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

Many physiological events require transient increases in cytosolic Ca(2+) concentrations. Ryanodine receptors (RyRs) are ion channels that govern the release of Ca(2+) 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.

Seeing the forest through the trees: towards a unified view on physiological calcium regulation of voltage-gated sodium channels.

Voltage-gated sodium channels (Na(V)s) underlie the upstroke of the action potential in the excitable tissues of nerve and muscle. After opening, Na(V)s rapidly undergo inactivation, a crucial process through which sodium conductance is negatively regulated. Disruption of inactivation by inherited mutations is an established cause of lethal cardiac arrhythmia, epilepsy, or painful syndromes. Intracellular calcium ions (Ca(2+)) modulate sodium channel inactivation, and multiple players have been suggested in this process, including the cytoplasmic Na(V) C-terminal region including two EF-hands and an IQ motif, the Na(V) domain III-IV linker, and calmodulin. Calmodulin can bind to the IQ domain in both Ca(2+)-bound and Ca(2+)-free conditions, but only to the DIII-IV linker in a Ca(2+)-loaded state. The mechanism of Ca(2+) regulation, and its composite effect(s) on channel gating, has been shrouded in much controversy owing to numerous apparent experimental inconsistencies. Herein, we attempt to summarize these disparate data and propose a novel, to our knowledge, physiological mechanism whereby calcium ions promote sodium current facilitation due to Ca(2+) memory at high-action-potential frequencies where Ca(2+) levels may accumulate. The available data suggest that this phenomenon may be disrupted in diseases where cytoplasmic calcium ion levels are chronically high and where targeted phosphorylation may decouple the Ca(2+) regulatory machinery. Many Na(V) disease mutations associated with electrical dysfunction are located in the Ca(2+)-sensing machinery and misregulation of Ca(2+)-dependent channel modulation is likely to contribute to disease phenotypes.

Molecular and structural characterization of the SH3 domain of AHI-1 in regulation of cellular resistance of BCR-ABL(+) chronic myeloid leukemia cells to tyrosine kinase inhibitors.

ABL tyrosine kinase inhibitor (TKI) therapy induces clinical remission in chronic myeloid leukemia (CML) patients but early relapses and later emergence of TKI-resistant disease remain problematic. We recently demonstrated that the AHI-1 oncogene physically interacts with BCR-ABL and JAK2 and mediates cellular resistance to TKI in CML stem/progenitor cells. We now show that deletion of the SH3 domain of AHI-1 significantly enhances apoptotic response of BCR-ABL(+) cells to TKIs compared to cells expressing full-length AHI-1. We have also discovered a novel interaction between AHI-1 and Dynamin-2, a GTPase, through the AHI-1 SH3 domain. The crystal structure of the AHI-1 SH3 domain at 1.53-Å resolution reveals that it adopts canonical SH3 folding, with the exception of an unusual C-terminal α helix. PD1R peptide, known to interact with the PI3K SH3 domain, was used to model the binding pattern between the AHI-1 SH3 domain and its ligands. These studies showed that an "Arg-Arg-Trp" stack may form within the binding interface, providing a potential target site for designing specific drugs. The crystal structure of the AHI-1 SH3 domain thus provides a valuable tool for identification of key interaction sites in regulation of drug resistance and for the development of small molecule inhibitors for CML.

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.

Talin autoinhibition is required for morphogenesis.

The establishment of a multicellular body plan requires coordinating changes in cell adhesion and the cytoskeleton to ensure proper cell shape and position within a tissue. Cell adhesion to the extracellular matrix (ECM) via integrins plays diverse, essential roles during animal embryogenesis and therefore must be precisely regulated. Talin, a FERM-domain containing protein, forms a direct link between integrin adhesion receptors and the actin cytoskeleton and is an important regulator of integrin function. Similar to other FERM proteins, talin makes an intramolecular interaction that could autoinhibit its activity. However, the functional consequence of such an interaction has not been previously explored in vivo. Here, we demonstrate that targeted disruption of talin autoinhibition gives rise to morphogenetic defects during fly development and specifically that dorsal closure (DC), a process that resembles wound healing, is delayed. Impairment of autoinhibition leads to reduced talin turnover at and increased talin and integrin recruitment to sites of integrin-ECM attachment. Finally, we present evidence that talin autoinhibition is regulated by Rap1-dependent signaling. Based on our data, we propose that talin autoinhibition provides a switch for modulating adhesion turnover and adhesion stability that is essential for morphogenesis.

The deletion of exon 3 in the cardiac ryanodine receptor is rescued by ß 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 ß 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 Ca²âº 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.

Crystal structures of the N-terminal domains of cardiac and skeletal muscle ryanodine receptors: insights into disease mutations.

Ryanodine receptors (RyRs) are channels governing the release of Ca(2+) from the sarcoplasmic or endoplasmic reticulum. They are required for the contraction of both skeletal (RyR1) and cardiac (RyR2) muscles. Mutations in both RyR1 and RyR2 have been associated with severe genetic disorders, but high-resolution data describing the disease variants in detail have been lacking. Here we present the crystal structures of the N-terminal domains of both RyR2 (1-217) and RyR1 (9-205) at 2.55 A and 2.9 A, respectively. The domains map in a hot spot region for disease mutations. Both structures consist of a core beta trefoil domain flanked by an alpha helix. Crystal structures of two RyR2 disease mutants, A77V (2.2 A) and V186M (1.7 A), show that the mutations cause distinct local changes in the surface of the protein. A RyR2 deletion mutant causes significant changes in the thermal stability. The disease positions highlight two putative binding interfaces required for normal RyR function.