The N-Terminal Region of the Ryanodine Receptor Affects Channel Activation

Mutations in the cardiac ryanodine receptor (RyR2), the ion channel responsible for release of calcium ions from intracellular stores into cytoplasm, are the cause of several inherited cardiac arrhythmias. At the molecular level, disease symptoms can be mimicked by domain peptides from mutation-prone regions of RyR2 that bind to RyR2 and activate it. Here we show that the domain peptide DPcpvtN2, corresponding to the central helix of the N-terminal region of RyR2, activates the RyR2 channel. Structural modeling of interaction between DPcpvtN2 and the N-terminal region of RyR2 in the closed and open conformation provided three plausible structures of the complex. Only one of them could explain the dependence of RyR2 activity on concentration of DPcpvtN2. The structure of the complex was at odds with the previously proposed “domain switch” mechanism of competition between domain peptides and ryanodine receptor domains. Likewise, in structural models of the N-terminal region, the conformational changes induced by DPcpvtN2 binding were different from those induced by mutation of central helix amino acids. The activating effect of DPcpvtN2 binding and of mutations in the central helix could be explained by their similar effect on the transition energy between the closed and open conformation of RyR2

A regulatory component of the human ryanodine receptor 2 N-terminus

Human cardiac ryanodine receptor (hRyR2) is a channel mediating Ca2+ release from the sarcoplasmic reticulum during excitation-contraction coupling. The N-terminal (1-655) and central (2100-2500) regions of hRyR2 are thought to be involved in regulating channel gating. Mutations linked to several heart diseases are clustered within these two, as well as in the channel pore-containing C-terminal regions. High resolution structures of key regions involved in the regulation of RyR2 activity could further the understanding of the gating mechanism of hRyR2 and of its malfunction in disease

Crystallization and preliminary X-ray diffraction analysis of two peptides from Alzheimer PHF in complex with the MN423 antibody Fab fragment

The major constituent of the Alzheimer's disease paired helical filaments (PHF) core is the intrinsically disordered protein (IDP) tau. Globular binding partners, e.g. monoclonal antibodies, can stabilize the fold of disordered tau in complexes. A previously published structure of a proteolytically generated tau fragment in a complex with the PHF-specific monoclonal antibody MN423 revealed a turn-like structure of the PHF core C-terminus [Sevcik et al. (2007). FEBS Lett. 581, 5872-5878]. To examine the structures of longer better-defined PHF segments, crystals of the MN423 Fab fragment were grown in the presence of two synthetic peptides derived from the PHF core C-terminus. For each, X-ray diffraction data were collected at 100 K at a synchrotron source and initial phases were obtained by molecular replacement

Contribution of Single Tryptophan Residues to the Fluorescence and Stability of Ribonuclease Sa

Ribonuclease Sa (RNase Sa) contains no tryptophan (Trp) residues. We have added single Trp residues to RNase Sa at sites where Trp is found in four other microbial ribonucleases, yielding the following variants of RNase Sa: Y52W, Y55W, T76W, and Y81W. We have determined crystal structures of T76W and Y81W at 1.1 and 1.0 Å resolution, respectively. We have studied the fluorescence properties and stabilities of the four variants and compared them to wild-type RNase Sa and the other ribonucleases on which they were based. Our results should help others in selecting sites for adding Trp residues to proteins. The most interesting findings are: 1), Y52W is 2.9 kcal/mol less stable than RNase Sa and the fluorescence intensity emission maximum is blue-shifted to 309 nm. Only a Trp in azurin is blue-shifted to a greater extent (308 nm). This blue shift is considerably greater than observed for Trp(71) in barnase, the Trp on which Y52W is based. 2), Y55W is 2.1 kcal/mol less stable than RNase Sa and the tryptophan fluorescence is almost completely quenched. In contrast, Trp(59) in RNase T1, on which Y55W is based, has a 10-fold greater fluorescence emission intensity. 3), T76W is 0.7 kcal/mol more stable than RNase Sa, indicating that the Trp side chain has more favorable interactions with the protein than the threonine side chain. The fluorescence properties of folded Y76W are similar to those of the unfolded protein, showing that the tryptophan side chain in the folded protein is largely exposed to solvent. This is confirmed by the crystal structure of the T76W which shows that the side chain of the Trp is only ∼7% buried. 4), Y81W is 0.4 kcal/mol less stable than RNase Sa. Based on the crystal structure of Y81W, the side chain of the Trp is 87% buried. Although all of the Trp side chains in the variants contribute to the unusual positive circular dichroism band observed near 235 nm for RNase Sa, the contribution is greatest for Y81W