The crystal structure of a cardiac calsequestrin filament reveals an atomic mechanism of calsequestrin-associated catecholaminergic polymorphic ventricular tachycardia

Calcium homeostasis is essential to cardiac and skeletal muscle physiology, where contractile function requires tight but dynamic control of calcium levels across different cellular compartments. The major calcium storage protein in muscle tissues is calsequestrin, a highly acidic protein responsible for buffering up to 50 % of total sarcoplasmic reticulum (SR) calcium. Mutations in cardiac calsequestrin cause a highly lethal familial arrhythmia, catecholaminergic polymorphic ventricular tachycardia (CPVT), while mutations in skeletal muscle calsequestrin have been linked to myopathies. Calsequestrin’s high density calcium storage is facilitated by homomultimerization of the protein into filaments, but a compelling atomic-resolution structure of a calsequestrin filament is lacking. This gap in knowledge has limited our understanding of calsequestrin biochemistry, SR calcium storage, and molecular mechanisms of calseqestrin-associated diseases. We report here a crystal structure of a cardiac calsequestrin filament with supporting mutation analysis by an in vitro filamentation assay. We also report and characterize a novel disease-associated mutation, S173I, which localizes to the structure’s filament-forming interface. In addition, we show that a previously reported dominant calsequestrin-associated CPVT mutation, K180R, maps to the same multimerization surface. Both mutations disrupt filamentation in vitro, suggesting a model where dominant disease arises from mutations that disrupt multimer formation. Finally, a ytterbium-derivatized structure pinpoints multiple credible calcium sites at filament-forming interfaces, explaining the atomic basis of calsequestrin filamentation in the presence of calcium. This work advances our basic understanding of calsequestrin biochemistry and also provides a unifying structure-function molecular mechanism by which dominant-acting calsequestrin mutations provoke lethal arrhythmias.


Introduction
The Ca 2+ ion is a ubiquitous chemical messenger in eukaryotic cells. Passive flows of calcium from regions of high concentration to low produce transient changes in intracellular calcium, which are then coupled to a diverse set of signal transduction pathways. In facilitation of its diverse roles as an intracellular messenger, calcium exhibits by far the largest concentration difference across compartments, from approximately 2 mM extracellularly to 100 nM intracellularly -a range of up to 5 orders of magnitude. Carafoli and Krebs (2016) This calcium gradient exists across the plasma membrane separating the cytosol from the extracellular space but also between the cytosol and the endoplasmic reticulum (ER) lumen, with the latter maintaining an ionic milieu resembling extracellular conditions. Direct physical coupling between the ER lumen and the extracellular space allows some degree of passive equilibration between these two compartmentsPrakriya and , so that the biochemical work of establishing and maintaining the potential is not done twice. activates the contractile apparatus is known as excitation-contraction coupling. Under conditions of high muscle loading, the calcium flows required by the contractile apparatus become much more substantial than other biological calcium fluxes. In order to minimize the energy expended per contractile cycle, muscle cells possess enhanced calcium buffering at the sites of storage and release, allowing a high total calcium content with much lower free calcium. Calsequestrin (CSQ) is a 44 kD highly acidic protein that serves as the principal calcium buffer of the sarcoplasmic reticulum (SR, a muscle-specific extension of the ER) in cardiac and skeletal muscle, storing up to 50 % of SR calcium in a bound state, with each calsequestrin monomer (1998) Knollmann et al. (2006) Calsequestrin is complexed to the SR calcium channel, the ryanodine receptor (RyR), thereby ensuring that calcium is stored at the site of its release.Bers (2004) Since their initial identification, in 1971, a substantial field of research has formed around calsequestrins. Calsequestrins (CASQ1 in skeletal muscle and CASQ2 in cardiac muscle) are highly homologous in structure and function, and 64 % identical in sequence, with skeletal muscle calsequestrin appearing to have higher calcium capacity.Park et al. (2004) Calcium-binding propensity is explained in large part by calsequestrin's remarkable fraction of negatively-charged acidic residues (26 % glutamate or aspartate in CASQ2 and 28 % in CASQ1, with corresponding average isoelectric points of 4.2 and 4.0, respectively). In both cardiac and skeletal muscle, calsequestrin localizes to the junctional SR (jSR) of muscle and forms multimers that are anchored to the luminal SR membrane. From low resolution electron micrographs of isolated skeletal muscle fibers, these multimers appear as a dense collection of filaments.Franzini-Armstrong et al. (1987) Perni et al. (2013) Anchoring of calsequestrin filaments occurs within a complex consisting of RyR and the single-pass transmembrane proteins triadin and junctin. Bers (2004) Extended homo-multimerization provides high density calcium storage, but multimerization appears to be essential for localization, too, in that multimerization-defective mutants are trafficked along the secretory pathway and (2016), none of these contain a filament-like assembly. All 16 prior structures reveal calsequestrin dimers that are nearly-identical to one another, but a search for dimer-to-dimer interfaces within and across these crystal unit cells reveals only weak crystallographic packing contacts that appear incompatible with robust biological multimerization. In addition, the observed dimer-to-dimer interfaces vary substantially from one lattice to another. Lack of mutagenesis studies supporting proposed oligomerization interfaces in the prior published structures further calls into question whether the relevant biological multimer has ever been observed. These prior studies have established that calsequestrins dimerize in a wide variety of conditions, with intra-dimer interactions that are largely the same across published structures, but the mechanism by which dimers assemble into higher order multimers (inter -dimer assembly) remains elusive.
A structure of a calsequestrin filament would advance our understanding of calsequestrin biology and permit a comprehensive mapping of disease-causing mutations to biologicallyrelevant multimerization interfaces. We report here an investigation of dominant-acting cardiac calsequestrin disease mutants that culminated in a new X-ray structure of cardiac calsequestrin, one that we believe reveals the biologically relevant filament-forming interface. We show that known dominant-acting mutants -one previously reported and one described for the first time in this study -map to the newly reported multimerization interface. Furthermore, we provide supporting biochemical analysis of likely calcium-binding sites where the protein 4/51 multimerizes. These findings fundamentally advance our understanding of the mechanism by which calsequestrin contributes to calcium homeostasis and provide insight into mechanisms of calsequestrin-associated familial diseases.

Multimerization
We encountered a proband from a family with CPVT-like tachy-arrhythmias and multiple cases of sudden, unexplained death at a young age ( Figure 1A). The proband presented at age 33 with a biventricular tachycardia on ECG. She underwent EP study that showed an inducible ventricular tachycardia with focal origination next to the anterior fascicle, and an inducible atypical AVNRT that was successfully ablated. Targeted sequencing of channelopathy genes (KCNQ1, KCNH2, SCN5A, ANK2, KCNE1, KCNE2, KNCJ2, CAV3, RYR2, and CASQ2) revealed only a heterozygously-carried isoleucine-for-serine substitution at position 173 in cardiac calsequestrin. The proband's son and multiple other family members report a tachycardic phenotype (ranging from self-reported palpitations to diagnosed episodes of tachycardia).
The family history is also notable for multiple cases of sudden cardiac death at a young age.
Overall, the distribution of affected individuals in the pedigree is potentially consistent with dominant inheritance. As the family did not consent to follow-up genetic testing or clinical phenotyping, we were unable to rigorously assess disease/mutation co-segregation.
The genetic evidence for pathogenicity of the S173I novel variant is not conclusive, but since known pathogenic point mutations in CASQ2 exhibit defective filamentation in a simple turbidity assay, we elected to investigate the S173I mutation biochemically. The turbidity assay for S173I reveals a profound decrease in multimerization rate ( Figure 1B). Minimal improvement in the multimerization rate is obsered in a non-physiologic 0 mM potassium condition ( Figure S1), demonstrating that the protein is intact but defective in multimerization. calsequestrin maintains distinct magnesium and calcium binding sites. Krause et al. (1991) Therefore, we investigated filamentation kinetics of the K180R mutant in the presence of magnesium. Extended incubation of the K180R mutant with magnesium (2 mM MgCl2) prior to addition of calcium yields a profound filamentation defect ( Figure 1D). In vivo, calsequestrin would likely encounter similar levels of free magnesium throughout the SR.
As the dominant inheritance pattern is classically associated with disease mutations that disrupt protein-protein interactions, we might expect to find dominant-acting disease mutations at cardiac calsequestrin's dimer or multimer interfaces. However, neither S173 nor K180 fall at credible, previously identified candidate multimerization interfaces: they are not near the intra-dimer interface, nor are they near candidate inter-dimer interfaces in the prior crystal structures. We therefore elected to pursue another structure in the belief that the biologically relevant multimer has not yet been observed.

Overview of The New Candidate Structure of a Calsequestrin Filament
We have determined a new crystal structure of human cardiac calsequestrin obtained from a full-length construct in a very low-pH (3-3.5) crystallization condition. The previously characterized calsequestrin dimer is again observed, but now in an arrangement that produces a closely-packed filament ( Figure 2). The new structure provides a compelling candidate for a biologically-plausible higher-order multimer. Crystallographic data collection and processing statistics are summarized in (Table S1) for the native structure as well as a ytterbium-soaked condition used to identify probable calcium-binding sites. The repeating unit of the native crystal resides in a higher-symmetry point group compared to prior calsequestrin structures (Table S2). The oligomer-forming contacts that exist between dimers are novel, differing significantly from all previously reported calsequestrin crystal structures (Table S4). The inter-dimer interface in our structure encompasses significantly greater buried surface area than previously observed (Table S4).
The cardiac calsequestrin monomer, like its skeletal calsequestrin equivalent, consists of an N terminal loop, 3 thioredoxin domains, and a disordered acidic tail. Within the dimer, there is two-fold symmetry with mutual exchange of N-terminal loops, as previously observed ( Figure 3A). In our structure, the dimers are stacked along a screw axis to form the filament, with each dimer rotated 90°with respect to its neighbors. Although the dimers are positioned at discrete 90°rotations with respect to one another, the underlying architecture of the filament is in fact helical at the level of thioredoxin domains ( Figure 3A). Thioredoxin domains II and larger diameter, consisting only of thioredoxin domain I, then winds around the inner double helix. All helices are left-handed, corresponding to the left-handed screw axis at the level of dimer-stacking.
The 3-helix configuration appears to promote the close packing of globular thioredoxin domains. Helical packing permits each domain to contact multiple other domains in multiple other protomers, which is in stark contrast to other reported candidate structures (PDB ID 1A8Y, rabbit skeletal muscle calsequestrin; PDB ID 1SJI, canine cardiac calsequestrin), which lack the helical pitch and have much more limited interacting surface area (Figure 4). The close-packing of the new filament candidate is starkly visible when thioredoxin domains are represented as equally-sized spheres centered at the domain center of mass (Figure 4, right-hand side).
Notably, our crystallization condition produced crystals only at a low pH (3-3.5, measured by applying a crystallization drop to litmus paper) resulting from the use of aged PEG reagents (presumably having undergone degradation to glycolic acid) or from direct addition of concentrated HCl. This restriction obtained irrespective of calcium in concentrations ranging from trace to 14 mM (higher calcium concentrations led to precipitation, likely due to the presence of sulfate, and were therefore not assessed). Given calsequestrin's remarkable overall acidity and low isoelectric point, an explanation for the role of low pH in promoting calsequestrin filamentation could be the neutralization of substantial negative charge on acidic side chains that extend across the filament interfaces.

Lanthanide Substitution Identifies Cation-Binding Sites that Participate in Dimer Formation
Prior work has identified putative calcium binding sites at the dimer interface of calse-questrinSanchez et al. (2012a). Our new filament structure permits us to examine the role of multivalent ions at all surfaces relevant for filamentation -both the intra-dimer surfaces as well as the inter-dimer surfaces responsible for higher-order multimers. To localize candidate calcium ligand sites within the context of the new filament structure, we collected data from a Ytterbium (Yb)-soaked crystal. (Table S1) From the anomalous map, we identified approximately 8 sites with strong Yb signal per CASQ2 chain (> 3.8 in the anomalous map and difference density in the Fo-Fc map). As the prior work to identify calsequestrin's calciumbinding sites was based largely on the indirect method of inference from metal coordination geometry with nearby side chains and waters, the use of Yb provides a direct approach to 7/51 . CC-BY 4.0 International license certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was not this version posted June 15, 2019. . https://doi.org/10.1101/672303 doi: bioRxiv preprint confirming the prior findings, as well as extending our understanding to the entire filament. We first assessed the presence of Yb at the intra-dimer interface. We identified several highoccupancy Yb sites, most of which are clustered in a narrow region between protomers, where they are coordinated by multiple, highly-conserved acidic residues ( Figure 5 and Figure S2).
Consistent with the location of a putative calcium ion in the prior study, we find a Yb atom coordinated primarily by E147 of chain A and D278 of chain B. In addition, we find a Yb atom coordinated primarily by E143 and E275, as well as another Yb atom coordinated by D310 and a putative sulfate ion within a solvent cavity enclosed by the dimer. These Yb-bridging sites stabilize the interaction between thioredoxin domain II from protomer A and thioredoxin domain III of protomer B. The interactions between domains result in close juxtaposition of acidic side chains ( Figure 5), requiring neutralization of the charge by the multivalent counterions or, in the case of the native structure, protonation. In the native structure, the E147/D278 interaction provides an example of a carboxyl-carboxylate bond stabilized by low pH.Sawyer and James (1982a) Krause et al. (1991) Anomalous signal is found at this site in the derivative structure, but consistently on just one side of the otherwise symmetric dimer. This subtle asymmetry, with a cation bound on one side and a carboxyl-carboxylate bond on the other, likely reflects a degree difference in occupancy rather than a sharp distinction, but it is a consistent feature of the density map across all 4 dimers in the crystal asymmetric unit and may be conformationally conducive to filament formation.
In both the native structure and Yb-soaked structure, the dimer subunits have undergone substantial inward rotation in comparison to the other high-resolution cardiac calsequestrin structure (PDB ID 1SJI) ( Figure 5B). The inward domain movement is produced largely by rotation of the monomer as a rigid body and results in a conformation where domains II and III are more closely packed. This conformational shift recapitulates a similar finding from the prior study where a 15°rotation within the skeletal calsequestrin dimer was observed for structures crystallized in a high-calcium buffer.Sanchez et al. (2012a) Upon inspection of all prior published calsequestrin structures, it is apparent that 6 prior calsequestrin structures belong to this "tightly-packed dimer" group, while the remaining 10 contain dimers that are more loosely-packed, without inward rotation of chains ( Figure S3). The more tightly-packed structures were all crystallized in the presence of multivalent cations (usually calcium), or in one case (PDB ID 2VAF) a monovalent cation at extremely high concentration ( 2 M NaCl. The other group were crystallized with no added multivalent cations, with the exception of PDB ID 3TRP, which contained calcium in the crystallization drop at approximately 5-fold lower than 8/51 . CC-BY 4.0 International license certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was not this version posted June 15, 2019. . https://doi.org/10.1101/672303 doi: bioRxiv preprint lowest concentration from the tightly-packed group. Within the tightly-packed group, there is a greater degree of conformational disorder in Domain I ( Figure S4). This is accompanied by a modest loss of contact in Domain I, while multiple hydrophobic side chains from Domains II and III that were not buried now obtain buried surface area ( Figure S5). Thus, our data provide additional evidence for conformational change within the dimer upon calcium-binding (likely induced by closer approximation of acidic side chains), independent confirmation of intra-dimer calcium binding sites, and an explanation for why an altered conformation of the dimer becomes energetically tolerable in the presence of calcium.

Cation-Binding Promotes Filament Formation by Dimer-Dimer Stacking
Like the dimer interface, the inter-dimer filament-forming interface is bridged by Yb sites surrounded by closely-apposed acidic side chains ( Figure 6 and Figure S6). In contrast to the dimer interface, where carboxyl-carboxylate bonds are still seen in the Yb-derivative structure, at the inter-dimer interface the sites of carboyxl-carboylate interaction have been throughly substituted with a cation. Identification of these coordination sites provides a testable model for the biological correctness of the new filament structure. To test this model, we mutated the aspartate or glutamate side chains involved in Yb binding to alanine and examined the effect of the mutations on filamentation kinetics. The most pronounced effect resulted from mutations targeting the symmetrical interaction involving E184 and E187 ( Figure 6A). The Yb site formed by these residues lies on the edge of a solvent cavity enclosed by the inter-dimer interface, partially shielded from exposure to the bulk solvent. Glutamates 184 and 187 belong to an alpha helix that provides a linkage between domains of the outer thioredoxin collar. This helix, belonging to thioredoxin domain 2, sits between thioredoxin I domains of different dimers and interacts with inter-dimer salt bridges on either side. Mutating E184 and E187 to alanine effectively prevents filamentation within the time period of observation.( Figure 6B). Alanine mutagenesis of the D50 residue that participates in a salt bridge with K180 at the N-terminal end of this helix produces a similar defect ( Figure 6C). Two more sites of strong Yb signal are notable. Residues D348 and D350 are oriented along with their symmetry mates to form a cluster of 4 acidic side chains that coordinate a single cation. As rendered in ( Figure 6A), this Yb site forms the base of the solvent cavity enclosed by the inter-dimer interface. Outside this cavity, on the fully solvent-exposed exterior of the filament, residues D351 and E357 and their symmetry mates form bidentate interactions with two Yb atoms, adopting conformations in which opposing acidic rotamers are bent away from one another, thereby alleviating electrostatic repulsion that would otherwise disrupt 9/51 . CC-BY 4.0 International license certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was not this version posted June 15, 2019. . https://doi.org/10.1101/672303 doi: bioRxiv preprint multimer formation ( Figure 6A). In summary, the bound cation at these sites appears to facilitate filamentation by neutralizing the abundant negative surface charge of calsequestrin.
Mutating these sites to alanine would be expected to relieve mutal repulsion of closely-packed acidic side chains. Consistent with this, alanine mutagenesis of these residues has a largely net neutral effect on filamentation kinetics ( Figure S7).
Intriguingly, the vast majority of Yb sites that we identify are located within a continuous solvent cavity that winds through the interior of the filament, forming a hollow core with solvent exposure at the dimer and inter-dimer interfaces. Each dimer contains a solvent pocket within its interior, with a second solvent cavity located at inter-dimer interface ( Figure 7A-B). The stacking of dimers forms a continuous solvent cavity along the entire length of the filament ( Figure 7). The calcium ions that would appear to be bound in the dimer's interior would constitute a separate store of calcium that is more slowly-mobilized than the highly-accessible pool of ions bound to the surface and the solvated acidic tail.

Interface
The newly observed filament structure permits us to assess the possible pathogenic mechanism of the S173I mutation that began this investigation. Remarkably, S173 occupies a critical position at the filament-forming interface -a charged pocket formed by the interaction of K87, S173, and D325 ( Figure 8A and Figure S8. This pocket, formed of residues from 3 different protomers and also 3 different thioredoxin domains, enforces an interaction between the 3 thioredoxin domains at a single site. This interaction explains the disruptive effect of the S173I mutation discussed above (turbidity assay from Figure 1). On the basis of the apparent importance of this site, as well as the absence of D325 in any other previously described candidate filament interface, we also mutated residue D325 to D325I. As expected, D325I exhibits similar profound loss of function in the turbidity assay ( Figure 8B).
The afore-mentioned K180 residue has recently been implicated in CPVTGray et al. (2016). Specifically, a lysine-to-arginine mutation at this position provided the first strong genetic evidence for a dominant CPVT-causing mutation in cardiac calsequestrin. Although this substitution is relatively conservative, the genetic evidence for the pathogenicity of K180R as a dominant mutation is compelling on the basis of a comprehensive segregation analysis in a large family. As shown in Figure 1, the K180R mutation also results in a filamentation defect.
Our new structure provides a mechanistic explanation for this effect. In the new structure, 10/51 . CC-BY 4.0 International license certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was not this version posted June 15, 2019. . https://doi.org/10.1101/672303 doi: bioRxiv preprint K180 participates in helix-stapling salt bridges at the E184/E187 putative calcium site/interface region ( Figure 6A) shown by us to be essential for robust filamentation.( Figure 6B) The high conservation of K180 in evolution ( Figure S9) and key role of K180 and its interacting partners in supporting calsequestrin filamentation provide additional support for the physiological relevance of our new structure.

Discussion
We report here a crystal structure of a calsequestrin filament. We also elucidate the biochemical basis of divalent cation-induced filamentation. Using Yb substitution, we confirm previous findings of intra-dimer cation binding sites, and we reveal additional inter-dimer cation sites that have a substantial effect on the rate of multimerization. Furthermore, we provide three forms of evidence in support of the biological relevance of the new structure: biophysical (buried surface area, all-by-all interactions of protomers and domains), biochemical (disruption of multimerization by targeted mutagenesis), and biomedical (disease mutations with dominant inheritance are located at the newly-identified filament-forming interfaces).
What is the proximal source of free energy for calsequestrin filamentation? The prevailing view has been that calsequestrin filamentation is driven by an increase in solvent entropy: ions are bound at filament interfaces, and solvent entropy increases as ion hydration shells are lost. Krause et al. (1991) Under this view, the net contribution from solvent entropy is much greater than the net enthalpic contribution from calcium binding after considering the energetic cost of dehydration. This hypothesis is complicated, however, by the fact that we observe stable filamentation in the near-absence of ligand (only trace concentrations of divalent cations were present in the low-pH crystallization condition). The low-pH filament is stabilized instead by carboxyl-carboxylate interactions between closely juxtaposed acidic residues. While increased solvent entropy likely contributes to filamentation, we postulate instead that protein conformational entropy is likely to be the major driving force. The structure we report is remarkably disordered by conventional B-factor metrics, and especially so in Domain I. In fact, all tightly-packed calsequestrin dimers (7 crystallographic results total, including this study) exhibit increased disorder in the solvent-exposed loops of Domain I ( Figure S4). The consistency of this observation within the group makes it less likely that conformational disorder within Domain I is a feature of our specific crystallization condition and more likely that it is a result of the dimer having adopted the tightly-packed conformation favorable for filamentation.

11/51
. CC-BY 4.0 International license certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was not this version posted June 15, 2019. . https://doi.org/10.1101/672303 doi: bioRxiv preprint Although low pH may not be necessary for filamentation, calsequestrin's pH-sensitivity prompts questions about the possible role of intra-luminal pH changes in the regulation of calcium uptake and release. Carboxyl-carboxylate side chain interactions modulated by prevailing pH are a common feature of calcium-binding proteins, wherein a shared proton provides a stabilizing interaction when the cation is unavailable. Proton efflux from the SR during calcium reuptake, by way of the SERCA pump's antiporter function, would increase the availability of calcium binding sites. Conversely, protein influx into the SR during calcium release -possibly via a second, independent proton transport pathway within the SERCA pumpEspinoza-Fonseca (2017) -could conceivably stabilize calsequestrin filaments until high levels of calcium are restored. Prior work showed a change in calsequestrin's intrinsic fluorescence at a pH of 6.0 Hidalgo et al. (1996), suggesting that dynamic effects on calsequestrin are not limited to the low pH regime used in our crystallization experiments. Since calsequestrin is present at high concentration and likely acts non-trivially as a proton buffer in its own right, effects due to total free proton concentration may manifest as protons move from an SR-based buffer system to one in the cytosol and back with little change in detectable pH. It is important to note that presumed regulatory of effects of dynamic SR pH are speculative and require further elucidation.
Strikingly, most known CASQ2 mutations that disrupt filamentation at the intra-dimer interface (e.g. R33Q, D307H, and P308L) are associated with recessive inheritance, yet the mutations investigated here (S173I, K180R) that disrupt filamentation at the inter-dimer interface are associated with dominant inheritance. The apparent discrepancy in inheritance patterns, mapping cleanly to distinct interfaces albeit with limited sampling, is notable. To 12/51 . CC-BY 4.0 International license certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was not this version posted June 15, 2019. . https://doi.org/10.1101/672303 doi: bioRxiv preprint date, the field has explained the recessive mechanism of intra-dimer interface mutations by way observing that calsequestrins have no classical ER retention signal: filamentation is necessary for calsequestrin to remain in the jSR. (2010) Knollmann (2010) In the setting of a heterozygously-carried mutation that inhibits dimer formation, mutant monomers are eliminated by trafficking, such that the pool of wild type protein would be largely unaffected. Under this model, filamentation defects overlap mechanistically with a class of calsequestrin-deficient conditions arising from null or hypomorphic alleles, all leading to a final common endpoint of decreased calcium-buffering capacity, and a resulting susceptibility to diastolic calcium leak.
In seeming contrast to this model, poisoning of multimerization in classical dominant fashion would require mutant-incorporating dimers to remain jSR-resident, where they could interfere with assembly. There is no reason, however, based on current understanding of secretory pathways, that the dimer would be any less susceptible than the monomer to export.
How, then, can the ER export mechanism be reconciled with dominant-acting filamentationdefect mutants? If filamentation defects are equivalent at the molecular level to calsequestrin deficiency, how do we explain the finding of filamentation-disrupting mutations with likely dominant inheritance? We would propose that all filamentation-defective mutants within unincoporated dimers are trafficked out of the jSR in much the same way that unincoporated mutant monomers are. The resulting disease mechanism for dominant mutations is therefore more complex than a classical dominant negative effect: for as long as mutant-containing dimers may be present in the jSR, they interfere with multimerization, but crucially, when they leave, they steal half of the total WT protein with them. The result is paradoxical and perhaps somewhat novel: insufficiency by way of underlying dominant negative biochemistry.
In sum, taking into account calsequestrin's demonstrated susceptibility to ER export, we can combine the present work with the existing rich body of calsequestrin research to neatly explain the inheritance puzzle associated with CPVT-causing calsequestrin mutations. Dimerdefective mutants produce monomers which are trafficked away, but the heterozygous state is rescued because WT protein is unaffected by the pool of defective monomeric protein.
In contrast, mutations that interfere with the inter-dimer interaction result in depletion of a substantial fraction of WT protein, as unincorporated dimers containing a mix of WT and mutant protein are continually lost. Future work should seek to confirm this hypothetical mechanism using cell biological investigative methods.

13/51
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Human Subjects
The patient included in the study provided informed consent as part of a research protocol approved by the University of California, San Francisco Committee on Human Research.

Cloning and Generation of Plasmids
Full-length cardiac calsequestrin was cloned from human cardiac mRNA by reverse transcription, PCR, A-tailing of a PCR product, and TA ligation. A clone lacking the signal peptide sequence was subcloned by PCR and Gibson Assembly into a T7-based bacterial overexpression vector (pET28a) in front of a 6His site and TEV protease cleavage sequence. Site-directed mutagenesis was performed to generate alanine mutants and CPVT mutants. All constructs were transformed in NEB Stable or XL-1 Blue E. coli, purified by miniprep, and retransformed into Rosetta (DE3)pLysS E. coli for overexpression. Selection for the pET28a vector was performed using 50 µg/L kanamycin. Selection for pLysS was performed by adding 25 µg/L chlorampenicol.

Molecular Cloning
The human cardiac calsequestrin coding sequence was obtained by RT-PCR. Subsequent cloning was performed by Gibson Assembly. The construct for protein expression was cloned into a pET28a-based expression vector. This construct excluded the N-terminal signal peptide but retained the remaining coding sequence, including the C-terminal acidic tail. Point mutants were generated using the protocol from the Q5 Site-Directed Mutagenesis Kit (New England BioLabs), using either the Q5 or Phusion polymerases. All constructs were verified by Sanger sequencing. Primers used for cloning and mutagenesis are provided in Table S1.

Expresssion and Purification of Cardiac Calsequestrin
pET28a-based expression constructs were miniprepped from a cloning strain and transformed into Rosetta (DE3)pLysS E. coli. Overnight starter cultures were used to inoculate large cultures (typically 750 mL of broth per 2.8 L flask), which were grown to OD 0.4 and then induced with 0.25 mM IPTG. Upon induction, temperature was reduced from 37 • C to 24 • C.
Cultures were grown for 6-9 hours post-induction or overnight and then spun down (optimal yields were obsered from shorter durations of growth). All cultures were grown in standard LB in 50 µg/L kanamycin and 25 µg/L chlorampenicol. Pellets were resuspended in lysis buffer (20 mM Tris pH 7.4, 500 mM NaCl, 10 mM imidazole, 1 EDTA-free protease inhibitor tablet per 50 mL) and frozen at −80 • C. Frozen suspensions were thawed, sonicated on ice (5 min at 1 s on/1 s off), and clarified (15,000 g, 45 min, 4 • C). The clarified supernatant was filtered (0.2 µm), and calsequestrin-containing fractions were isolated by IMAC using a 5 mL HisTrap Alanine mutants and the D325I mutant were purified as described above, except that phosphate IMAC buffers were employed (Buffer A: phosphate buffer at pH 7.4, 500 mM NaCl, 10 mM imidazole; Buffer B: phosphate buffer at pH 7.4, 500 mM NaCl, 300 mM imidazole), and an on-column high-salt wash was performed (phosphate buffer at pH 7.4, 2 M NaCl). In addition, the TEV protease dialysis buffer used for these purifications contained 100 mM NaCl.

Crystallization of Cardiac Calsequestrin (Native Conditions)
Crystallization screens were carried out in 96-well hanging-drop format and monitored using a Formulatrix Rock Imager automated imaging system. Conditions conducive to crystal growth were optimized and then reproduced in a 24-well format. The best diffraction was obtained by mixing thawed protein (10-20 mg mL −1 in 20 mM HEPES pH 7.3, 400-500 mM NaCl) 1:1 with 15% PEG 4000 and 400 mM Li 2 SO 4 . The pH of the PEG 4000 solution used to produce the best-diffracting crystals was tested by litmus paper and found to be approximately 3-3.5.
Despite the presence of 20 mM HEPES in the protein reagent, the pH of the drops in which 15/51 . CC-BY 4.0 International license certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was not this version posted June 15, 2019. . https://doi.org/10.1101/672303 doi: bioRxiv preprint cystals grew was controlled by the PEG and remained 3-3.5. Freshly-made PEGs were incompatible with calsequestrin crystal growth except when concentrated HCl was added to the mother liquor, producing crystals similar to those observed with benchtop-aged PEGs. Interestingly, only unbuffered conditions yielded crystals. Multiple attempts to grow crystals at a buffered low pH (using acetate or glycine-based buffers) failed.

Ytterbium Soak
We initially attempted to identify calcium sites using anomalous signal from calcium (CaCl 2 ) added to the crystallization condition described above. We were unsuccessful, likely due to a combination of several factors. The calcium absorption edge is unreachable with conventional tunable x-ray sources and in normal atmosphere; thus it can only be approached, with resulting weakened anomalous signal. In addition, calsequestrin has an average K d for calcium of 1 mM. Thus occupancy at a typical site would be expected to be lower as compared to other calcium-binding proteins. Presence of sulfate in the crystallization condition limited calcium concentrations to approximately 14 mM and below, above which a precipitate was observed.
Although this limit is above the K d , it was insufficient for robust anomalous signal. Crystals of calsequestrin that formed in trace calcium were therefore soaked in YbCl 3 . Hanging drops containing calsequestrin crystals were uncovered and an Eppendorf Microloader was used to inject 2 µL drops (1 µL protein and 1 µL mother liquor) with 200 µL of 2 M YbCl 3 . Data were collected within 5 minutes with no back-soaking.

Crystal Data Collection and Structure Determination
Hanging drops were uncovered and submerged in a drop of Parabar 10312 (previously known as Paratone). For Yb-soaks, Yb was quickly injected prior to application of the oil. Crystals were looped and pulled through the oil. Excess oil was blotted away, and the loop was mounted directly into the cryostream of the Tom-Alber-Tron endstation at ALS beamline 8.3.1. Frames were collected at 1.116 keV (1.386 keV for Yb-soaked crystals) using the endstation's Pilatus3 S 6M detector using a strategy designed to balance redunancy against radiation damage. (2010). For the Yb-complexed dataset, data were processed as above but with preservation of anomalous signal (no merging of Friedel pairs). The refined native structure was used a molecular replacement model, and a solution was found in space group P4 3 2 1 2 containing a dimer in the AU. This solution refined poorly. The Yb-complexed dataset was reprocessed in P1, and a molecular replacement solution was found in P1 with 16 chains in the AU.
Refinement of this model was tested using ZanudaLebedev and Isupov (2014), and the best R-free was found to be in space group P12 1 1. The Yb-complexed dataset was reprocessed in space group P12 1 1, and a molecular replacement solution was found with 8 chains in the AU.
This solution refined well. The anomalous map was used in refinement to place Yb atoms in the structure.

Turbidity Assays
Recombinant protein samples were thawed, diluted in 2 mL-3 mL of Turbidity Assay Buffer (15 mM Tris pH 7.4, 20 mM NaCl, 85 mM KCl) and dialyzed in Turbidity Assay Buffer plus 10 mM EDTA. Samples were then redialyzed overnight in the same buffer without EDTA.
Approximately half of the sample was then dialyzed in Zero-Potassium Turbidity Assay Buffer (15 mM Tris pH 7.4, 20 mM NaCl). Protein A280 was measured in triplicate (Nanodrop) using the appropriate matching buffer as background, and protein was diluted to 2.25 µM in a 140 µL volume in half-area wells of a µClear 96-well plate. The plate was covered, and protein in the wells was allowed to equilibrate on the benchtop for 20 minutes. The turbidity assay was performed using a BioTek Synergy 2 plate reader equipped with reagent injectors. Seven µL of 20 mM CaCl 2 solution was injected into each well, and the plate underwent shaking for 20 s. Absorbance at 350 nm was monitored for 45 min. The protocol was performed in plate synchronized mode for consistent well-to-well timing. A 100 mM ion-selective electrode calcium standard (Sigma, cat no. ) stock solution was used for all CaCl 2 dilutions.   (A) Pedigree of a large extended family with the S173I mutation and a CPVT-like phenotype. CABG = coronary artery bypass graft; CAD = coronary artery disease; CHD = congenital heart disease; ETT = exercise treadmill test; MI = myocardial infarction; SCD = suddent cardiac death; SIDS = sudden infant death syndrome. (B) Multimerization kinetics of the S173I mutant observed using a turbidity assay after addition of 1 mM CaCl 2 to purified protein (pH 7.4, 20 mM NaCl, 85 mM KCl). (C) Multimerization kinetics of the K180R mutant observed using a turbidity assay (same conditions as in B). (D) Multimerization kinetics of the K180R mutant observed using a turbidity assay (same conditions as in B, but with 2 mM MgCl 2 added prior to calcium).

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. CC-BY 4.0 International license certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was not this version posted June 15, 2019. . https://doi.org/10.1101/672303 doi: bioRxiv preprint       . The cardiac calsequestrin S173 residue is located at a highly hydrophilic 3-protomer interface within the inter-dimer contact region. Disruption of this hydrophilic pocket by hydrophobic substitution leads to filamentation defect. (A) Inter-dimer interface with with interface residues in the S173 region rendered as spheres. the closeup panel shows a charged pocket at the inter-dimer interface where the CPVT-associated S173I mutation is found. In this pocket, 3 different thioredoxin domains from 3 distinct chains interact (K87, S173, D325).  Overlays of tightly-packed dimers from published calsequestrin structures onto the tightly-packed cardiac calsequestrin dimer from this study (6OVW) Overlays of loosely-packed dimers from published calsequestrin structures onto the tightly-packed cardiac calsequestrin dimer from this study (6OVW) Figure S3. Overlays of dimers from published calsequestrin structures (ligher orange and green) onto the tightly-packed dimer from this study (darker orange and green) reveals two distinct conformational groupings. The more tightly-packed conformation with inwardly-rotated chains is correlated with presence of neutralizing cations. Dimers are aligned on one chain (chain A arbitrarily chosen) to show degree of inward rotation in the other chain.

Figure S8
Native map at 1.5 Figure S8. Electron density map for the S173 region at the cardiac calsequestrin inter-dimer interface.

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. CC-BY 4.0 International license certified by peer review) is the author/funder. It is made available under a . CC-BY 4.0 International license certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was not this version posted June 15, 2019. . https://doi.org/10.1101/672303 doi: bioRxiv preprint Table S3. Comparison of buried surface area (BSA) at dimer interfaces across all published calsequestrin structures. All calsequestrin dimers observed to date have the same symmetry but can be divided into two classes based on rigid body rotations of the chains (see Figure 5 for additional details).