Crystal structure of Mg2+- and Ca2+-bound Gla domain of factor IX complexed with binding protein

Factor IX is an indispensable protein required in the blood coagulation cascade. It binds to the surface of phospholipid membrane by means of a gamma-carboxyglutamic acid (Gla) domain situated at the N terminus. Recently, we showed that physiological concentrations of Mg2+ ions affect the native conformation of the Gla domain and in doing so augment the biological activity of factor IXa and binding affinity with its binding protein even in the presence of Ca2+ ions. Here we report on the crystal structures of the Mg2+/Ca2+-bound and Ca2+-bound (Mg2+-free) factor IX Gla domain (IXGD1-46) in complex with its binding protein (IX-bp) at 1.55 and 1.80 A resolutions, respectively. Three Mg2+ and five Ca2+ ions were bound in the Mg2+/Ca2+-bound IXGD1-46, and the Mg2+ ions were replaced by Ca2+ ions in Mg2+-free IXGD1-46. Comparison of Mg2+/Ca2+-bound with Ca2+-bound structures of the complexes showed that Mg2+ ion, which formed a bridge between IXGD1-46 and IX-bp, forced IXGD1-46 to rotate 4 degrees relative to IX-bp and hence might be the cause of a more tight interaction between the molecules than in the case of the Mg2+-free structure. The results clearly suggest that Mg2+ ions are required to maintain native conformation and in vivo function of factor IX Gla domain during blood coagulation.     

Structural evidence for non-canonical binding of Ca2+ to a canonical EF-hand of a conventional myosin

We have previously identified a single inhibitory Ca2+-binding site in the first EF-hand of the essential light chain of Physarum conventional myosin (Farkas, L., Malnasi-Csizmadia, A., Nakamura, A., Kohama, K., and Nyitray, L. (2003) J. Biol. Chem. 278, 27399-27405). As a general rule, conformation of the EF-hand-containing domains in the calmodulin family is "closed" in the absence and "open" in the presence of bound cations; a notable exception is the unusual Ca2+-bound closed domain in the essential light chain of the Ca2+-activated scallop muscle myosin. Here we have reported the 1.8 A resolution structure of the regulatory domain (RD) of Physarum myosin II in which Ca2+ is bound to a canonical EF-hand that is also in a closed state. The 12th position of the EF-hand loop, which normally provides a bidentate ligand for Ca2+ in the open state, is too far in the structure to participate in coordination of the ion. The structure includes a second Ca2+ that only mediates crystal contacts. To reveal the mechanism behind the regulatory effect of Ca2+, we compared conformational flexibilities of the liganded and unliganded RD. Our working hypothesis, i.e. the modulatory effect of Ca2+ on conformational flexibility of RD, is in line with the observed suppression of hydrogen-deuterium exchange rate in the Ca2+-bound form, as well as with results of molecular dynamics calculations. Based on this evidence, we concluded that Ca2+-induced change in structural dynamics of RD is a major factor in Ca2+-mediated regulation of Physarum myosin II activity.     

A model for human cardiac troponin C and for modulation of its Ca2+ affinity by drugs

Calcium sensitizers are drugs which increase force development in striated muscle by sensitizing myofilaments to Ca2+. This can happen by increasing Ca2+ affinity of the regulatory domain of Ca2+ binding protein troponin C. High resolution crystal structures of two calcium binding proteins, calmodulin (Babu et al.: J. Mol. Biol. 203:191-204, 1988) and skeletal troponin C (Satyshur et al.: J. Biol. Chem. 263:1628-1647, 1988; Herzber et al.: J. Mol. Biol. 203:761-779, 1988), have recently been published. This makes it possible to model in detail the calcium-sensitizing action of drugs on troponin C. In this study a model of human cardiac troponin C in three-calcium state has been constructed. When calcium is bound to calcium site II of cardiac troponin C an open conformation of the protein results, which has a hydrophobic pocket surrounded by a few polar side chains. Complexation of three drugs, trifluoperazine, bepridil, and pimobendan, to the hydrophobic pocket is studied using energy minimization techniques. Two different binding modes are found, which differ in the location of a strong electrostatic interaction. In analogy with the crystal structure of skeletal troponin C it is hypothezed that in cardiac troponin C an interaction occurs between Gln-50 and Asp-88, which has a long-range effect on calcium binding. The binding modes of drugs, where a strong interaction with Asp-88 exists, can effectively prevent the interaction between Asp-88 and Gln-50 in the protein, and are proposed to be responsible for the calcium-sensitizing properties of the studied drugs.     

CD, 1H NMR and molecular modeling studies of the interaction of Ca2+ with substance P and Ala7-substance P in a non-polar solvent.

The biologically relevant conformation of substance P is likely to be dictated by the lipid milieu wherein the hormone would interact with its receptor. Assuming that specific constraints to the hormone structure may be imparted by its interaction with Ca2+ ions in the low dielectric lipid medium, the interaction of substance P and its inactive analog, Ala7-substance P, has been characterized in a lipid-mimetic solvent. Circular dichroism (CD) and NMR spectral methods were employed to study the conformation of the free and Ca2+-bound forms of the peptides and the conformational changes that occur on Ca2+ binding. The results show that both peptides assume a helical structure in the non-polar solvent used, a mixture of acetonitrile and trifluoroethanol. The N-terminal region is, however, less ordered in the analog peptide compared with the native hormone. Ca2+ addition causes significant conformational changes in both the peptides. However, while substance P binds two Ca2+ ions in a cooperative manner, Ala7-substance P binds only one Ca2+ ion with a relatively weaker affinity. Computations of the minimum-energy conformations of the free and Ca2+-bound peptides were performed using interproton distances derived from nuclear Overhauser enhancement spectra of the two peptides, as well as the information provided by changes in proton chemical shifts caused by Ca2+ addition. Taken together, the results of this study suggest that differences in the interaction of substance P and Ala7-substance P with Ca2+ in the non-polar milieu, which in turn leads to differences in their Ca2+-bound conformations, may be the basis for the differences in their biological potencies.     

Solution structure of the chicken skeletal muscle troponin complex via small-angle neutron and X-ray scattering

Troponin is a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle by participating in a series of conformational events within the actin-based thin filament. Troponin is a heterotrimeric complex consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT). Ternary troponin complexes have been produced by assembling recombinant chicken skeletal muscle TnC, TnI and the C-terminal portion of TnT known as TnT2. A full set of small-angle neutron scattering data has been collected from TnC-TnI-TnT2 ternary complexes, in which all possible combinations of the subunits have been deuterated, in both the +Ca2+ and -Ca2+ states. Small-angle X-ray scattering data were also collected from the same troponin TnC-TnI-TnT2 complex. Guinier analysis shows that the complex is monomeric in solution and that there is a large change in the radius of gyration of TnI when it goes from the +Ca2+ to the -Ca2+ state. Starting with a model based on the human cardiac troponin crystal structure, a rigid-body Monte Carlo optimization procedure was used to yield models of chicken skeletal muscle troponin, in solution, in the presence and in the absence of regulatory calcium. The optimization was carried out simultaneously against all of the scattering data sets. The optimized models show significant differences when compared to the cardiac troponin crystal structure in the +Ca2+ state and provide a structural model for the switch between +Ca2+ and -Ca2+ states. A key feature is that TnC adopts a dumbbell conformation in both the +Ca2+ and -Ca2+ states. More importantly, the data for the -Ca2+ state suggest a long extension of the troponin IT arm, consisting mainly of TnI. Thus, the troponin complex undergoes a large structural change triggered by Ca2+ binding.     

Structural change of troponin C molecule and its domains upon Ca2+ binding in the presence of Mg2+ ions measured by a solution X-ray scattering technique

A small-angle X-ray scattering study on troponin C showed that two domains of the molecule move closer to each other and the molecule shrinks along its long axis upon Ca2+ binding in the absence of Mg2+ ions (Fujisawa, T., Ueki, T., & Iida S. (1988) J. Biochem. 105, 377-383). When Mg2+ ions bind to troponin-C, the radius of gyration changes from 27.8 to 24.3 A and the average radius of gyration of the two domains is estimated to be 15.1 A. These radii indicate that the distance between the centers of the two domains is 38.1 A. Such a change is analogous to the previous result for troponin C with two Ca2+ ions bound at the high-affinity sites. Thus, the structural behavior of troponin C molecule is essentially the same when Ca2+/Mg2+ ions bind to its high-affinity sites. On the other hand, the effect of Ca2+ binding to the low-affinity sites in the presence of Mg2+ ions is quite different from the previous result. The binding of Ca2+ ions causes a dimerization of troponin C molecules with an apparent constant of 511 M-1. Such a characteristic behavior, implying the occurrence of a surface property change, may be related to the physiological role of troponin C molecule in the muscle. The scattering experiments on the tryptic fragments of troponin C also had interesting and important results: the C-domain shrinks, with the radius of gyration changing from 17.0 to 14.9 A while the N-domain swells from 13.9 to 15.0 A upon Ca2+ binding. Such an opposite change is consistent with the results of circular dichroism and spectroscopic studies of the domains.     

Troponin T core structure and the regulatory NH2-terminal variable region

The conserved central and COOH-terminal regions of troponin T (TnT) interact with troponin C, troponin I, and tropomyosin to regulate striated muscle contraction. Phylogenic data show that the NH2-terminal region has evolved as an addition to the conserved core structure of TnT. This NH2-terminal region does not bind other thin filament proteins, and its sequence is hypervariable between fiber type and developmental isoforms. Previous studies have demonstrated that NH2-terminal modifications alter the COOH-terminal conformation of TnT and thin filament Ca2+-activation, yet the functional core structure of TnT and the mechanism of NH2-terminal modulation are not well understood. To define the TnT core structure and investigate the regulatory role of the NH2-terminal variable region, we investigated two classes of model TnT molecules: (1) NH2-terminal truncated cardiac TnT and (2) chimera proteins consisting of an acidic or basic skeletal muscle TnT NH2-terminus spliced to the cardiac TnT core. Deletion of the TnT hypervariable NH2-terminus preserved binding to troponin I and tropomyosin and sustained cardiac muscle contraction in the heart of transgenic mice. Further deletion of the conserved central region diminished binding to tropomyosin. The reintroduction of differently charged NH2-terminal domains in the chimeric molecules produced long-range conformational changes in the central and COOH-terminal regions to alter troponin I and tropomyosin binding. Similar NH2-terminal charge effects are demonstrated in naturally occurring cardiac TnT isoforms, indicating a physiological significance. These results suggest that the hypervariable NH2-terminal region modulates the conformation and function of the TnT core structure to fine-tune muscle contractility.     

Refined crystal structure of troponin C from turkey skeletal muscle at 2.0 A resolution

The crystal structure of troponin C from turkey skeletal muscle has been refined at 2.0 A resolution (1 A = 0.1 nm). The resulting crystallographic R factor (R = sigma[[Fo[-[Fc[[/sigma[Fo[, where [Fo[ and [Fc[ are the observed and calculated structure factor amplitudes) is 0.155 for the 8054 reflections with intensities I greater than or equal to 2 sigma(I) within the 10 A to 2.0 A resolution range. With 66% of the residues in helical conformation, troponin C provides a good sample for helix analysis. The mean alpha-helix dihedral angles (phi, psi = -62 degrees, -42 degrees) agree with values observed for helical regions in other proteins. The helices are all curved and/or kinked. In particular, the 31 amino acid long inter-domain helix is smoothly curved, with a rather large radius of curvature of 137 A. Helix packing is different in the Ca2+-free domain (N-terminal) and the Ca2+-bound domain (C-terminal). The inter-helix angles for the two helix-loop-helix motifs in the regulatory domain are 133 degrees and 151 degrees, whereas the value for the two motifs in the C-terminal domain is 110 degrees, as observed in the EF-hands of parvalbumin. These differences affect the packing of the respective hydrophobic cores of each domain, in particular the disposition of aromatic rings. Pairwise arrangement of Ca2+-binding loops is common to both states, but the conformation is markedly different. Conversion of one to the other can be achieved by small cumulative changes of main-chain dihedral angles. The integrity of loop structure is maintained by numerous electrostatic interactions. Both salt bridges and carboxyl-carboxylate interactions are observed in TnC. There are more intramolecular (9) than intermolecular (1) salt bridges. Carboxyl-carboxylate interactions occur because the pH of the crystals is 5.0 and there is a multitude of aspartate and glutamate residues. One is intramolecular and four are intermolecular. Polar side-chain interactions occur more commonly with main-chain carbonyls and amides than with other polar side-chains. These interactions are mostly short range, and are similar to those observed in other proteins with one exception: negatively charged side-chains interact more frequently with main-chain carbonyl oxygen atoms. However, out of 19 such interactions, 10 involve oxygen atoms of the Ca2+ ligands. These unfavorable interactions are compensated by the favorable interactions with the Ca2+ ions and with main-chain amides. They are a trivial consequence of the tight fold of the Ca2+-binding loops.     

Common structural framework of the two Ca2+/Mg2+ binding loops of troponin C and other Ca2+ binding proteins

The refinement of the crystal structure of turkey skeletal muscle troponin C at 2.2-A resolution reveals that the two calcium binding loops that are occupied by Ca2+ ions adopt conformations very similar to those of the two homologous loops of parvalbumin and to that of loop III-IV of the intestinal calcium binding protein. This specific fold assures suitable spatial positioning of the Ca2+ ligands. It consists of two reverse turns, one located at each end of the loop, and four Asx turns (a cyclic hydrogen-bonded structure involving an oxygen of the side chain of residue n and the main-chain amide nitrogen of residue n + 2) whenever such a side chain coordinates to the metal ion. The fifth Ca2+ coordination position in both loops of troponin C is occupied by a water molecule that is within hydrogen-bonding distance of an aspartic acid, thus mediating indirect interaction between the cation and the negatively charged carboxylate. The same loop framework is conserved in the two Ca2+ binding loops of parvalbumin and loop III-IV of the intestinal Ca2+ binding protein in spite of the variability in the nature of the side chains at equivalent positions. The disposition of the Ca2+ and of its coordinating water molecule relative to the protein main chain is conserved in all these cases.     

Structural determinants for Ca2+ and phosphatidylinositol 4,5-bisphosphate binding by the C2A domain of rabphilin-3A

Rabphilin-3A is a neuronal C2 domain tandem containing protein involved in vesicle trafficking. Both its C2 domains (C2A and C2B) are able to bind phosphatidylinositol 4,5-bisphosphate, a key player in the neurotransmitter release process. The rabphilin-3A C2A domain has previously been shown to bind inositol-1,4,5-trisphosphate (IP3; phosphatidylinositol 4,5-bisphosphate headgroup) in a Ca2+-dependent manner with a relatively high affinity (50 microm) in the presence of saturating concentrations of Ca2+. Moreover, IP3 and Ca2+ binding to the C2A domain mutually enhance each other. Here we present the Ca2+-bound solution structure of the C2A domain. Structural comparison with the previously published Ca2+-free crystal structure revealed that Ca2+ binding induces a conformational change of Ca2+ binding loop 3 (CBL3). Our IP3 binding studies as well as our IP3-C2A docking model show the active involvement of CBL3 in IP3 binding, suggesting that the conformational change on CBL3 upon Ca2+ binding enables the interaction with IP3 and vice versa, in line with a target-activated messenger affinity mechanism. Our data provide detailed structural insight into the functional properties of the rabphilin-3A C2A domain and reveal for the first time the structural determinants of a target-activated messenger affinity mechanism.     

Solution structure and fluctuation of the Mg(2+)-bound form of calmodulin C-terminal domain

Calmodulin (CaM) is a Ca(2+)-binding protein that functions as a ubiquitous Ca(2+)-signaling molecule, through conformational changes from the "closed" apo conformation to the "open" Ca(2+)-bound conformation. Mg(2+) also binds to CaM and stabilizes its folded structure, but the NMR signals are broadened by slow conformational fluctuations. Using the E104D/E140D mutant, designed to decrease the signal broadening in the presence of Mg(2+) with minimal perturbations of the overall structure, the solution structure of the Mg(2+)-bound form of the CaM C-terminal domain was determined by multidimensional NMR spectroscopy. The Mg(2+)-induced conformational change mainly occurred in EF hand IV, while EF-hand III retained the apo structure. The helix G and helix H sides of the binding sequence undergo conformational changes needed for the Mg(2+) coordination, and thus the helices tilt slightly. The aromatic rings on helix H move to form a new cluster of aromatic rings in the hydrophobic core. Although helix G tilts slightly to the open orientation, the closed conformation is maintained. The fact that the Mg(2+)-induced conformational changes in EF-hand IV and the hydrophobic core are also seen upon Ca(2+) binding suggests that the Ca(2+)-induced conformational changes can be divided into two categories, those specific to Ca(2+) and those common to Ca(2+) and Mg(2+).     

Roles for the troponin tail domain in thin filament assembly and regulation. A deletional study of cardiac troponin T

Striated muscle contraction is regulated by Ca2+ binding to troponin, which has a globular domain and an elongated tail attributable to the NH2-terminal portion of the bovine cardiac troponin T (TnT) subunit. Truncation of the bovine cardiac troponin tail was investigated using recombinant TnT fragments and subunits TnI and TnC. Progressive truncation of the troponin tail caused progressively weaker binding of troponin-tropomyosin to actin and of troponin to actin-tropomyosin. A sharp drop-off in affinity occurred with NH2-terminal deletion of 119 rather than 94 residues. Deletion of 94 residues had no effect on Ca2+-activation of the myosin subfragment 1-thin filament MgATPase rate and did not eliminate cooperative effects of Ca2+ binding. Troponin tail peptide TnT1-153 strongly promoted tropomyosin binding to actin in the absence of TnI or TnC. The results show that the anchoring function of the troponin tail involves interactions with actin as well as with tropomyosin and has comparable importance in the presence or absence of Ca2+. Residues 95-153 are particularly important for anchoring, and residues 95-119 are crucial for function or local folding. Because striated muscle regulation involves switching among the conformational states of the thin filament, regulatory significance for the troponin tail may arise from its prominent contribution to the protein-protein interactions within these conformations.     

Distance distributions and anisotropy decays of troponin C and its complex with troponin I

We used frequency domain measurements of fluorescence resonance energy transfer to recover the distribution of distances between Met 25 and Cys 98 in rabbit skeletal troponin C. These residues were labeled with dansylaziridine as energy donor and 5-(iodoacetamido)eosin as acceptor and are located on the N- and C-terminal lobes of the two-domain protein, respectively. We developed a procedure to correct for the fraction of the sample that was incompletely labeled with the acceptor independent of chemical data. At pH 7.5 and in the presence of Mg2+, the mean distance was near 15 A with a half-width of the distribution of 15 A; when Mg2+ was replaced by Ca2+, the mean distance increased to 22 A with a decrease in the half-width by 4 A. Similar but less pronounced differences in the mean distance and half-width between samples containing Mg2+ and Ca2+ were also observed with troponin C complexed to troponin I. The results suggest that the conformation of troponin C is altered by Ca2+ binding to the Ca(2+)-specific sites and displacing bound Mg2+ at the Ca2+/Mg2+ sites. This alteration may play an important role in Ca2+ signaling in muscle. At pH 7.5, the anisotropy decays of the donor-labeled troponin C showed two components, with the long rotational correlation time (12 ns) reflecting the overall motion of the protein. When the pH was lowered from 7.5 to 5.2, the mean distribution distance of apotroponin C increased from 22 to 32 A and the half-width decreased by a factor of 2 from 13 to 7 A. The long correlation time of apotroponin C increased to 19 ns at the acidic pH. These results are discussed in terms of a model in which skeletal troponin C is a dimer at low pH and enable comparison of the solution conformation of the protein at neutral pH with a crystal structure obtained at pH 5.2. While the conformation of the monomeric unit of troponin C dimer at pH 5.2 is extended and consistent with the crystal structure, the conformation at neutral pH is likely more compact than the crystal structure predicts.     

Structural basis of the Ca2+ inhibitory mechanism of Drosophila Na+/Ca2+ exchanger CALX and its modification by alternative splicing

The Na(+)/Ca(2+) exchanger CALX promotes Ca(2+) efflux in Drosophila sensory neuronal cells to facilitate light-mediated Ca(2+) homeostasis. CALX activity is negatively regulated by specific Ca(2+) interaction within its two intracellular Ca(2+) regulatory domains CBD1 and CBD2, yet how the Ca(2+) binding is converted to molecular motion to operate the exchanger is unknown. Here, we report crystal structures of the entire Ca(2+) regulatory domain CBD12 from two alternative splicing isoforms, CALX 1.1 and 1.2, exhibiting distinct regulatory Ca(2+) dependency. The structures show an open V-shaped conformation with four Ca(2+) ions bound on the CBD domain interface, confirmed by LRET analysis. The structures together with Ca(2+)-binding analysis support that the Ca(2+) inhibition of CALX is achieved by interdomain conformational changes induced by Ca(2+) binding at CBD1. The conformational difference between the two isoforms also indicates that alternative splicing adjusts the interdomain orientation angle to modify the Ca(2+) regulatory property of the exchangers.     

Structural basis for Ca2+-regulated muscle relaxation at interaction sites of troponin with actin and tropomyosin

Troponin and tropomyosin on actin filaments constitute a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle through a series of conformational changes within the actin-based thin filament. Troponin consists of three subunits: an inhibitory subunit (TnI), a Ca2+-binding subunit (TnC), and a tropomyosin-binding subunit (TnT). Ca2+-binding to TnC is believed to weaken interactions between troponin and actin, and triggers a large conformational change of the troponin complex. However, the atomic details of the actin-binding sites of troponin have not been determined. Ternary troponin complexes have been reconstituted from recombinant chicken skeletal TnI, TnC, and TnT2 (the C-terminal region of TnT), among which only TnI was uniformly labelled with 15N and/or 13C. By applying NMR spectroscopy, the solution structures of a "mobile" actin-binding domain (approximately 6.1 kDa) in the troponin ternary complex (approximately 52 kDa) were determined. The mobile domain appears to tumble independently of the core domain of troponin. Ca2+-induced changes in the chemical shift and line shape suggested that its tumbling was more restricted at high Ca2+ concentrations. The atomic details of interactions between actin and the mobile domain of troponin were defined by docking the mobile domain into the cryo-electron microscopy (cryo-EM) density map of thin filament at low [Ca2+]. This allowed the determination of the 3D position of residue 133 of TnI, which has been an important landmark to incorporate the available information. This enabled unique docking of the entire globular head region of troponin into the thin filament cryo-EM map at a low Ca2+ concentration. The resultant atomic model suggests that troponin interacted electrostatically with actin and caused the shift of tropomyosin to achieve muscle relaxation. An important feature is that the coiled-coil region of troponin pushed tropomyosin at a low Ca2+ concentration. Moreover, the relationship between myosin and the mobile domain on actin filaments suggests that the latter works as a fail-safe latch.     

Thermodynamic study of domain organization in troponin C and calmodulin

Intramolecular melting of troponin C, calmodulin and their proteolytic fragments has been studied microcalorimetrically at various concentrations of monovalent and divalent ions. It is shown by thermodynamic analysis of the experimentally determined excess heat capacity function that the four calcium-binding domains in these two related proteins are not integrated into a single co-operative system, as would be the case if they formed a common hydrophobic core in the molecule, but still interact with each other in a very specific way. There is a positive interaction between domains I and II, which is so strong that they actually form a single co-operative block. The interaction between domains III and IV is positive also, although much less pronounced, while the interaction between the pairs of domains (I and II) and (III and IV) is negative, as if they repel each other. The structure of the co-operative block of domains I and II at room temperature does not depend noticeably on the ionic conditions, which influence its stability to a small extent only. The same applies to domain IV of calmodulin, but in troponin C this domain is unstable in the absence of divalent ions, in solutions of low ionic strength. In both proteins, the least stable is domain III, which forms a compact ordered structure at room temperature only in the presence of Ca2+. In troponin C, calcium ions can be replaced by magnesium ions, although the compact structure of domain III formed by these two ions does not seem to be quite identical. Thus, at conditions close to physiological, with regard to temperature and ionic strength, the removal of free Ca2+ from the solution induces in both proteins a reversible transition of domain III to the non-compact disordered state. This dramatic Ca2+-induced change in the domain III conformation in troponin C and calmodulin might play a key role in the functioning of these proteins as a Ca2+-controlled switch in the molecular mechanisms of living systems.     

Structures of the MthK RCK domain and the effect of Ca2+ on gating ring stability

MthK is a Ca2+-gated K+ channel from Methanobacterium autotrophicum. The crystal structure of the MthK channel in a Ca2+-bound open state was previously determined at 3.3 A and revealed an octameric gating ring composed of eight intracellular ligand-binding RCK (regulate the conductance of K+) domains. It was suggested that Ca2+ binding regulates the gating ring conformation, which in turn leads to the opening and closing of the channel. However, at 3.3 AA resolution, the molecular details of the structure are not well defined, and many of the conclusions drawn from that structure were hypothetical. Here we have presented high resolution structures of the MthK RCK domain with and without Ca2+ bound from three different crystals. These structures revealed a dimeric architecture of the RCK domain and allowed us to visualize the Ca2+ binding and protein-protein contacts at atomic detail. The dimerization of RCK domains is also conserved in other RCK-regulated K+ channels and transporters, suggesting that the RCK dimer serves as a basic unit in the gating ring assembly. A comparison of these dimer structures confirmed that the dimer interface is indeed flexible as suggested previously. However, the conformational change at the flexible interface is of an extent smaller than the previously hypothesized gating ring movement, and a reconstruction of these dimers into octamers by applying protein-protein contacts at the fixed interface did not generate enclosed gating rings. This indicated that there is a high probability that the previously defined fixed interface may not be fixed during channel gating. In addition to the structural studies, we have also carried out biochemical analyses and have shown that near physiological pH, isolated RCK domains form a stable octamer in solution, supporting the notion that the formation of octameric gating ring is a functionally relevant event in MthK gating. Additionally, our stability studies indicated that Ca2+ binding stabilizes the RCK domains in this octameric state.     

Dynamics of Ca2+-saturated calmodulin D129N mutant studied by multiple molecular dynamics simulations

Fifteen independent 1-nsec MD simulations of fully solvated Ca(2+) saturated calmodulin (CaM) mutant D129N were performed from different initial conditions to provide a sufficient statistical basis to gauge the significance of observed dynamical properties. In all MD simulations the four Ca(2+) ions remained in their binding sites, and retained a single water ligand as observed in the crystal structure. The coordination of Ca(2+) ions in EF-hands I, II, and III was sevenfold. In EF-hand IV, which was perturbed by the mutation of a highly conserved Asp129, an anomalous eightfold Ca(2+) coordination was observed. The Ca(2+) binding loop in EF-hand II was observed to dynamically sample conformations related to the Ca(2+)-free form. Repeated MD simulations implicate two well-defined conformations of Ca(2+) binding loop II, whereas similar effect was not observed for loops I, III, and IV. In 8 out of 15 MD simulations Ca(2+) binding loop II adopted an alternative conformation in which the Thr62 >C=O group was displaced from the Ca(2+) coordination by a water molecule, resulting in the Ca(2+) ion ligated by two water molecules. The alternative conformation of the Ca(2+) binding loop II appears related to the "closed" state involved in conformational exchange previously detected by NMR in the N-terminal domain fragment of CaM and the C-terminal domain fragment of the mutant E140Q. MD simulations suggest that conformations involved in microsecond exchange exist partially preformed on the nanosecond time scale.     

An interaction-based analysis of calcium-induced conformational changes in Ca2+ sensor proteins.

Calcium sensor proteins translate transient increases in intracellular calcium levels into metabolic or mechanical responses, by undergoing dramatic conformational changes upon Ca2+ binding. A detailed analysis of the calcium binding-induced conformational changes in the representative calcium sensors calmodulin (CaM) and troponin C was performed to obtain insights into the underlying molecular basis for their response to the binding of calcium. Distance difference matrices, analysis of interresidue contacts, comparisons of interhelical angles, and inspection of structures using molecular graphics were used to make unbiased comparisons of the various structures. The calcium-induced conformational changes in these proteins are dominated by reorganization of the packing of the four helices within each domain. Comparison of the closed and open conformations confirms that calcium binding causes opening within each of the EF-hands. A secondary analysis of the conformation of the C-terminal domain of CaM (CaM-C) clearly shows that CaM-C occupies a closed conformation in the absence of calcium that is distinct from the semi-open conformation observed in the C-terminal EF-hand domains of myosin light chains. These studies provide insight into the structural basis for these changes and into the differential response to calcium binding of various members of the EF-hand calcium-binding protein family. Factors contributing to the stability of the Ca2+-loaded open conformation are discussed, including a new hypothesis that critical hydrophobic interactions stabilize the open conformation in Ca2+ sensors, but are absent in "non-sensor" proteins that remain closed upon Ca2+ binding. A role for methionine residues in stabilizing the open conformation is also proposed.     

Structural basis for Ca(2+)-induced activation of human PAD4

Peptidylarginine deiminase 4 (PAD4) is a Ca(2+)-dependent enzyme that catalyzes the conversion of protein arginine residues to citrulline. Its gene is a susceptibility locus for rheumatoid arthritis. Here we present the crystal structure of Ca(2+)-free wild-type PAD4, which shows that the polypeptide chain adopts an elongated fold in which the N-terminal domain forms two immunoglobulin-like subdomains, and the C-terminal domain forms an alpha/beta propeller structure. Five Ca(2+)-binding sites, none of which adopt an EF-hand motif, were identified in the structure of a Ca(2+)-bound inactive mutant with and without bound substrate. These structural data indicate that Ca(2+) binding induces conformational changes that generate the active site cleft. Our findings identify a novel mechanism for enzyme activation by Ca(2+) ions, and are important for understanding the mechanism of protein citrullination and for developing PAD-inhibiting drugs for the treatment of rheumatoid arthritis.