Consequences of Sequential Ca2+ Occupancy for the Structure and Dynamics of CalbindinD9K: Computational Simulations and Comparison to Experimental Determinations in Solution

Abstract

Molecular dynamics (MD) simulations of the structures of calbindin_D9K (CAB) with different occupancies of the two Ca²⁺ binding sites were carried out to gain insight into structural and energetic consequences of sequential Ca²⁺ binding. The aim of the study is to identify effects of Ca-binding site occupancy that relate to the properties and functions of Ca-binding proteins containing EF-hand motifs. Two different models of solvation were employed, one defined by a linear, distance dependent dielectric permittivity (ε = r) and inclusion only of the 36 crystallographically observed water molecules, and the other with the protein immersed in a 9Å shell of explicit waters and ε = 1. Experimental results from x-ray crystallography, and insights from NMR and from measurements of hydrogen exchange rates in these systems served as tests and guides for assessing the quality, validity and mechanistic interpretation of the results from the computational study. The results of the MD simulations agree very well with earlier experimental observations that the structure of calbindin_D9k is rather insensitive to removal of Ca²⁺, and indicate that this insensitivity is not dependent on the order in which the ions are removed. The calculated values of the electrostatic potentials at the Ca²⁺ binding sites are very similar, in agreement with the small differences in the measured microscopic binding constants. Details of the dynamic mechanisms of molecular flexibility revealed by the MD simulations are also in good agreement with experimental findings, including the local properties identified from comparisons of hydrogen exchange rates in various parts of the structures of sequentially occupied forms of CAB. Estimation of the changes in configurational entropy from the rms fluctuations in the structures of CAB at various levels of Ca²⁺ occupancy in the EF-hands, supports earlier suggestions relating the dynamic properties of the protein to the observed cooperativity in the binding of Ca²⁺. The computational approaches and the results of the MD simulations are evaluated in relation to the study of effects of Ca²⁺ occupancy in calmodulin and troponin C where ion binding determines function and is known to trigger significant changes in structural and dynamic properties.     

Heterogeneous behavior of metalloproteins toward metal ion binding and selectivity: insights from molecular dynamics studies

About one-third of the existing proteins require metal ions as cofactors for their catalytic activities and structural complexities. While many of them bind only to a specific metal, others bind to multiple (different) metal ions. However, the exact mechanism of their metal preference has not been deduced to clarity. In this study, we used molecular dynamics (MD) simulations to investigate whether a cognate metal (bound to the structure) can be replaced with other similar metal ions. We have chosen seven different proteins (phospholipase A₂, sucrose phosphatase, pyrazinamidase, cysteine dioxygenase (CDO), plastocyanin, monoclonal anti-CD4 antibody Q425, and synaptotagmin 1 C₂B domain) bound to seven different divalent metal ions (Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Ba²⁺, and Sr²⁺, respectively). In total, 49 MD simulations each of 50 ns were performed and each trajectory was analyzed independently. Results demonstrate that in some cases, cognate metal ions can be exchanged with similar metal ions. On the contrary, some proteins show binding affinity specifically to their cognate metal ions. Surprisingly, two proteins CDO and plastocyanin which are known to bind Fe²⁺ and Cu²⁺, respectively, do not exhibit binding affinity to any metal ion. Furthermore, the study reveals that in some cases, the active site topology remains rigid even without cognate metals, whereas, some require them for their active site stability. Thus, it will be interesting to experimentally verify the accuracy of these observations obtained computationally. Moreover, the study can help in designing novel active sites for proteins to sequester metal ions particularly of toxic nature.     

Simulation of the effect of an external GHz electric field on the potential energy profile of Ca2+ ions in the selectivity filter of the CaVAb channel

Ca_V channels are transmembrane proteins that mediate and regulate ion fluxes across cell membranes, and they are activated in response to action potentials to allow Ca²⁺ influx. Since ion channels are composed of charge or polar groups, an external alternating electric field may affect the ion‐selective membrane transport and the performance of the channel. In this article, we have investigated the effect of an external GHz electric field on the dynamics of calcium ions in the selectivity filter of the Ca_VAb channel. Molecular dynamics (MD) simulations and the potential of mean force (PMF) calculations were carried out, via the umbrella sampling method, to determine the free energy profile of Ca²⁺ ions in the Ca_VAb channels in presence and absence of an external field. Exposing Ca_VAb channel to 1, 2, 3, 4, and 5 GHz electric fields increases the depth of the potential energy well and this may result in an increase in the affinity and strength of Ca²⁺ ions to binding sites in the selectivity filter the channel. This increase of strength of Ca²⁺ ions binding in the selectivity filter may interrupt the mechanism of Ca²⁺ ion conduction, and leads to a reduction of Ca²⁺ ion permeation through the Ca_VAb channel.     

Coupling of Calcium and Substrate Binding through Loop Alignment in the Outer-Membrane Transporter BtuB

In Gram-negative bacteria, TonB-dependent outer-membrane transporters bind large, scarce organometallic substrates with high affinity preceding active transport. The cobalamin transporter BtuB requires the additional binding of two Ca²⁺ ions before substrate binding can occur, but the underlying molecular mechanism is unknown. Using the crystallographic structures available for different bound states of BtuB, we have carried out extended molecular dynamics simulations of multiple functional states of BtuB to address the role of Ca²⁺ in substrate recruitment. We find that Ca²⁺ binding both stabilizes and repositions key extracellular loops of BtuB, optimizing interactions with the substrate. Interestingly, replacement by Mg²⁺ abolishes this effect, in accordance with experiments. Using a set of new force-field parameters developed for cyanocobalamin, we also simulated the substrate-bound form of BtuB, where we observed interactions not seen in the crystal structure between the substrate and loops previously found to be important for binding and transport. Based on our results, we suggest that the large size of cobalamin compared to other TonB-dependent transporter substrates explains the requirement of Ca²⁺ binding for high-affinity substrate recruitment in BtuB.     

Predicting Ca2+‐binding sites using refined carbon clusters

Identifying Ca²⁺‐binding sites in proteins is the first step toward understanding the molecular basis of diseases related to Ca²⁺‐binding proteins. Currently, these sites are identified in structures either through X‐ray crystallography or NMR analysis. However, Ca²⁺‐binding sites are not always visible in X‐ray structures due to flexibility in the binding region or low occupancy in a Ca²⁺‐binding site. Similarly, both Ca²⁺ and its ligand oxygens are not directly observed in NMR structures. To improve our ability to predict Ca²⁺‐binding sites in both X‐ray and NMR structures, we report a new graph theory algorithm (MUG^C) to predict Ca²⁺‐binding sites. Using carbon atoms covalently bonded to the chelating oxygen atoms, and without explicit reference to side‐chain oxygen ligand co‐ordinates, MUG^C is able to achieve 94% sensitivity with 76% selectivity on a dataset of X‐ray structures composed of 43 Ca²⁺‐binding proteins. Additionally, prediction of Ca²⁺‐binding sites in NMR structures was obtained by MUG^C using a different set of parameters, which were determined by the analysis of both Ca²⁺‐constrained and unconstrained Ca²⁺‐loaded structures derived from NMR data. MUG^C identified 20 of 21 Ca²⁺‐binding sites in NMR structures inferred without the use of Ca²⁺ constraints. MUG^C predictions are also highly selective for Ca²⁺‐binding sites as analyses of binding sites for Mg²⁺, Zn²⁺, and Pb²⁺ were not identified as Ca²⁺‐binding sites. These results indicate that the geometric arrangement of the second‐shell carbon cluster is sufficient not only for accurate identification of Ca²⁺‐binding sites in NMR and X‐ray structures but also for selective differentiation between Ca²⁺ and other relevant divalent cations. © Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Towards understanding the unbound state of drug compounds: Implications for the intramolecular reorganization energy upon binding

There has been an explosion of structural information for pharmaceutical compounds bound to biological targets, but the conformations and dynamics of compounds free in solution are poorly characterized, if at all. Yet, knowledge of the unbound state is essential to understand the fundamentals of molecular recognition, including the much debated conformational intramolecular reorganization energy of a compound upon binding (ΔE_Reorg). Also, dependable observation of the unbound compounds is important for ligand-based drug discovery, e.g. with pharmacophore modelling. Here, these questions are addressed with long (⩾0.5 μs) state-of-the-art molecular dynamics (MD) simulations of 26 compounds (including 7 approved drugs) unbound in explicit solvent. These compounds were selected to be chemically diverse, with a range of flexibility, and good quality bioactive X-ray structures. The MD-simulated free compounds are compared to their bioactive structure and conformers generated with ad hoc sampling in vacuo or with implicit generalized Born (GB) aqueous solvation models. The GB conformational models clearly depart from those obtained in explicit solvent, and suffer from conformational collapse almost as severe as in vacuo. Thus, the global energy minima in vacuo or with GB are not suitable representations of the unbound state, which can instead be extensively sampled by MD simulations. Many, but not all, MD-simulated compounds displayed some structural similarity to their bioactive structure, supporting the notion of conformational pre-organization for binding. The ligand–protein complexes were also simulated in explicit solvent, to estimate ΔE_Reorg as an enthalpic difference ΔH_Reorg between the intramolecular energies in the bound and unbound states. This fresh approach yielded ΔH_Reorg values ⩽ 6 kcal/mol for 18 out of 26 compounds. For three particularly polar compounds 15 ⩽ ΔH_Reorg ⩽ 20 kcal/mol, supporting the notion that ΔH_Reorg can be substantial. Those large ΔH_Reorg values correspond to a redistribution of electrostatic interactions upon binding. Overall, the study illustrates how MD simulations offer a promising avenue to characterize the unbound state of medicinal compounds.     

Structure and Calcium Binding Properties of a Neuronal Calcium-Myristoyl Switch Protein,Visinin-Like Protein 3

Visinin-like protein 3 (VILIP-3) belongs to a family of Ca²⁺-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca²⁺ binding, characterize Ca²⁺-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca²⁺ bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (K_D = 0.52 μM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca²⁺-bound VILIP-3, whereas Ca²⁺-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues (E26, Y64, V68), which are distinct from myristate contacts seen in other Ca²⁺-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C₁₄ methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins (recoverin, NCS-1, and GCAP1). We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity.     

Structural and energetic parameters of Ca2+ binding to peptides and proteins

The characteristics of Ca²⁺-binding sites and of the structural reorganization induced by Ca²⁺-binding in storage proteins and ion carriers are being studied as models for molecular mechanisms in Ca²⁺ channels and in Ca²⁺-dependent modulatory proteins. A first step in the study was the development of energy parameters for Ca²⁺ compatible with those in the CHARMM package of computer simulation software. Such parameters were obtained from an analytical fit to the potential surface for [(Ca)(OCH₂)₄]²⁺ calculated with an ab initio molecular orbital method. The resulting parametrization was tested for the hexapeptide cyclo-(Pro-Gly)₃, and a 75 residue long calcium binding protein from bovine intestine (ICaBP). The geometrical parameters calculated for the hexapeptide and its 2:1 complex with Ca²⁺ were in good agreement with experimental data from crystallography and nmr. Similarly, the structure of ICaBP optimized with CHARMM using the new Ca²⁺ parameters showed good agreement with the x-ray structure both in the local structures of the calcium-binding sites and in the overall shape of the protein.     

Microsecond Molecular Dynamics Simulations of Mg2+- and K+- Bound E1 Intermediate States of the Calcium Pump

We have performed microsecond molecular dynamics (MD) simulations to characterize the structural dynamics of cation-bound E1 intermediate states of the calcium pump (sarcoendoplasmic reticulum Ca²⁺-ATPase, SERCA) in atomic detail, including a lipid bilayer with aqueous solution on both sides. X-ray crystallography with 40 mM Mg²⁺ in the absence of Ca²⁺ has shown that SERCA adopts an E1 structure with transmembrane Ca²⁺-binding sites I and II exposed to the cytosol, stabilized by a single Mg²⁺ bound to a hybrid binding site I′. This Mg²⁺-bound E1 intermediate state, designated E1•Mg²⁺, is proposed to constitute a functional SERCA intermediate that catalyzes the transition from E2 to E1•2Ca²⁺ by facilitating H⁺/Ca²⁺ exchange. To test this hypothesis, we performed two independent MD simulations based on the E1•Mg²⁺ crystal structure, starting in the presence or absence of initially-bound Mg²⁺. Both simulations were performed for 1 µs in a solution containing 100 mM K⁺ and 5 mM Mg²⁺ in the absence of Ca²⁺, mimicking muscle cytosol during relaxation. In the presence of initially-bound Mg²⁺, SERCA site I′ maintained Mg²⁺ binding during the entire MD trajectory, and the cytosolic headpiece maintained a semi-open structure. In the absence of initially-bound Mg²⁺, two K⁺ ions rapidly bound to sites I and I′ and stayed loosely bound during most of the simulation, while the cytosolic headpiece shifted gradually to a more open structure. Thus MD simulations predict that both E1•Mg²⁺ and E•2K⁺ intermediate states of SERCA are populated in solution in the absence of Ca²⁺, with the more open 2K⁺-bound state being more abundant at physiological ion concentrations. We propose that the E1•2K⁺ state acts as a functional intermediate that facilitates the E2 to E1•2Ca²⁺ transition through two mechanisms: by pre-organizing transport sites for Ca²⁺ binding, and by partially opening the cytosolic headpiece prior to Ca²⁺ activation of nucleotide binding.     

Combining NMR ensembles and molecular dynamics simulations provides more realistic models of protein structures in solution and leads to better chemical shift prediction

While chemical shifts are invaluable for obtaining structural information from proteins, they also offer one of the rare ways to obtain information about protein dynamics. A necessary tool in transforming chemical shifts into structural and dynamic information is chemical shift prediction. In our previous work we developed a method for 4D prediction of protein ¹H chemical shifts in which molecular motions, the 4th dimension, were modeled using molecular dynamics (MD) simulations. Although the approach clearly improved the prediction, the X-ray structures and single NMR conformers used in the model cannot be considered fully realistic models of protein in solution. In this work, NMR ensembles (NMRE) were used to expand the conformational space of proteins (e.g. side chains, flexible loops, termini), followed by MD simulations for each conformer to map the local fluctuations. Compared with the non-dynamic model, the NMRE+MD model gave 6–17% lower root-mean-square (RMS) errors for different backbone nuclei. The improved prediction indicates that NMR ensembles with MD simulations can be used to obtain a more realistic picture of protein structures in solutions and moreover underlines the importance of short and long time-scale dynamics for the prediction. The RMS errors of the NMRE+MD model were 0.24, 0.43, 0.98, 1.03, 1.16 and 2.39 ppm for ¹Hα, ¹HN, ¹³Cα, ¹³Cβ, ¹³CO and backbone ¹⁵N chemical shifts, respectively. The model is implemented in the prediction program 4DSPOT, available at .     

Atomic-Level Mechanisms for Phospholamban Regulation of the Calcium Pump

We performed protein pKa calculations and molecular dynamics (MD) simulations of the calcium pump (sarcoplasmic reticulum Ca²⁺-ATPase (SERCA)) in complex with phospholamban (PLB). X-ray crystallography studies have suggested that PLB locks SERCA in a low-Ca²⁺-affinity E2 state that is incompatible with metal-ion binding, thereby blocking the conversion toward a high-Ca²⁺-affinity E1 state. Estimation of pKa values of the acidic residues in the transport sites indicates that at normal intracellular pH (7.1–7.2), PLB-bound SERCA populates an E1 state that is deprotonated at residues E309 and D800 yet protonated at residue E771. We performed three independent microsecond-long MD simulations to evaluate the structural dynamics of SERCA-PLB in a solution containing 100 mM K⁺ and 3 mM Mg²⁺. Principal component analysis showed that PLB-bound SERCA lies exclusively along the structural ensemble of the E1 state. We found that the transport sites of PLB-bound SERCA are completely exposed to the cytosol and that K⁺ ions bind transiently (≤5 ns) and nonspecifically (nine different positions) to the two transport sites, with a total occupancy time of K⁺ in the transport sites of 80%. We propose that PLB binding to SERCA populates a novel (to our knowledge) E1 intermediate, E1⋅H⁺₇₇₁. This intermediate serves as a kinetic trap that controls headpiece dynamics and depresses the structural transitions necessary for Ca²⁺-dependent activation of SERCA. We conclude that PLB-mediated regulation of SERCA activity in the heart results from biochemical and structural transitions that occur primarily in the E1 state of the pump.     

Auxiliary Ca2+ binding sites can influence the structure of CIB1

Recent X-ray crystal structures and solution NMR spectroscopy data for calcium- and integrin-binding protein 1 (CIB1) have all revealed a common EF-hand domain structure for the protein. However, the orientation of the two protein domains, the oligomerization state, and the conformations of the N- and C-terminal extensions differ among the structures. In this study, we examine whether the binding of glutathione or auxiliary Ca²⁺ ions as observed in the crystal structures, occur in solution, and whether these interactions can influence the structure or dimerization of CIB1. In addition, we test the potential phosphatase activity of CIB1, which was hypothesized based on the glutathione binding site geometry observed in one of the crystal structures of the protein. Biophysical and biochemical experiments failed to detect glutathione binding, protein dimerization, or phosphatase activity for CIB1 under several solution conditions. However, our data identify low affinity (K_d, 10⁻²M) Ca²⁺ binding events that influence the structures of the N- and C-terminal extensions of CIB1 under high (300 mM) Ca²⁺ crystallization conditions. In addition to providing a rationale for differences amongst the various solution and crystal structures of CIB1, our results show that the impact of low affinity Ca²⁺ binding events should be considered when analyzing and interpreting protein crystallographic structures determined in the presence of very high Ca²⁺ concentrations.     

Structural effects of divalent calcium cations on the α7 nicotinic acetylcholine receptor: A molecular dynamics simulation study

The α7 subtype of neuronal nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein that is vital to various neurological functions, including modulation of neurotransmitter release. A relatively high concentration of extracellular Ca²⁺ in the neuronal environment is likely to exert substantial structural and functional influence on nAChRs, which may affect their interactions with agonists and antagonists. In this work, we employed atomistic molecular dynamics (MD) simulations to examine the effects of elevated Ca²⁺ on the structure and dynamics of α7 nAChR embedded in a model phospholipid bilayer. Our results suggest that the presence of Ca²⁺ in the α7 nAChR environment results in closure of loop C-in the extracellular ligand-binding domain, a motion normally associated with agonist binding and receptor activation. Elevated Ca²⁺ also alters the conformation of key regions of the receptor, including the inter-helical loops, pore-lining helices and the “gate” residues, and causes partial channel opening in the absence of an agonist, leading to an attendant reduction in the free energy of Ca²⁺ permeation through the pore as elucidated by umbrella sampling simulations. Overall, the structural and permeability changes in α7 nAChR suggest that elevated Ca²⁺ induces a partially activated receptor state that is distinct from both the resting and the agonist-activated states. These results are consistent with the notion that divalent ions can serve as a potentiator of nAChRs, resulting in a higher rate of receptor activation (and subsequent desensitization) in the presence of agonists, with possible implications for diseases involving calcium dysregulation.     

Comparison of the effects of calcium and magnesium on the conformation of tetra cycline in Me2SO solution

The effects of anhydrous Ca(NO₃)₂ on the ¹³C nuclear magnetic resonance (nmr) and circular dichroism spectra of tetracycline in Me₂SO-d₆ solution have been investigated in order to make a comparison with the results of a previous study in which the effects of Mg(NO₃)₂ were determined under the same conditions. The results of experiments described in this article provide strong evidence that Ca²⁺ and Mg²⁺ bind tetracycline at the same sites in Me₂SO and that both ions induce the same change in molecular conformation of tetracycline upon binding. The Ca²⁺ complex, in contrast to the Mg²⁺ complex, has a lifetime that is short on the nmr time scale.     

Investigating dual Ca2+ modulation of the ryanodine receptor 1 by molecular dynamics simulation

The ryanodine receptors (RyR) are essential to calcium signaling in striated muscles. A deep understanding of the complex Ca²⁺-activation/inhibition mechanism of RyRs requires detailed structural and dynamic information for RyRs in different functional states (eg, with Ca²⁺ bound to activating or inhibitory sites). Recently, high-resolution structures of the RyR isoform 1 (RyR1) were solved by cryo-electron microscopy, revealing the location of a Ca²⁺ binding site for activation. Toward elucidating the Ca²⁺-modulation mechanism of RyR1, we performed extensive molecular dynamics simulation of the core RyR1 structure in the presence and absence of activating and solvent Ca²⁺ (total simulation time is >5 μs). In the presence of solvent Ca²⁺, Ca²⁺ binding to the activating site enhanced dynamics of RyR1 with higher inter-subunit flexibility, asymmetric inter-subunit motions, outward domain motions and partial pore dilation, which may prime RyR1 for subsequent channel opening. In contrast, the solvent Ca²⁺ alone reduced dynamics of RyR1 and led to inward domain motions and pore contraction, which may cause inhibition. Combining our simulation with the map of disease mutation sites in RyR1, we constructed a wiring diagram of key domains coupled via specific hydrogen bonds involving the mutation sites, some of which were modulated by Ca²⁺ binding. The structural and dynamic information gained from this study will inform future mutational and functional studies of RyR1 activation and inhibition by Ca²⁺.     

Improved model of hydrated calcium ion for molecular dynamics simulations using classical biomolecular force fields

Calcium ions (Ca²⁺) play key roles in various fundamental biological processes such as cell signaling and brain function. Molecular dynamics (MD) simulations have been used to study such interactions, however, the accuracy of the Ca²⁺ models provided by the standard MD force fields has not been rigorously tested. Here, we assess the performance of the Ca²⁺ models from the most popular classical force fields AMBER and CHARMM by computing the osmotic pressure of model compounds and the free energy of DNA–DNA interactions. In the simulations performed using the two standard models, Ca²⁺ ions are seen to form artificial clusters with chloride, acetate, and phosphate species; the osmotic pressure of CaAc₂ and CaCl₂ solutions is a small fraction of the experimental values for both force fields. Using the standard parameterization of Ca²⁺ ions in the simulations of Ca²⁺‐mediated DNA–DNA interactions leads to qualitatively wrong outcomes: both AMBER and CHARMM simulations suggest strong inter‐DNA attraction whereas, in experiment, DNA molecules repel one another. The artificial attraction of Ca²⁺ to DNA phosphate is strong enough to affect the direction of the electric field‐driven translocation of DNA through a solid‐state nanopore. To address these shortcomings of the standard Ca²⁺ model, we introduce a custom model of a hydrated Ca²⁺ ion and show that using our model brings the results of the above MD simulations in quantitative agreement with experiment. Our improved model of Ca²⁺ can be readily applied to MD simulations of various biomolecular systems, including nucleic acids, proteins and lipid bilayer membranes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 752–763, 2016.     

Molecular dynamic simulations study of the effect of salt valency on structure and thermodynamic solvation behaviour of anionic polyacrylate PAA in aqueous solutions

The effect of salt concentration and valency on intermolecular structure and solvation thermodynamic properties of aqueous solution containing polyacrylicacid (PAA) chains and multi-valent salts calcium chloride (CaCl₂) and aluminium chloride (AlCl₃) as a function of charge density was investigated using atomistic molecular dynamic simulations with explicit solvent. Salt-free solution favours the self-association of uncharged (acidic form) PAA chains facilitated by inter-chain hydrogen bonds. The ionised (charged) PAA chains are not associated in salt-free aqueous solutions and undergo self-association in the salt solutions due to bridging effect induced by condensed salt ions in agreement with scattering investigations available in literature. The collapse behaviour of PAA in presence of CaCl₂ and re-expansion behaviour of PAA chains in case of AlCl₃ salt solutions are observed. The rigidity of PAA chains decrease with increase in salt concentration, in agreement with experimental results available in literature. The trivalent salt favours relatively the greater extent of shrinking of PAA chains as well as inter-chain interactions as compared to divalent salts as evident from radius-of-gyration, H-bond and pair-wise solvation enthalpy data. The conformation and hydration behaviour of the acid form of PAA chains are not significantly altered by added salt ions. The hydration behaviour of ionised PAA chains is significantly reduced by added salts due to screening effect of the condensed salt ions. The pair correlation functions of solutions species such as Ca²⁺, Al³⁺, Na⁺ and Cl^? with respect to PAA oxygen show the greater affinity of PAA units with the higher valency Al³⁺ ions over Ca²⁺ and Na⁺ in solution. With increase in concentration of AlCl₃ and CaCl₂ salts, a decrease in effective charge density of ionised PAA chains is observed from the existence of unfavourable PAA–water, PAA–Ca²⁺ and PAA–Al³⁺ interactions.     

Residues important for K+ ion transport in the K+-dependent Na+-Ca2+ exchanger (NCKX2)

K⁺-dependent Na⁺-Ca²⁺ exchangers (NCKXs) play an important role in Ca²⁺ homeostasis in many tissues. NCKX proteins are bi-directional plasma membrane Ca²⁺-transporters which utilize the inward Na⁺ and outward K⁺ gradients to move Ca²⁺ ions into and out of the cytosol (4Na⁺:1Ca²⁺ + 1 K⁺). In this study, we carried out scanning mutagenesis of all the residues of the highly conserved α-1 and α-2 repeats of NCKX2 to identify residues important for K⁺ transport. These structural elements are thought to be critical for cation transport. Using fluorescent intracellular Ca²⁺-indicating dyes, we measured the K⁺ dependence of transport carried out by wildtype or mutant NCKX2 proteins expressed in HEK293 cells and analyzed shifts in the apparent binding affinity (K_m) of mutant proteins in comparison with the wildtype exchanger. Of the 93 residue substitutions tested, 34 were found to show a significant shift in the external K⁺ ion dependence of which 16 showed an increased affinity to K⁺ ions and 18 showed a decreased affinity and hence are believed to be important for K⁺ ion binding and transport. We also identified 8 residue substitutions that resulted in a partial loss of K⁺ dependence. Our biochemical data provide strong support for the cation binding sites identified in a homology model of NCKX2 based on crystal structures reported for distantly related archaeal Na⁺-Ca²⁺ exchanger NCX_Mj. In addition, we compare our results here with our previous studies that report on residues important for Ca²⁺ and Na⁺ binding. Supported by CIHR MOP-81327.     

Comparison of protein solution structures refined by molecular dynamics simulation in vacuum,with a generalized Born model,and with explicit water

The inclusion of explicit solvent water in molecular dynamics refinement of NMR structures ought to provide the most physically meaningful accounting for the effects of solvent on structure, but is computationally expensive. In order to evaluate the validity of commonly used vacuum refinements and of recently developed continuum solvent model methods, we have used three different methods to refine a set of NMR solution structures of a medium sized protein, Escherichia coliglutaredoxin 2, from starting structures calculated using the program DYANA. The three different refinement protocols used molecular dynamics simulated annealing with the program AMBER in vacuum (VAC), including a generalized Born (GB) solvent model, and a full calculation including explicit solvent water (WAT). The structures obtained using the three methods of refinements were very similar, a reflection of their generally well-determined nature. However, the structures refined with the generalized Born model were more similar to those from explicit water refinement than those refined in vacuum. Significant improvement was seen in the percentage of backbone dihedral angles in the most favored regions of , space and in hydrogen bond pattern for structures refined with the GB and WAT models, compared with the structures refined in vacuum. The explicit water calculation took an average of 200 h of CPU time per structure on an SGI cluster, compared to 15–90 h for the GB calculation (depending on the parameters used) and 2 h for the vacuum calculation. The generalized Born solvent model proved to be an excellent compromise between the vacuum and explicit water refinements, giving results comparable to those of the explicit water calculation. Some improvement for and angle distribution and hydrogen bond pattern can also be achieved by energy minimizing the vacuum structures with the GB model, which takes a much shorter time than MD simulations with the GB model.     

Binding of calcium is sensed structurally and dynamically throughout the second calcium‐binding domain of the sodium/calcium exchanger

We report the effects of Ca²⁺ binding on the backbone relaxation rates and chemical shifts of the AD and BD splice variants of the second Ca²⁺‐binding domain (CBD2) of the sodium–calcium exchanger. Analysis of the Ca²⁺‐induced chemical shifts perturbations yields similar K_D values of 16–24 μM for the two CBD2‐AD Ca²⁺‐binding sites, and significant effects are observed up to 20 Å away. To quantify the Ca²⁺‐induced chemical shift changes, we performed a comparative analysis of eight Ca²⁺‐binding proteins that revealed large differences between different protein folds. The CBD2 ¹⁵N relaxation data show the CBD2‐AD Ca²⁺ coordinating loops to be more rigid in the Ca²⁺‐bound state as well as to affect the FG‐loop located at the opposite site of the domain. The equivalent loops of the CBD2‐BD splice variant do not bind Ca²⁺ and are much more dynamic relative to both the Ca²⁺‐bound and apo forms of CBD2‐AD. A more structured FG‐loop in CBD2‐BD is suggested by increased S² order parameter values relative to both forms of CBD2‐AD. The chemical shift and relaxation data together indicate that, in spite of the small structural changes, the Ca²⁺‐binding event is felt throughout the molecule. The data suggest that the FG‐loop plays an important role in connecting the Ca²⁺‐binding event with the other cytosolic domains of the NCX, in line with in vivo and in vitro biochemical data as well as modeling results that connect the CBD2 FG‐loop with the first Ca²⁺‐binding domain of NCX. Proteins 2010. © 2010 Wiley‐Liss, Inc.