Ching-Chieh Tung

The amino-terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule

Many physiological events require transient increases in cytosolic Ca2+ concentrations. Ryanodine receptors (RyRs) are ion channels that govern the release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. Mutations in RyRs can lead to severe genetic conditions that affect both cardiac and skeletal muscle, but locating the mutated residues in the full-length channel structure has been difficult. Here we show the 2.5 Å resolution crystal structure of a region spanning three domains of RyR type 1 (RyR1), encompassing amino acid residues 1–559. The domains interact with each other through a predominantly hydrophilic interface. Docking in RyR1 electron microscopy maps unambiguously places the domains in the cytoplasmic portion of the channel, forming a 240-kDa cytoplasmic vestibule around the four-fold symmetry axis. We pinpoint the exact locations of more than 50 disease-associated mutations in full-length RyR1 and RyR2. The mutations can be classified into three groups: those that destabilize the interfaces between the three amino-terminal domains, disturb the folding of individual domains or affect one of six interfaces with other parts of the receptor. We propose a model whereby the opening of a RyR coincides with allosterically coupled motions within the N-terminal domains. This process can be affected by mutations that target various interfaces within and across subunits. The crystal structure provides a framework to understand the many disease-associated mutations in RyRs that have been studied using functional methods, and will be useful for developing new strategies to modulate RyR function in disease states.

Crystallographic basis for calcium regulation of sodium channels

Voltage-gated sodium channels underlie the rapid regenerative upstroke of action potentials and are modulated by cytoplasmic calcium ions through a poorly understood mechanism. We describe the 1.35 Å crystal structure of Ca2+-bound calmodulin (Ca2+/CaM) in complex with the inactivation gate (DIII-IV linker) of the cardiac sodium channel (NaV1.5). The complex harbors the positions of five disease mutations involved with long Q-T type 3 and Brugada syndromes. In conjunction with isothermal titration calorimetry, we identify unique inactivation-gate mutations that enhance or diminish Ca2+/CaM binding, which, in turn, sensitize or abolish Ca2+ regulation of full-length channels in electrophysiological experiments. Additional biochemical experiments support a model whereby a single Ca2+/CaM bridges the C-terminal IQ motif to the DIII-IV linker via individual N and C lobes, respectively. The data suggest that Ca2+/CaM destabilizes binding of the inactivation gate to its receptor, thus biasing inactivation toward more depolarized potentials.

Disease mutations in the ryanodine receptor N-terminal region couple to a mobile intersubunit interface.

Ryanodine receptors are large channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of RyR mutations can cause cardiac and skeletal muscle disorders, yet detailed mechanisms explaining their effects have been lacking. Here we compare pseudo-atomic models and propose that channel opening coincides with widening of a cytoplasmic vestibule formed by the N-terminal region, thus altering an interface targeted by 20 disease mutations. We solve crystal structures of several disease mutants that affect intrasubunit domain–domain interfaces. Mutations affecting intrasubunit ionic pairs alter relative domain orientations, and thus couple to surrounding interfaces. Buried disease mutations cause structural changes that also connect to the intersubunit contact area. These results suggest that the intersubunit contact region between N-terminal domains is a prime target for disease mutations, direct or indirect, and we present a model whereby ryanodine receptors and inositol-1,4,5-trisphosphate receptors are activated by altering domain arrangements in the N-terminal region.

The Deletion of Exon 3 in the Cardiac Ryanodine Receptor Is Rescued by β Strand Switching

Mutations in the cardiac Ryanodine Receptor (RYR2) are linked to triggered arrhythmias. Removal of exon 3 results in a severe form of catecholaminergic polymorphic ventricular tachycardia (CPVT). This exon encodes secondary structure elements that are crucial for folding of the N-terminal domain (NTD), raising the question of why the deletion is neither lethal nor confers a loss of function. We determined the 2.3 Å crystal structure of the NTD lacking exon 3. The removal causes a structural rescue whereby a flexible loop inserts itself into the β trefoil domain and increases thermal stability. The exon 3 deletion is not tolerated in the corresponding RYR1 domain. The rescue shows a novel mechanism by which RYR2 channels can adjust their Ca²⁺ release properties through altering the structure of the NTD. Despite the rescue, the deletion affects interfaces with other RYR2 domains. We propose that relative movement of the NTD is allosterically coupled to the pore region.

The cardiac ryanodine receptor N-terminal region contains an anion binding site that is targeted by disease mutations.

Ryanodine receptors (RyRs) are calcium release channels located in the membrane of the endoplasmic and sarcoplasmic reticulum and play a major role in muscle excitation-contraction coupling. The cardiac isoform (RyR2) is the target for >150 mutations that cause catecholaminergic polymorphic ventricular tachycardia (CPVT) and other conditions. Here, we present the crystal structure of the N-terminal region of RyR2 (1-547), an area encompassing 29 distinct disease mutations. The protein folds up in three individual domains, which are held together via a central chloride anion that shields repulsive positive charges. Several disease mutant versions of the construct drastically destabilize the protein. The R420Q disease mutant causes CPVT and ablates chloride binding. The mutation results in reorientations of the first two domains relative to the third domain. These conformational changes likely activate the channel by destabilizing intersubunit interactions that are disrupted upon channel opening.

Paramagnetic Ligand Tagging To Identify Protein Binding Sites

Transient biomolecular interactions are the cornerstones of the cellular machinery. The identification of the binding sites for low affinity molecular encounters is essential for the development of high affinity pharmaceuticals from weakly binding leads but is hindered by the lack of robust methodologies for characterization of weakly binding complexes. We introduce a paramagnetic ligand tagging approach that enables localization of low affinity protein–ligand binding clefts by detection and analysis of intermolecular protein NMR pseudocontact shifts, which are invoked by the covalent attachment of a paramagnetic lanthanoid chelating tag to the ligand of interest. The methodology is corroborated by identification of the low millimolar volatile anesthetic interaction site of the calcium sensor protein calmodulin. It presents an efficient route to binding site localization for low affinity complexes and is applicable to rapid screening of protein–ligand systems with varying binding affinity.

Structural insights into binding of STAC proteins to voltage-gated calcium channels

Significance Skeletal muscle contraction is a tightly orchestrated event that starts with the depolarization of the T-tubular membrane. At the center is a functional and mechanical coupling between two membrane proteins: L-type voltage-gated calcium channels, located in the plasma membrane, and ryanodine receptors, located in the membrane of the sarcoplasmic reticulum. How exactly these proteins associate has remained a mystery, but recent reports have highlighted a key role for the STAC3 adaptor protein in this process. Here, we provide structural snapshots of the three STAC isoforms and identify a cytosolic loop of two CaV isoforms as a functional interaction site. A mutation linked to Native American myopathy is at the interface and abolishes the interaction. Excitation–contraction (EC) coupling in skeletal muscle requires functional and mechanical coupling between L-type voltage-gated calcium channels (CaV1.1) and the ryanodine receptor (RyR1). Recently, STAC3 was identified as an essential protein for EC coupling and is part of a group of three proteins that can bind and modulate L-type voltage-gated calcium channels. Here, we report crystal structures of tandem-SH3 domains of different STAC isoforms up to 1.2-Å resolution. These form a rigid interaction through a conserved interdomain interface. We identify the linker connecting transmembrane repeats II and III in two different CaV isoforms as a binding site for the SH3 domains and report a crystal structure of the complex with the STAC2 isoform. The interaction site includes the location for a disease variant in STAC3 that has been linked to Native American myopathy (NAM). Introducing the mutation does not cause misfolding of the SH3 domains, but abolishes the interaction. Disruption of the interaction via mutations in the II–III loop perturbs skeletal muscle EC coupling, but preserves the ability of STAC3 to slow down inactivation of CaV1.2.

Crystallographic insight into the evolutionary origins of xyloglucan endotransglycosylases and endohydrolases.

The xyloglucan endotransglycosylase/hydrolase (XTH) gene family encodes enzymes of central importance to plant cell wall remodeling. The evolutionary history of plant XTH gene products is incompletely understood vis-à-vis the larger body of bacterial endoglycanases in Glycoside Hydrolase Family 16 (GH16). To provide molecular insight into this issue, high-resolution X-ray crystal structures and detailed enzyme kinetics of an extant transitional plant endoglucanase (EG) were determined. Functionally intermediate between plant XTH gene products and bacterial licheninases of GH16, Vitis vinifera EG16 (VvEG16) effectively catalyzes the hydrolysis of the backbones of two dominant plant cell wall matrix glycans, xyloglucan (XyG) and β(1,3)/β(1,4)-mixed-linkage glucan (MLG). Crystallographic complexes with extended oligosaccharide substrates reveal the structural basis for the accommodation of both unbranched, mixed-linked (MLG) and highly decorated, linear (XyG) polysaccharide chains in a broad, extended active-site cleft. Structural comparison with representative bacterial licheninases, a xyloglucan endotranglycosylase (XET), and a xyloglucan endohydrolase (XEH) outline the functional ramifications of key sequence deletions and insertions across the phylogenetic landscape of GH16. Although the biological role(s) of EG16 orthologs remains to be fully resolved, the present biochemical and tertiary structural characterization provides key insight into plant cell wall enzyme evolution, which will continue to inform genomic analyses and functional studies across species.

The voltage-gated sodium channel EF-hands form an interaction with the III-IV linker that is disturbed by disease-causing mutations

Voltage-gated sodium channels (NaV) are responsible for the rapid depolarization of many excitable cells. They readily inactivate, a process where currents diminish after milliseconds of channel opening. They are also targets for a multitude of disease-causing mutations, many of which have been shown to affect inactivation. A cluster of disease mutations, linked to Long-QT and Brugada syndromes, is located in a C-terminal EF-hand like domain of NaV1.5, the predominant cardiac sodium channel isoform. Previous studies have suggested interactions with the III-IV linker, a cytosolic element directly involved in inactivation. Here we validate and map the interaction interface using isothermal titration calorimetry (ITC) and NMR spectroscopy. We investigated the impact of various disease mutations on the stability of the domain, and found that mutations that cause misfolding of the EF-hand domain result in hyperpolarizing shifts in the steady-state inactivation curve. Conversely, mutations in the III-IV linker that disrupt the interaction with the EF-hand domain also result in large hyperpolarization shifts, supporting the interaction between both elements in intact channels. Disrupting the interaction also causes large late currents, pointing to a dual role of the interaction in reducing the population of channels entering inactivation and in stabilizing the inactivated state.

β Strand Switching: A Novel Structural Rescue Mechanism in a Δexon3 Cardiac Ryanodine Receptor Mutant

The contraction of cardiac muscle requires release of Ca2+ from the sarcoplasmic reticulum through the cardiac ryanodine receptor (RyR2). Several mutations in RyR2 are linked to inherited disorders, including triggered cardiac arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT) that may lead to sudden cardiac death. A severe form of CPVT is caused by removal of an entire third exon (Δexon3) of RyR2. The 35 deleted residues form secondary structure elements which are crucial in folding of the N-terminal domain, raising the question of why the deletion is neither lethal nor confers a loss-of-function phenotype. A 2.3A crystal structure shows that the removal results in a structural rescue: an otherwise flexible loop compensates for the loss by inserting itself into the β trefoil domain and increases the thermal stability. The other β strands in the domain show increased mobility to accommodate a sequence that bears no similarity to the deleted exon. The exon3 deletion is not tolerated in the corresponding RyR1 domain. The rescue shows a novel mechanism by which RyR2 channels can adjust their Ca2+ release properties through altering the structure of an individual domain.