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.

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(α) atom movements of up to 8 Šupon 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.

Contribution of hydrogen bonds to protein stability.

Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein.

Human cardiac ryanodine receptor: preparation, crystallization and preliminary X-ray ANALysis of the N-terminal region.

Human ryanodine receptor 2 (hRyR2) is a calcium ion channel present in the membrane of the sarcoplasmic reticulum of cardiac myocytes that mediates release of calcium ions from the sarcoplasmic reticulum stores during excitation- contraction coupling. Disease-causing mutations of hRyR2 are clustered into N-terminal (amino acids 1-600), central (amino acids 2100-2500) and C-terminal (amino acids 3900-5000) regions. These regions are believed to be involved in regulation of channel gating. The N-terminal region of hRyR2 has been implicated in regulating basal channel activity by interaction with the central hRyR2 region. This paper reports preparation, crystallization and preliminary X-ray analysis of recombinant hRyR2(1-606) N-terminal fragment. Soluble hRyR2(1-606) was expressed in Escherichia coli. Purification conditions were optimized using thermal shift assay. The quality and stability of the sample was probed by dynamic light scattering. A monomeric protein showing over 95% purity was obtained. The protein was crystallized by the hanging drop vapor-diffusion method. Diffraction data with resolution 2.39 Å were collected and processed.

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.

Monoclonal antibody MN423 as a stable mold facilitates structure determination of disordered tau protein.

Flexibility of intrinsically disordered tau protein is important for performing its functions. It is believed that alteration of the flexibility is instrumental to the assembly of tau protein into paired helical filaments (PHF) in tauopathies. Tau flexibility represents the main obstacle for structure determination of its conformation in physiology and/or pathology. We have alleviated this inherited difficulty by using specific monoclonal antibodies as tau protein surrogate binding partners. In this work we compare two "antibody mold structures": (1) X-ray structure of the free form of the Alzheimer's disease PHF core-specific antibody MN423 and (2) previously solved structure of the complex of MN423 with the PHF core C-terminal tau peptide. We found that MN423 combining site is in both structures identical. As a consequence, recombinant tau assumes in the complex a fold determined by the antibody combining site. Obtained results show that MN423 functions as a molecular mold for the PHF core segment, and opens the way for structure determination of other PHF core segments providing that other conformation-specific antibodies are available. Data from in silico docking of tau peptide into antibody mold, obtained in this study, show that biochemical data and computational approaches provide results comparable to X-ray crystallography.

Bioinformatic mapping and production of recombinant N-terminal domains of human cardiac ryanodine receptor 2.

We report the domain analysis of the N-terminal region (residues 1-759) of the human cardiac ryanodine receptor (RyR2) that encompasses one of the discrete RyR2 mutation clusters associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1) and arrhythmogenic right ventricular dysplasia (ARVD2). Our strategy utilizes a bioinformatics approach complemented by protein expression, solubility analysis and limited proteolytic digestion. Based on the bioinformatics analysis, we designed a series of specific RyR2 N-terminal fragments for cloning and overexpression in Escherichia coli. High yields of soluble proteins were achieved for fragments RyR2(1-606)xHis(6), RyR2(391-606)xHis(6), RyR2(409-606)xHis(6), Trx.RyR2(384-606)xHis(6), TrxxRyR2(391-606)xHis(6) and Trx.RyR2(409-606)xHis(6). The folding of RyR2(1-606)xHis(6) was analyzed by circular dichroism spectroscopy resulting in alpha-helix and beta-sheet content of approximately 23% and approximately 29%, respectively, at temperatures up to 35 degrees C, which is in agreement with sequence based secondary structure predictions. Tryptic digestion of the largest recombinant protein, RyR2(1-606)xHis(6), resulted in the appearance of two specific subfragments of approximately 40 and 25 kDa. The 25 kDa fragment exhibited greater stability. Hybridization with anti-His(6).Tag antibody indicated that RyR2(1-606)xHis(6) is cleaved from the N-terminus and amino acid sequencing of the proteolytic fragments revealed that digestion occurred after residues 259 and 384, respectively.

Structure of RNase Sa2 complexes with mononucleotides--new aspects of catalytic reaction and substrate recognition.

Although the mechanism of RNA cleavage by RNases has been studied for many years, there remain aspects that have not yet been fully clarified. We have solved the crystal structures of RNase Sa2 in the apo form and in complexes with mononucleotides. These structures provide more details about the mechanism of RNA cleavage by RNase Sa2. In addition to Glu56 and His86, which are the principal catalytic residues, an important role in the first reaction step of RNA cleavage also seems to be played by Arg67 and Arg71, which are located in the phosphate-binding site and form hydrogen bonds with the oxygens of the phosphate group of the mononucleotides. Their positive charge very likely causes polarization of the bonds between the oxygens and the phosphorus atom, leading to electron deficiency on the phosphorus atom and facilitating nucleophilic attack by O2' of the ribose on the phosphorus atom, leading to cyclophosphate formation. The negatively charged Glu56 is in position to attract the proton from O2' of the ribose. Extended molecular docking of mononucleotides, dinucleotides and trinucleotides into the active site of the enzyme allowed us to better understand the guanosine specificity of RNase Sa2 and to predict possible binding subsites for the downstream base and ribose of the second and third nucleotides.

Structure solution of misfolded conformations adopted by intrinsically disordered Alzheimer's tau protein.

Until now it was impossible to obtain atomic structure of intrinsically disordered protein (IDP) tau and/or its assembly in Alzheimer's paired helical filaments as neither of them could have been prepared in the form amenable to X-ray or NMR techniques. Using IDP tau property to attain regular tertiary structure after binding events during self-assembly or when complexed with its target we propose monoclonal antibodies as surrogate tau protein binding partners to form complexes and crystals for structure solution by X-ray technique.

X-ray structure of the PHF core C-terminus: insight into the folding of the intrinsically disordered protein tau in Alzheimer's disease.

The major constituent of Alzheimer's disease paired helical filaments (PHF) core is intrinsically disordered protein (IDP) tau. In spite of a considerable effort, insoluble character of PHF together with inherent physical properties of IDP tau have precluded so far reconstruction of PHF 3D structure by X-ray crystallography or NMR spectroscopy. Here we present first crystallographic study of PHF core C-terminus. Using monoclonal antibody MN423 specific to the tertiary structure of the PHF core, the in vivo PHF structure was imprinted into recombinant core PHF tau. Crystallization of the complex led to determination of the structure of the core PHF tau protein fragment 386TDHGAE391 at 1.65A resolution. Structural analysis suggests important role of the core PHF C-terminus for PHF assembly. It is reasonable to expect that this approach will help to reveal the structural principles underlying the tau protein assembly into PHF and possibly will facilitate rationale drug design for inhibition of Alzheimer neurofibrillary changes.

Preparation, crystallization and preliminary X-ray analysis of the Fab fragment of monoclonal antibody MN423, revealing the structural aspects of Alzheimer's paired helical filaments.

Monoclonal antibody (mAb) MN423 recognizes Alzheimer's disease specific conformation of tau protein assembled into paired helical filaments (PHF). Since the three-dimensional structure of PHF is currently unavailable, the structure of MN423 binding site could provide important information about PHF conformation with the consequences for the Alzheimer's disease prevention and cure. Fab fragment of MN423 was prepared and purified. We have identified two different conditions for crystallization of the Fab fragment that yielded two crystal forms. They diffracted to 3.0 and 1.6 A resolution with four and one molecule in the asymmetric unit, respectively. Both crystal forms belonged to the space group P2(1) with unit cell parameters a = 76.4 A, b = 138.4 A, c = 92.4 A, beta = 101.9 degrees , and a = 71.5 A, b = 36.8 A, c = 85.5 A, beta = 113.9 degrees .

Intrinsically disordered proteins in the neurodegenerative processes: formation of tau protein paired helical filaments and their analysis.

1. Several intrinsically disordered proteins (IDPs) play principal role in the neurodegenerative processes of various types. Among them, alpha-synuclein is involved in Parkinson's disease, prion protein in transmissible spongiform encephalopathies, and tau protein in Alzheimer's disease (AD) and related tauopathies. Neuronal damage in AD is accompanied by the presence of tau protein fibrils composed of paired helical filaments (PHF). 2. Tau protein represents a typical IDP. IDPs do not exhibit any stable secondary structure in the free form, but they are able to fold after binding to targets and contain regions with large propensity to adopt a defined type of secondary structure. Binding-folding event at tau protein leading to PHF generation is believed to happen in the course of tauopathies. 3. Detailed molecular topology of PHF formation is unknown. There are evidences about the cross-beta structure in PHF core; however the precise arrangement of the tau polypeptide chain is unclear. In this review we summarize current attempts at in vitro PHF reconstruction and the development of methods for PHF structure determination. The emphasis is put on the monoclonal antibodies used as structural molecular probes for research on the role of IDPs in pathogenesis of neurodegenerative diseases.

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 (alpha-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 A and that of the enzyme-acarbose complex at 1.6 A 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 alpha-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.

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 A 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 Trp71 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, Trp59 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 approximately 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.

Crystal structure reveals two alternative conformations in the active site of ribonuclease Sa2.

Three different strains of Streptomyces aureofaciens produce the homologous ribonucleases Sa, Sa2 and Sa3. The crystal structures of ribonuclease Sa (RNase Sa) and its complexes with mononucleotides have previously been reported at high resolution. Here, the structures of two crystal forms (I and II) of ribonuclease Sa2 (RNase Sa2) are presented at 1.8 and 1.5 A resolution. The structures were determined by molecular replacement using the coordinates of RNase Sa as a search model and were refined to R factors of 17.5 and 15.0% and R(free) factors of 21.8 and 17.2%, respectively. The asymmetric unit of crystal form I contains three enzyme molecules, two of which have similar structures to those seen for ribonuclease Sa, with Tyr87 at the bottom of their active sites. In the third molecule, Tyr87 has moved substantially: the CA atom moves almost 5 A and the OH of the side chain moves 10 A, inserting itself into the active site of a neighbouring molecule at a similar position to that observed for the nucleotide base in RNase Sa complexes. The asymmetric unit of crystal form II contains two Sa2 molecules, both of which are similar to the usual Sa structures. In one molecule, two main-chain conformations were modelled in the alpha-helix. Finally, a brief comparison is made between the conformations of the Sa2 molecules and those of 34 independent molecules taken from 20 structures of ribonuclease Sa and two independent molecules taken from two structures of ribonuclease Sa3 in various crystal forms.

Actin-binding domain of mouse plectin. Crystal structure and binding to vimentin.

Plectin, a large and widely expressed cytolinker protein, is composed of several subdomains that harbor binding sites for a variety of different interaction partners. A canonical actin-binding domain (ABD) comprising two calponin homology domains (CH1 and CH2) is located in proximity to its amino terminus. However, the ABD of plectin is unique among actin-binding proteins as it is expressed in the form of distinct, plectin isoform-specific versions. We have determined the three-dimensional structure of two distinct crystalline forms of one of its ABD versions (pleABD/2alpha) from mouse, to a resolution of 1.95 and 2.0 A. Comparison of pleABD/2alpha with the ABDs of fimbrin and utrophin revealed structural similarity between plectin and fimbrin, although the proteins share only low sequence identity. In fact, pleABD/2alpha has been found to have the same compact fold as the human plectin ABD and the fimbrin ABD, differing from the open conformation described for the ABDs of utrophin and dystrophin. Plectin harbors a specific binding site for intermediate filaments of various types within its carboxy-terminal R5 repeat domain. Our experiments revealed an additional vimentin-binding site of plectin, residing within the CH1 subdomain of its ABD. We show that vimentin binds to this site via the amino-terminal part of its rod domain. This additional amino-terminal intermediate filament protein binding site of plectin may have a function in intermediate filament dynamics and assembly, rather than in linking and stabilizing intermediate filament networks.

X-ray structure of two crystalline forms of a streptomycete ribonuclease with cytotoxic activity.

Ribonuclease (RNase) Sa3 is secreted by the Gram-positive bacterium Streptomyces aureofaciens. The enzyme catalyzes the cleavage of RNA on the 3' side of guanosine residues. Here, x-ray diffraction analysis was used to determine the three-dimensional structure of two distinct crystalline forms of RNase Sa3 to a resolution of 2.0 and 1.7 A. These two structures are similar to each other as well as to that of a homolog, RNase Sa. All of the key active-site residues of RNase Sa (Asn(42), Glu(44), Glu(57), Arg(72), and His(88)) are located in the putative active site of RNase Sa3. Also herein, RNase Sa3 is shown to be toxic to human erythroleukemia cells in culture. Like onconase, which is an amphibian ribonuclease in Phase III clinical trials as a cancer chemotherapeutic, RNase Sa3 is not inhibited by the cytosolic ribonuclease inhibitor protein. Thus, a prokaryotic ribonuclease can be toxic to mammalian cells.

Atomic resolution data reveal flexibility in the structure of RNase Sa.

Ribonuclease from Streptomyces aureofaciens, the bacterial source for the industrial production of chlorotetracycline, is a guanylate endoribonuclease (RNase Sa; EC 3.1.27.3) which hydrolyses the phosphodiester bonds of single-stranded RNA at the 3'-side of guanosine nucleotides with high specificity. The structure of the enzyme was previously refined at atomic resolution (1.2 A) using room-temperature data. Here, the RNase Sa structure refined against 1.0 A data collected at cryogenic temperature is reported. There are two surface loops in molecule A and one in molecule B for which two main-chain conformations are modelled: these loops contain active-site residues. The separation for most of the corresponding main-chain atoms in the two conformations is about 0.8 A, with a maximum of 2.5 A. The two regions of dual conformation represent the most important differences in comparison with the structure determined at room temperature, where the corresponding loops have one conformation only but the largest degree of anisotropy. The flexibility of the loops observed in the structure of RNase Sa is directly linked to the need for the active site to interact productively with substrates and/or inhibitors.

Purification, crystallization and preliminary X-ray analysis of the plectin actin-binding domain.

Plectin is an abundantly expressed cytoskeletal crosslinking protein of enormous size (>500 kDa) and multiple functions. It represents one of the many members of a large family of actin-binding proteins. The actin-binding domain of mouse plectin was expressed in Escherichia coli and purified to homogeneity. Crystals of the actin-binding domain of plectin were prepared by the hanging-drop method. They belong to space group P2(1), with unit-cell parameters a = 55.92, b = 108.92, c = 63.75 A, beta = 115.25 degrees. Data from a single crystal were collected to 2.0 A resolution at room temperature using synchrotron radiation at EMBL, Hamburg. The asymmetric unit contains two molecules of the protein, which corresponds to V(M) = 3.06 A(3) Da(-1) and a solvent content of 60%. The structure was solved by the molecular-replacement method. In addition, the preparation of selenomethionine-derivative crystals is described.