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Walter J. Chazin - One of the best experts on this subject based on the ideXlab platform.
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relating form and function of EF Hand calcium binding proteins
Accounts of Chemical Research, 2011Co-Authors: Walter J. ChazinAbstract:The EF Hand, a helix-loop-helix structure, is one of the most common motifs found in animal genomes, and EF-Hand Ca(2+)-binding proteins (EFCaBPs) are widely distributed throughout the cell. However, researchers remain confounded by a lack of understanding of how peptide sequences code for specific functions and by uncertainty about the molecular mechanisms that enable EFCaBPs to distinguish among many diverse cellular targets. Such knowledge could dEFine the roles of EFCaBPs in health and disease and ultimately enable control or even design of Ca(2+)-dependent functions in medicine and biotechnology. In this Account, we describe our structural and biochemical research designed to understand the sequence-to-function relationship in EFCaBPs. The first structural goal was to dEFine conformational changes induced by binding Ca(2+), and our group and others established that solution NMR spectroscopy is well suited for this task. We pinpointed residues critical to the differences in Ca(2+) response of calbindin D(9k) and calmodulin (CaM), homologous EFCaBPs from different functional classes, by using direct structure determination with site-directed mutagenesis and protein engineering. Structure combined with biochemistry provided the foundation for identifying the fundamental mechanism of cooperativity in the binding of Ca(2+) ions: this cooperativity provides EFCaBPs with the ability to detect the relatively small changes in concentration that constitute Ca(2+) signals. Using calbindin D(9k) as a model system, studies of the structure and fast time scale dynamics of each of the four ion binding states in a typical EF-Hand domain provided direct evidence that site-site communication lowers the free energy cost of reorganization for binding the second ion. Our work has also extended models of how EFCaBPs interact with their cellular targets. We determined the unique dimeric architecture of S100 proteins, a specialized subfamily of EFCaBPs found exclusively in vertebrates. We described the implications for how these proteins transduce signals and went on to characterize interactions with peptide fragments of important cellular targets. Studies of the CaM homolog centrin revealed novel characteristics of its binding of Ca(2+) and its interaction with its cellular target Kar1. These results provided clear examples of how subtle differences in sequence fine-tune EFCaBPs to interact with their specific targets. The structural approach stands at a critical crossroad, shifting in emphasis from descriptive structural biochemistry to integrated biology and medicine. We present our dual-molecular-switch model for Ca(2+) regulation of gating functions of voltage-gated sodium channels in which both CaM and an intrinsic EF-Hand domain serve as coupled Ca(2+) sensors. A second example involves novel EFCaBP extracellular function, that is, the role of S100A8/S100A9 heterodimer in the innate immune response to bacterial pathogens. A mechanism for the antimicrobial activity of S100A8/S100A9 was discovered. We describe interactions of S100A8/S100A9 and S100B with the cell surface receptor for advanced glycation end products. Biochemical and structural studies are now uncovering the mechanisms by which EFCaBPs work and are helping to dEFine their biological activities, while simultaneously expanding knowledge of the roles of these proteins in normal cellular physiology and the pathology of disease.
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the EF Hand domain a globally cooperative structural unit
Protein Science, 2009Co-Authors: Melanie R Nelson, Walter J. Chazin, Eva Thulin, Patricia A Fagan, Sture ForsenAbstract:EF-Hand Ca2+-binding proteins participate in both modulation of Ca2+ signals and direct transduction of the ionic signal into downstream biochemical events. The range of biochemical functions of these proteins is correlated with differences in the way in which they respond to the binding of Ca2+. The EF-Hand domains of calbindin D9k and calmodulin are homologous, yet they respond to the binding of calcium ions in a drastically different manner. A series of comparative analyses of their structures enabled the development of hypotheses about which residues in these proteins control the calcium-induced changes in conformation. To test our understanding of the relationship between protein sequence and structure, we specifically designed the F36G mutation of the EF-Hand protein calbindin D9k to alter the packing of helices I and II in the apoprotein. The three-dimensional structure of apo F36G was determined in solution by nuclear magnetic resonance spectroscopy and showed that the design was successful. Surprisingly, significant structural perturbations also were found to extend far from the site of mutation. The observation of such long-range EFfects provides clear evidence that four-helix EF-Hand domains should be treated as a single globally cooperative unit. A hypothetical mechanism for how the long-range EFfects are transmitted is described. Our results support the concept of energetic and structural coupling of the key residues that are crucial for a protein's fold and function.
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Solution NMR structure of the C-terminal EF-Hand domain of human cardiac sodium channel Nav1.5
Journal of Biological Chemistry, 2008Co-Authors: Benjamin Chagot, Franck Potet, Jeffrey R. Balser, Walter J. ChazinAbstract:Abstract The voltage-gated sodium channel NaV1.5 is responsible for the initial upstroke of the action potential in cardiac tissue. Levels of intracellular calcium modulate inactivation gating of NaV1.5, in part through a C-terminal EF-Hand calcium binding domain. The significance of this structure is underscored by the fact that mutations within this domain are associated with specific cardiac arrhythmia syndromes. In an EFfort to elucidate the molecular basis for calcium regulation of channel function, we have determined the solution structure of the C-terminal EF-Hand domain using multidimensional heteronuclear NMR. The structure confirms the existence of the four-helix bundle common to EF-Hand domain proteins. However, the location of this domain is shifted with respect to that predicted on the basis of a consensus 12-residue EF-Hand calcium binding loop in the sequence. This finding is consistent with the weak calcium affinity reported for the isolated EF-Hand domain; high affinity binding is observed only in a construct with an additional 60 residues C-terminal to the EF-Hand domain, including the IQ motif that is central to the calcium regulatory apparatus. The binding of an IQ motif peptide to the EF-Hand domain was characterized by isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The peptide binds between helices I and IV in the EF-Hand domain, similar to the binding of target peptides to other EF-Hand calcium-binding proteins. These results suggest a molecular basis for the coupling of the intrinsic (EF-Hand domain) and extrinsic (calmodulin) components of the calcium-sensing apparatus of NaV1.5.
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target selectivity in EF Hand calcium binding proteins
Biochimica et Biophysica Acta, 2004Co-Authors: Shibani Bhattacharya, Christopher G Bunick, Walter J. ChazinAbstract:Abstract EF-Hand calcium binding proteins have remarkable sequence homology and structural similarity, yet their response to binding of calcium is diverse and they function in a wide range of biological processes. Knowledge of the fine-tuning of EF-Hand protein sequences to optimize specific biochemical properties has been significantly advanced over the past 10 years by determination of atomic resolution structures. These data lay the foundation for addressing how functional selectivity is generated from a generic ionic signal. This review presents current ideas about the structural mechanisms that provide the selectivity of different EF-Hand proteins for specific cellular targets, using S100 and calmodulin family proteins to demonstrate the critical concepts. Three factors contribute significantly to target selectivity: molecular architecture, response to binding of Ca 2+ ions, and the characteristics of target binding surfaces. Comparisons of calmodulin and S100 proteins provide insights into the role these factors play in facilitating the variety of binding configurations necessary for recognizing a diverse set of targets.
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An EF-Hand in the sodium channel couples intracellular calcium to cardiac excitability
Nature Structural & Molecular Biology, 2004Co-Authors: Tammy L Wingo, Walter J. Chazin, Vikas N Shah, Mark E Anderson, Terry P Lybrand, Jeffrey R. BalserAbstract:Sodium channels initiate the electrical cascade responsible for cardiac rhythm, and certain life-threatening arrhythmias arise from Na^+ channel dysfunction. We propose a novel mechanism for modulation of Na^+ channel function whereby calcium ions bind directly to the human cardiac Na^+ channel (hH1) via an EF-Hand motif in the C-terminal domain. A functional role for Ca^2+ binding was identified electrophysiologically, by measuring Ca^2+-induced modulation of hH1. A small hH1 fragment containing the EF-Hand motif was shown to form a structured domain and to bind Ca^2+ with affinity characteristic of calcium sensor proteins. Mutations in this domain reduce Ca^2+ affinity in vitro and the inactivation gating EFfects of Ca^2+ in electrophysiology experiments. These studies reveal the molecular basis for certain forms of long QT syndrome and other arrhythmia-producing syndromes, and suggest a potential pharmacological target for antiarrhythmic drug design.
Robert H Kretsinger - One of the best experts on this subject based on the ideXlab platform.
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structural and functional diversity of EF Hand proteins evolutionary perspectives
Protein Science, 2017Co-Authors: Hiroshi Kawasaki, Robert H KretsingerAbstract:We have classified 865 sequences of EF-Hand proteins from five proteomes into 156 subfamilies. These subfamilies were put into six groups. Evolutionary relationships among subfamilies and groups were analyzed from the inferred ancestral sequence for each subfamily. CTER, CPV, and PEF groups arose from a common EF-lobe (pair of adjacent EF-Hands). They have two or more EF-lobes; the relative positions of their EF-lobes differ from each other. Comparisons of the ancestral sequences and the inferred structures of the EF-lobes of these groups indicate that the mutual positions of EF-lobes were established soon after divergence of an EF-lobe for each group and bEFore the duplication and fusion of EF-lobe gene(s). These ancestral sequences reveal that some subfamilies in low similarity and isolated groups did not evolve from the EF-lobe precursor, even if their conformations are similar to the canonical EF-Hand. This is an example of convergent evolution.
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structural differences among subfamilies of EF Hand proteins a view from the pseudo two fold symmetry axis
Proteins, 2014Co-Authors: Hiroshi Kawasaki, Robert H KretsingerAbstract:We have analyzed the conformations of EF-lobes, adjacent pairs of EF-Hand domains, in a coordinate system based on the approximate two-fold (z) axis that relates the two EF-Hands. Two parameters - dE(o), the azimuthal angle between the y-axis and the projection of the offset vector to helix E onto the yz-plane, and δdF(o), the difference angle between the two helices (F1 and F2) of odd and even domains--characterize the openness of a single EF-Hand domain and of an EF-lobe, respectively. We describe and compare values of dE(o) and of δdF(o) for EF-Hand proteins of five subfamilies--CTER, CPV, S100, PARV, CALP--in calci- and apo- forms, with and without bound target proteins. Each subfamily has characteristic changes associated with binding calcium and/or target proteins.
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structural differences among subfamilies of EF Hand proteins a view from the pseudo two fold symmetry axis
Proteins, 2014Co-Authors: Hiroshi Kawasaki, Robert H KretsingerAbstract:We have analyzed the conformations of EF-lobes, adjacent pairs of EF-Hand domains, in a coordinate system based on the approximate two-fold (z) axis that relates the two EF-Hands. Two parameters - dE(o), the azimuthal angle between the y-axis and the projection of the offset vector to helix E onto the yz-plane, and δdF(o), the difference angle between the two helices (F1 and F2) of odd and even domains—characterize the openness of a single EF-Hand domain and of an EF-lobe, respectively. We describe and compare values of dE(o) and of δdF(o) for EF-Hand proteins of five subfamilies—CTER, CPV, S100, PARV, CALP—in calci- and apo- forms, with and without bound target proteins. Each subfamily has characteristic changes associated with binding calcium and/or target proteins. Proteins 2014; 82:2915–2924. © 2014 Wiley Periodicals, Inc.
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classification and evolution of EF Hand proteins
Biometals, 1998Co-Authors: Hiroshi Kawasaki, Susumu Nakayama, Robert H KretsingerAbstract:Forty-five distinct subfamilies of EF-Hand proteins have been identified. They contain from two to eight EF-Hands that are recognizable by amino acid sequence as being statistically similar to other EF-Hand domains. All proteins within one subfamily are congruent to one another, i.e. the dendrogram computed from one of the EF-Hand domains is similar, within statistical error, to the dendrogram computed from another(s) domain. Thirteen subfamilies--including Calmodulin, Troponin C, Essential light chain, Regulatory light chain--rEFerred to collectively as CTER, are congruent with one another. They appear to have evolved from a single ur-domain by two cycles of gene duplication and fusion. The subfamilies of CTER subsequently evolved by gene duplications and speciations. The remaining 32 subfamilies do not show such general patterns of congruence; however, some--such as S100, intestinal calcium binding protein (calbindin 9 kd), and trichohylin--do not form congruent clusters of subfamilies. Nearly all of the domains 1, 3, 5, and 7 are most similar to other ODD domains. Correspondingly the EVEN numbered domains of all 45 subfamilies most closely resemble EVEN domains of other subfamilies. Many sequence and chemical characteristics do not show systemic trends by subfamily or species of host organisms; such homoplasy is widespread. Eighteen of the subfamilies are heterochimeric; in addition to multiple EF-Hands they contain domains of other evolutionary origins.
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evolution of the EF Hand family of proteins
Annual Review of Biophysics and Biomolecular Structure, 1994Co-Authors: Susumu Nakayama, Robert H KretsingerAbstract:CONTENTS OV ERVIEW . . . . . . . . . . . ... . . . . ... . . . . .. .. . . . . .. . .. . .. . . . .... . .. .. 473 CH ARACTERISTICS OF EF-H AND PROTEINS . . . . . . . . . . . . . . . . . . . . . . . . . 474 EF-Hand Domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 47 4 Calcium Coordination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 4 Pairs of Domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479 Calcium as a Secondary Messenger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480 CLASSIFI CATION ... ......... 48 1 Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481 Congruence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 3 Domain Grouping and Branching Order . . . . .. . . . . . . . . . . . . . . . . . .. . . . . .. . 486 Encoding DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486 Subfamilies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488 INTRONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499 CHROMOSOMAL DISTRIBUTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 CALMOD ULIN CONTRADICTION . ... .. ........ ... ... ... . 500 CON CLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
Zenon Grabarek - One of the best experts on this subject based on the ideXlab platform.
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x ray structures of magnesium and manganese complexes with the n terminal domain of calmodulin insights into the mechanism and specificity of metal ion binding to an EF Hand
Biochemistry, 2012Co-Authors: F T Senguen, Zenon GrabarekAbstract:Calmodulin (CaM), a member of the EF-Hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca(2+) in the presence of an ~1000-fold higher concentration of cellular Mg(2+). To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg(2+), Mn(2+), and Zn(2+). In contrast to Ca(2+), which induces domain opening in CaM, octahedrally coordinated Mg(2+) and Mn(2+) stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn(2+) ions bind at the protein surface and do not compete with Ca(2+). The relative positions of bound Mg(2+) and Mn(2+) within the EF-Hand loops are similar to those of Ca(2+); however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca(2+) is critical for domain opening, does not bind directly to either Mn(2+) or Mg(2+), and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-Hand β-scaffold. The structures suggest that Mg(2+) contributes to the switching off of calmodulin activity and possibly other EF-Hand proteins at the resting levels of Ca(2+). The Mg(2+)-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change.
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insights into modulation of calcium signaling by magnesium in calmodulin troponin c and related EF Hand proteins
Biochimica et Biophysica Acta, 2011Co-Authors: Zenon GrabarekAbstract:The Ca(2+)-binding helix-loop-helix structural motif called "EF-Hand" is a common building block of a large family of proteins that function as intracellular Ca(2+)-receptors. These proteins respond specifically to micromolar concentrations of Ca(2+) in the presence of ~1000-fold excess of the chemically similar divalent cation Mg(2+). The intracellular free Mg(2+) concentration is tightly controlled in a narrow range of 0.5-1.0mM, which at the resting Ca(2+) levels is sufficient to fully or partially saturate the Ca(2+)-binding sites of many EF-Hand proteins. Thus, to convey Ca(2+) signals, EF-Hand proteins must respond differently to Ca(2+) than to Mg(2+). In this review the structural aspects of Mg(2+) binding to EF-Hand proteins are considered and interpreted in light of the recently proposed two-step Ca(2+)-binding mechanism (Grabarek, Z., J. Mol. Biol., 2005, 346, 1351). It is proposed that, due to stereochemical constraints imposed by the two-EF-Hand domain structure, the smaller Mg(2+) ion cannot engage the ligands of an EF-Hand in the same way as Ca(2+) and dEFaults to stabilizing the apo-like conformation of the EF-Hand. It is proposed that Mg(2+) plays an active role in the Ca(2+)-dependent regulation of cellular processes by stabilizing the "off state" of some EF-Hand proteins, thereby facilitating switching off their respective target enzymes at the resting Ca(2+) levels. TherEFore, some pathological conditions attributed to Mg(2+) dEFiciency might be related to excessive activation of underlying Ca(2+)-regulated cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
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structural basis for diversity of the EF Hand calcium binding proteins
Journal of Molecular Biology, 2006Co-Authors: Zenon GrabarekAbstract:The calcium binding proteins of the EF-Hand super-family are involved in the regulation of all aspects of cell function. These proteins exhibit a great diversity of composition, structure, Ca2+-binding and target interaction properties. Here, our current understanding of the Ca2+-binding mechanism is assessed. The structures of the EF-Hand motifs containing 11–14 amino acid residues in the Ca2+-binding loop are analyzed within the framework of the recently proposed two-step Ca2+-binding mechanism. A hypothesis is put forward that in all EF-Hand proteins the Ca2+-binding and the resultant conformational responses are governed by the central structure connecting the Ca2+-binding loops in the two-EF-Hand domain. This structure, named EFβ-scaffold, dEFines the position of the bound Ca2+, and coordinates the function of the N-terminal (variable and flexible) with the C-terminal (invariable and rigid) parts of the Ca2+-binding loop. It is proposed that the nature of the first ligand of the Ca2+-binding loop is an important determinant of the conformational change. Additional factors, including the interhelical contacts, the length, structure and flexibility of the linker connecting the EF-Hand motifs, and the overall energy balance provide the fine-tuning of the Ca2+-induced conformational change in the EF-Hand proteins.
Hiroshi Kawasaki - One of the best experts on this subject based on the ideXlab platform.
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structural and functional diversity of EF Hand proteins evolutionary perspectives
Protein Science, 2017Co-Authors: Hiroshi Kawasaki, Robert H KretsingerAbstract:We have classified 865 sequences of EF-Hand proteins from five proteomes into 156 subfamilies. These subfamilies were put into six groups. Evolutionary relationships among subfamilies and groups were analyzed from the inferred ancestral sequence for each subfamily. CTER, CPV, and PEF groups arose from a common EF-lobe (pair of adjacent EF-Hands). They have two or more EF-lobes; the relative positions of their EF-lobes differ from each other. Comparisons of the ancestral sequences and the inferred structures of the EF-lobes of these groups indicate that the mutual positions of EF-lobes were established soon after divergence of an EF-lobe for each group and bEFore the duplication and fusion of EF-lobe gene(s). These ancestral sequences reveal that some subfamilies in low similarity and isolated groups did not evolve from the EF-lobe precursor, even if their conformations are similar to the canonical EF-Hand. This is an example of convergent evolution.
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structural differences among subfamilies of EF Hand proteins a view from the pseudo two fold symmetry axis
Proteins, 2014Co-Authors: Hiroshi Kawasaki, Robert H KretsingerAbstract:We have analyzed the conformations of EF-lobes, adjacent pairs of EF-Hand domains, in a coordinate system based on the approximate two-fold (z) axis that relates the two EF-Hands. Two parameters - dE(o), the azimuthal angle between the y-axis and the projection of the offset vector to helix E onto the yz-plane, and δdF(o), the difference angle between the two helices (F1 and F2) of odd and even domains--characterize the openness of a single EF-Hand domain and of an EF-lobe, respectively. We describe and compare values of dE(o) and of δdF(o) for EF-Hand proteins of five subfamilies--CTER, CPV, S100, PARV, CALP--in calci- and apo- forms, with and without bound target proteins. Each subfamily has characteristic changes associated with binding calcium and/or target proteins.
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structural differences among subfamilies of EF Hand proteins a view from the pseudo two fold symmetry axis
Proteins, 2014Co-Authors: Hiroshi Kawasaki, Robert H KretsingerAbstract:We have analyzed the conformations of EF-lobes, adjacent pairs of EF-Hand domains, in a coordinate system based on the approximate two-fold (z) axis that relates the two EF-Hands. Two parameters - dE(o), the azimuthal angle between the y-axis and the projection of the offset vector to helix E onto the yz-plane, and δdF(o), the difference angle between the two helices (F1 and F2) of odd and even domains—characterize the openness of a single EF-Hand domain and of an EF-lobe, respectively. We describe and compare values of dE(o) and of δdF(o) for EF-Hand proteins of five subfamilies—CTER, CPV, S100, PARV, CALP—in calci- and apo- forms, with and without bound target proteins. Each subfamily has characteristic changes associated with binding calcium and/or target proteins. Proteins 2014; 82:2915–2924. © 2014 Wiley Periodicals, Inc.
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classification and evolution of EF Hand proteins
Biometals, 1998Co-Authors: Hiroshi Kawasaki, Susumu Nakayama, Robert H KretsingerAbstract:Forty-five distinct subfamilies of EF-Hand proteins have been identified. They contain from two to eight EF-Hands that are recognizable by amino acid sequence as being statistically similar to other EF-Hand domains. All proteins within one subfamily are congruent to one another, i.e. the dendrogram computed from one of the EF-Hand domains is similar, within statistical error, to the dendrogram computed from another(s) domain. Thirteen subfamilies--including Calmodulin, Troponin C, Essential light chain, Regulatory light chain--rEFerred to collectively as CTER, are congruent with one another. They appear to have evolved from a single ur-domain by two cycles of gene duplication and fusion. The subfamilies of CTER subsequently evolved by gene duplications and speciations. The remaining 32 subfamilies do not show such general patterns of congruence; however, some--such as S100, intestinal calcium binding protein (calbindin 9 kd), and trichohylin--do not form congruent clusters of subfamilies. Nearly all of the domains 1, 3, 5, and 7 are most similar to other ODD domains. Correspondingly the EVEN numbered domains of all 45 subfamilies most closely resemble EVEN domains of other subfamilies. Many sequence and chemical characteristics do not show systemic trends by subfamily or species of host organisms; such homoplasy is widespread. Eighteen of the subfamilies are heterochimeric; in addition to multiple EF-Hands they contain domains of other evolutionary origins.
Ilkka Kilpelainen - One of the best experts on this subject based on the ideXlab platform.
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nmr assignments secondary structure and global fold of calerythrin an EF Hand calcium binding protein from saccharopolyspora erythraea
Protein Science, 2008Co-Authors: Helena Aitio, Arto Annila, Torbjorn Drakenberg, Eva Thulin, Sami Heikkinen, Ilkka KilpelainenAbstract:Calerythrin is a 20 kDa calcium-binding protein isolated from gram-positive bacterium Saccharopolyspora erythraea. Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF-Hand calcium-binding proteins (SCPs), and therEFore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain 1H, 13C, and 15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]-edited TOCSY, and HCCH-TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N-H residual dipolar couplings were measured from a spin-state selective [1H, 15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF-Hand motifs with characteristic helix-loop-helix patterns were observed. Three of these are typical calcium-binding EF-Hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium-binding proteins from the sandworm Nereis diversicolor and the amphioxus Branchiostoma lanceolatum.
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nmr solution structure of calerythrin an EF Hand calcium binding protein from saccharopolyspora erythraea
FEBS Journal, 2003Co-Authors: Helena Tossavainen, Perttu Permi, Arto Annila, Ilkka Kilpelainen, Torbjorn DrakenbergAbstract:The structure of calerythrin, a prokaryotic 20 kDa calcium-binding protein has been determined by solution NMR spectroscopy. Distance, dihedral angle, J coupling, secondary chemical shift, residual dipolar coupling and radius of gyration restraints reveal four EF-Hand motifs arranged in a compact globular structure. A tight turn in the middle of the amino acid sequence brings the two halves, each comprising a pair of EF-Hands, close together. The structural similarity between calerythrin and the eukaryotic sarcoplasmic calcium-binding proteins is notable.