Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C atom movements of up to 8 A ° upon channel gating, and predicts the location of the leucine– isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface

Structure of the complex of a yeast glucoamylase with acarbose reveals the presence of a raw starch binding site on the catalytic domain

Most glucoamylases (α-1,4-d-glucan glucohydrolase, EC 3.2.1.3) have structures consisting of both a catalytic and a starch binding domain. The structure of a glucoamylase from Saccharomycopsis fibuligera HUT 7212 (Glu), determined a few years ago, consists of a single catalytic domain. The structure of this enzyme with the resolution extended to 1.1 Å and that of the enzyme–acarbose complex at 1.6 Å resolution are presented here. The structure at atomic resolution, besides its high accuracy, shows clearly the influence of cryo-cooling, which is manifested in shrinkage of the molecule and lowering the volume of the unit cell. In the structure of the complex, two acarbose molecules are bound, one at the active site and the second at a site remote from the active site, curved around Tyr464 which resembles the inhibitor molecule in the 'sugar tongs' surface binding site in the structure of barley α-amylase isozyme 1 complexed with a thiomalto-oligosaccharide. Based on the close similarity in sequence of glucoamylase Glu, which does not degrade raw starch, to that of glucoamylase (Glm) from S. fibuligera IFO 0111, a raw starch-degrading enzyme, it is reasonable to expect the presence of the remote starch binding site at structurally equivalent positions in both enzymes. We propose the role of this site is to fix the enzyme onto the surface of a starch granule while the active site degrades the polysaccharide. This hypothesis is verified here by the preparation of mutants of glucoamylases Glu and Glm

Structural insights into the human RyR2 N terminal region involved in cardiac arrhythmias

Human ryanodine receptor 2 hRyR2 mediates calcium release from the sarcoplasmic reticulum, enabling cardio myocyte contraction. The N terminal region of hRyR2 amino acids 1 606 is the target of gt;30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near physiological conditions, as well as a homology model of the hRyR2 N terminal region, are presented. The N terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix amino acids 410 437 plays a prominent stabilizing role. Importantly, the anion binding site reported for the mouse RyR2 N terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix R420W, I419F and I419F R420W which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N terminus adopts a globular shape with a prominent tail that is likely to involve residues 545 606, which are unresolved in the crystal structure. Docking the N terminal domains into cryo electron microscopy maps of the closed and open RyR1 conformations reveals C[alpha] atom movements of up to 8 upon channel gating, and predicts the location of the leucine isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surfac