 2+ sired free Ca -concentration and a free Mg concentration at 1 mM, were implies that a significant amount of Ca /CaM could preassociate calculated using pCa Calculator (39). with the channel in resting conditions, in addition to apoCaM 2+ Using a liquid-handling robot (Microlab STARlet; Hamilton), we employed a preassociation. As several mutations decrease the Ca affinity, 2D titration assay in which CaM was serially diluted in different Ca2+-buffered 2+ they would affect both the degree of preassociated Ca /CaM, and solutions containing a constant amount of TAMRA-labeled Ca 1.2 IQ peptide. 2+ V affect Ca binding to preassociated apoCaM, resulting in a more The interaction between CaM and CaV1.2 IQ was monitored by changes in the complex effect on overall CDI. fluorescence anisotropy at 25 °C, using a fluorescence polarization plate These structures provide snapshots of disease-causing muta- reader (Infinite M1000; Tecan) and excitation and emission wavelengths at tions in CaM. Because CaM regulates multiple ion channels, it is 530 and 582 nm (5- and 20-nm bandwidths, respectively). The use of 384-well likely that the same mutations perturb the action of other cardiac plates (Cat. #3575; Corning) allowed for 24 titration points for CaM at 2+ – channels, with the structural distortions observed in this study 16 different [Ca ]free, resulting in 16 protein peptide titration curves for forming the same cause. For example, the cardiac RyR2 is also each plate (SI Appendix, Fig. S1). Curve fitting is described in SI Appendix, Supplementary Methods. regulated by CaM (32–36), and several studies show that some mutations in the present study also affect its function (11, 13– 2+ Crystallization, Data Collection, and Structure Solution. Crystallization condi- 16). In the presence of Ca , D95V CaM is still able to bind tions are shown in SI Appendix. After flash-freezing, diffraction data were either full-length RyR2 or the major CaM binding peptide of collected at the Stanford Synchrotron Radiation Lightsource beamline 9-2 + RyR2 with high affinity (13, 16), indicating that the inherently (N97S Ca2 /CaM) and the Advanced Photon Source beamline 23ID-B (D95V, 2+ + distorted D95V Ca /CaM structure can also interact with pep- N97I, and F141L Ca2 /CaM:IQ complexes) and processed using XDS (41). tides other than the IQ domain. Interestingly, F141L was found Structures were solved by molecular replacement using Phenix (42); and as the divergent mutant, as one study found it to confer an extra using, as search models, wild-type Ca2+/CaM (PDB ID code 1CLL) for N97S + + Ca2 /CaM, Ca2 /N-lobe (PDB ID code 2BE6), and apo/C-lobe (PDB ID code inhibitory effect on RyR2, in contrast to other CaM disease mu- + + 4CDK) for D95V Ca2 /CaM:IQ domain, D95V Ca2 /CaM:IQ domain for N97I tations (16). Further studies with either RyR2 CaM-binding pep- + + Ca2 /CaM:IQ domain, and wild-type Ca2 /CaM:IQ domain (PDB ID code 2BE6) tides (37) or full-length RyR2 will be required to determine the + for F141L Ca2 /CaM:IQ domain. The structures were refined using COOT (43)

precise structural impact of the disease mutations on RyR2 (38). BIOCHEMISTRY and Phenix (42). No residues are in disallowed regions of the Ramachandran + As these studies are performed on isolated IQ domains, not plot. Final models contain four molecules (N97S Ca2 /CaM) or two complexes intact channels, it is likely that several factors, such as the in- 2+ 2+ (D95V, F141L, and N97I Ca /CaM:IQ) in the asymmetric unit. Figures were herent affinities between CaM, Ca , and IQ, are altered, as well prepared using PyMOL (Schrödinger). Statistics are shown in SI Appendix, as the precise interaction between CaM and the channel. Cry- Table S3. Structures were deposited in the PDB database with ID codes oelectron microscopy (cryo-EM) studies on full-length channels 6DAD, 6DAE, 6DAF, and 6DAH. can shed more light on this, but thus far no reconstructions are available for full-length CaV1.2 and most cytosolic elements, Isothermal Titration Calorimetry. Samples were dialyzed against 10 mM including the IQ domain, have remained unresolved in cryo-EM Hepes, pH 7.4, and 10 mM CaCl2. The IQ peptide (GenScript) corresponds to α – reconstructions of the related Ca 1.1 (39, 40). High-quality cryo- human CaV1.2 (CaV 1c77) residues 1611 1644. Experiments were conducted V μ EM studies on Ca 1.2 in complex with CaM will be required to on an ITC200 (MicroCal) at 25 °C. Titrations consisted of 20 injections of 2 L V μ μ enhance our insights into the mechanism of CDI and the impact of 75- M C-lobe into the cell containing 11 M IQ peptide. Data were pro- cessed with MicroCal Origin 7.0. For data fitting, the active IQ peptide of disease mutations on this process. concentration was adjusted by a factor of 0.6 to achieve a binding stoichi- ∼ Methods ometry of 1. Control experiments injecting titrant into buffer were used to adjust the baseline of each experiment. Cloning, Expression, and Purification. Full-length human CaM, CaM C-lobe Protein expression and purification for NMR. Wild-type, D95V, and F141L CaM – α (residues 79 148), and the IQ domain of human CaV1.2 (CaV 1c77; residues were expressed from a pMAL vector, containing an N-terminal MBP and a TEV 1611–1641) were cloned as previously described (22) into a modified cleavage site, in E. coli Rosetta-2 (DE3) cells (Novagen) first grown in LB media

pET28 vector containing an N-terminal hexahistidine tag, maltose-binding to an OD600 of ∼0.5. The LB culture was centrifuged at 4,000 × g for 10 min, protein (MBP), and cleavage site for the tobacco etch virus (TEV) protease. and pelleted cells added to standard M9 minimal media (Cold Spring Harbor 15 13 Full-length human CaM for cocrystallization with the IQ domain was cloned protocols) containing N-ammonium sulfate and D-[U- C] glucose to an OD600 into pEGST without affinity tag. The D95V, N97I, N97S, and F141L mutations of ∼0.05. The M9 culture was induced at OD600 ∼0.5 by addition of 1 mM IPTG were produced by QuikChange (Stratagene). and incubated at 25 °C for 18 h. The cells were lysed by sonication in lysis Proteins were expressed at 37 °C in Escherichia coli Rosetta (DE3) pLacI buffer (20 mM Tris·HCl, 50 mM NaCl, pH 7.5, and 1 mM β-mercaptoethanol) with 1 mg/mL lysozyme and 0.34% (vol/vol) protease inhibitor mixture (Sigma (Novagen) grown in 2×YT media and induced at OD600 of ∼0.6 by addition × of 0.4 mM isopropyl-β-D-thiogalactoside (IPTG) for 3 h. For cocrystalization, Aldrich). The lysate was centrifuged at 30,000 g at 4 °C for 45 min and the the complex of CaM and IQ domain was coexpressed and copurified. Cells supernatant applied to a 120-mL amylose column (New England Biolabs), · were lysed by sonication in buffer A (250 mM NaCl, 10 mM Hepes, pH 7.4, washed with 5 CV buffer A (20 mM Tris HCl, 200 mM NaCl, pH 7.5), and eluted −1 −1 with 100% (vol/vol) buffer B (buffer A and 10 mM maltose). The fusion protein and 10 mM CaCl2) with 25 μgmL DNase I, 25 μgmL lysozyme, and 1 mM was cleaved with recombinant TEV protease overnight at 4 °C. The sample was phenylmethylsulphonyl fluoride. The lysate was applied to a 10 mL HisTrap then applied to a 50-mL Q-Sepharose anion exchange column equilibrated in FF column (GE), washed with 10 column volumes (CV) of buffer A, and eluted buffer C (20 mM Tris·HCl, 50 mM NaCl, pH 7.5), washed with 3 CV 30% (vol/vol) with 30% (vol/vol) buffer B (250 mM NaCl, 500 mM imidazole, pH 7.4, and buffer D (20 mM Tris·HCl, 500 mM NaCl, pH 7.5), and then eluted with a linear 10 mM CaCl ). The protein was dialyzed against buffer C (10 mM NaCl, 2 gradient from 30 to 100% (vol/vol) buffer D. The eluted protein was concen- 10 mM Hepes, pH 7.4, and 10 mM CaCl )for∼12 h at 4 °C and simultaneously 2 trated to 2 mL. Apo samples were produced by adding EDTA to a final con- + cleaved with recombinant TEV protease. The sample was applied to a 25-mL centration of 10 mM, whereas Ca2 loaded samples were obtained by adding amylose column (New England Biolabs), and the flow-through applied to a CaCl to a final concentration of 5 mM. The sample was applied to a HiLoad – 2 Hiload HQ column (GE) with a linear gradient of 0 45% buffer D (1 M NaCl, 16/60 Superdex 200 pg (GE Healthcare) using buffer E (2 mM Hepes, 100 mM 10 mM Hepes, pH 7.4, and 10 mM CaCl2). Residual MBP was removed by KCl, pH 6.5). The molecular weight of the purified protein was verified by passage of the purified material through a 5-mL talon column (GE), and the MALDI-TOF mass spectrometry and the concentration was determined by flow-through applied to a HiLoad 16/60 Superdex 75 prep grade (GE) in absorption at 280 nm. The protein buffer was exchanged to 2 mM Hepes, buffer A. For crystallography, the protein buffer was exchanged to 25 mM 10 mM KCl, pH 6.5. NaCl, 10 mM Hepes, pH 7.4, and 10 mM CaCl2. For ITC, the protein buffer NMR experiments. All NMR spectra, if not specified otherwise, were recorded

was exchanged to 10 mM Hepes, pH 7.4, and 10 mM CaCl2. on a BRUKER AVIII-600 MHz spectrometer equipped with a CPP-TCI probe.

Wang et al. PNAS Latest Articles | 9of10 Downloaded by guest on September 29, 2021 Spectra were acquired at 298.1 K. BRUKER TopSpin 3.5pl6 was used for (Menlo Park), and at the Canadian Light Source (Saskatoon, SK, Canada), recording and processing. A full description is provided in SI Appendix, which is supported by the Natural Sciences and Engineering Research Council Supplementary Methods. of Canada, the National Research Council Canada, the Canadian Institutes of Structures and restraints are deposited in the PDB databank (PDB ID codes Health Research, the Province of Saskatchewan, Western Economic Diversification 6GDL and 6GDK). NMR assignments and relaxation data were deposited in Canada, and the University of Saskatchewan. This work is funded by Canadian the BioMagResBank (accession nos. 34263 and 34262). Institutes of Health Research Operating Grant PJT-148632 (to F.V.P.). M.T.O. was supported by research grants from the Obel Family Foundation, Novo Nordisk Foundation Grants NNF15OC0012345 and NNF16OC0023344, Lundbeck Foun- ACKNOWLEDGMENTS. We thank Oleksandr Grachov, Oscar Mejias Gomez, and Morten Bjerring for expert technical assistance; and Frans A. Mulder for dation Grant R151-2013-14432, and Danish Council for Independent Research valuable discussions. Use of the 950-MHz spectrometer at the Danish Center Grant DFF-4181-00447. C.H. and R.W. acknowledge Grant DFF-1323-00344 for Ultrahigh-Field NMR Spectroscopy, funded by the Danish Ministry of from the Danish Council for Independent Research and Grant NNF15OC0016186 Higher Education and Science Grant AU-2010-612-181, is acknowledged. We from the Novo Nordisk Foundation. The NMR laboratory at Aalborg Univer- also thank the support staff at the Advanced Photon Source (Chicago) GM/CA- sity is supported by the Obel Family Foundation, SparNord, and Carlsberg CAT beamline 23-ID-D, the Stanford Synchrotron Radiation Lightsource Foundations.

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