Conformation Of Calmodulin Research Articles

Measurement and Differentiation of Ligand-Induced Calmodulin Conformations by Dual Polarization Interferometry

In early drug discovery, knowledge about ligand-induced conformational changes and their influence on protein activity greatly aids the identification of lead candidates for medicinal chemistry efforts. Efficiently acquiring such information remains a challenge in the initial stages of lead finding. Here we investigated the application of dual polarization interferometry (DPI) as a method for the real-time characterization of low molecular weight (LMW) ligands that induce conformational changes. As a model system we chose calmodulin (CaM), which undergoes large and distinct structural rearrangements in response to calcium ion and small molecule inhibitors such as trifluoperazine (TFP). We measured concentration-dependent mass, thickness, and density responses of an immobilized CaM protein layer, which correlated directly with binding and conformational events. Calcium ion binding to CaM induced an increase in thickness (≤0.05 nm) and decrease in density (≤-0.03 g/cm(3)) whereas TFP induced an increase in both thickness (≤0.05 nm) and density (≤0.01 g/cm(3)). The layer measurements reported here show how DPI can be used to assess and differentiate ligands with distinct structural modes of action.

Characterisation of Calmodulin Structural Transitions by Ion Mobility Mass Spectrometry

This study demonstrates the ability of travelling wave ion mobility-mass spectrometry to measure collision cross-sections of ions in the negative mode, using a calibration based approach. Here, negative mode ion mobility-mass spectrometry was utilised to understand structural transitions of calmodulin upon Ca2+ binding and complexation with model peptides melittin and the plasma membrane Ca2+ pump C20W peptide. Coexisting calmodulin conformers were distinguished on the basis of their mass and cross-section, and identified as relatively folded and unfolded populations, with good agreement in collision cross-section to known calmodulin geometries. Titration of calcium tartrate to physiologically relevant Ca2+ levels provided evidence for intermediately metalated species during the transition from apo- to holo-calmodulin, with collision cross-section measurements indicating that higher Ca2+ occupancy is correlated with more compact structures. The binding of two representative peptides which exemplify canonical compact (melittin) and extended (C20W) peptide-calmodulin binding models has also been interrogated by ion mobility mass spectrometry. Peptide binding to calmodulin involves intermediates with metalation states from 1–4 Ca2+, which demonstrate relatively collapsed structures, suggesting neither the existence of holo-calmodulin or a pre-folded calmodulin conformation is a prerequisite for binding target peptides or proteins. The biological importance of the different metal unsaturated calmodulin complexes, if any, is yet to be understood.

Mapping the Binding and Calmodulin-Dependent Activation of Nitric Oxide Synthase Isozymes

Calmodulin (CaM) is a ubiquitous cytosolic Ca 2+ -binding protein that can bind and activate numerous intracellular target proteins including the nitric oxide synthase (NOS) enzymes. There are three different isoforms of NOS found in mammals - neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) - with all three isozymes capable of producing the free radical, nitric oxide (•NO). At elevated cellular Ca 2+ concentrations, CaM is able to bind and activate nNOS and eNOS. In contrast, the iNOS isozyme is transcriptionally regulated and binds to CaM in the absence of Ca 2+ . To further investigate the differences in the association of CaM to the Ca 2+ -dependent and Ca 2+ - independent NOS isoforms, a variety of CaM mutants were employed. These included CaM-troponin C chimeras, CaM EF hand pair proteins, CaM mutants incapable of binding to Ca 2+ , methionine oxidized CaM mutants, phosphomimetic CaM mutants and central linker CaM mutants. By characterizing these CaM proteins using a variety of biochemical techniques, we have mapped the binding and the CaM-dependent activation of the NOS enzymes to study their effect on the different electron transfer steps within the NOS enzymes. In addition, fluorescence and Forster resonance energy transfer (FRET) were used to monitor the orientation and conformation of CaM when bound to the NOS peptides and enzymes. Combining these cumulative results, a working model describing the CaM-dependent regulation of iNOS is proposed.

Subtle pH differences trigger single residue motions for moderating conformations of calmodulin

This study reveals the essence of ligand recognition mechanisms by which calmodulin (CaM) controls a variety of Ca(2+) signaling processes. We study eight forms of calcium-loaded CaM each with distinct conformational states. Reducing the structure to two degrees of freedom conveniently describes main features of the conformational changes of CaM via simultaneous twist-bend motions of the two lobes. We utilize perturbation-response scanning (PRS) technique, coupled with molecular dynamics simulations. PRS is based on linear response theory, comprising sequential application of directed forces on selected residues followed by recording the resulting protein coordinates. We analyze directional preferences of the perturbations and resulting conformational changes. Manipulation of a single residue reproduces the structural change more effectively than that of single/pairs/triplets of collective modes of motion. Our findings also give information on how the flexible linker acts as a transducer of binding information to distant parts of the protein. Furthermore, by perturbing residue E31 located in one of the EF hand motifs in a specific direction, it is possible to induce conformational change relevant to five target structures. Independently, using four different pK(a) calculation strategies, we find this particular residue to be the charged residue (out of a total of 52), whose ionization state is most sensitive to subtle pH variations in the physiological range. It is plausible that at relatively low pH, CaM structure is less flexible. By gaining charged states at specific sites at a pH value around 7, such as E31 found in the present study, local conformational changes in the protein will lead to shifts in the energy landscape, paving the way to other conformational states. These findings are in accordance with Fluorescence Resonance Energy Transfer (FRET) measured shifts in conformational distributions towards more compact forms with decreased pH. They also corroborate mutational studies and proteolysis results which point to the significant role of E31 in CaM dynamics.

Narrowing the conformational space sampled by two-domain proteins with paramagnetic probes in both domains

Calmodulin is a two-domain protein which in solution can adopt a variety of conformations upon reorientation of its domains. The maximum occurrence (MO) of a set of calmodulin conformations that are representative of the overall conformational space possibly sampled by the protein, has been calculated from the paramagnetism-based restraints. These restraints were measured after inclusion of a lanthanide binding tag in the C-terminal domain to supplement the data obtained by substitution of three paramagnetic lanthanide ions to the calcium ion in the second calcium binding loop of the N-terminal domain. The analysis shows that the availability of paramagnetic restraints arising from metal ions placed on both domains, reduces the MO of the conformations to different extents, thereby helping to identify those conformations that can be mostly sampled by the protein.

Hidden conformations in protein structures

Prediction of interactions between protein residues (contact map prediction) can facilitate various aspects of 3D structure modeling. However, the accuracy of ab initio contact prediction is still limited. As structural genomics initiatives move ahead, solved structures of homologous proteins can be used as multiple templates to improve contact prediction of the major conformation of an unsolved target protein. Furthermore, multiple templates may provide a wider view of the protein's conformational space. However, successful usage of multiple structural templates is not straightforward, due to their variable relevance to the target protein, and because of data redundancy issues. We present here an algorithm that addresses these two limitations in the use of multiple structure templates. First, the algorithm unites contact maps extracted from templates sharing high sequence similarity with each other in a fashion that acknowledges the possibility of multiple conformations. Next, it weights the resulting united maps in inverse proportion to their evolutionary distance from the target protein. Testing this algorithm against CASP8 targets resulted in high precision contact maps. Remarkably, based solely on structural data of remote homologues, our algorithm identified residue-residue interactions that account for all the known conformations of calmodulin, a multifaceted protein. Therefore, employing multiple templates, which improves prediction of contact maps, can also be used to reveal novel conformations. As multiple templates will soon be available for most proteins, our scheme suggests an effective procedure for their optimal consideration. A Perl script implementing the WMC algorithm described in this article is freely available for academic use at http://tau.ac.il/~haimash/WMC.

Exploring NMR ensembles of calcium binding proteins: Perspectives to design inhibitors of protein-protein interactions

BackgroundDisrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding.ResultsIn order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin) into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces.ConclusionsNMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions.

Molecular Recognition Mechanism of Calmodulin Examined by Perturbation-Response Scanning

Calmodulin (CaM) has a pivotal role as an intracellular Ca2+ receptor that is involved in calcium signaling pathways in eukaryotic cells [1,2]. Binding of Ca2+ and proteins or small organic molecules to CaM induces large conformational changes that are distinct to each interacting partner [1,3,4]. To design drugs that inhibit Ca2+-CaM formation, the molecular binding mechanism must be thoroughly understood.In this study, we use a new tool called perturbation-response scanning that is based on systematically exerting directed forces on the residues of the protein [5] and recording the changes in fluctuation profiles as the response. Different conformations of CaM are investigated to locate domains or key residues controlling ligand binding and release. We perform 200 ns long MD simulations on the apo form of CaM. The simulation is divided into chunks which resemble different conformations of the apo form. Using the cross-correlation matrices obtained from these chunks as the kernel in linear response, we determine the residues whose perturbation yields the experimentally determined displacement profiles of the apo and holo forms. We find that it is possible to induce the different conformational changes relevant to the binding of five different ligands, by perturbing varying ligand binding residues, and/or residues on the distant helices in the single apo-form. The findings thus give information on how the flexible linker region acts as a transducer of binding information to distant parts of the protein. This new tool enables us to reveal the essence of ligand recognition mechanisms by which CaM controls a wide variety of Ca2+ signaling processes.1. Ikura M. et.al., PNAS 103:1159-1164.2. Fallon J.L., et.al Structure 11:1303-1307.3. Vandonselaar M.,et.al., Struct.Biol. 1:795-801.4. Shepherd C.M., Vogel H.J., Biophys.J. 87:780-791.5. Atilgan C., Atilgan A.R., PloS Comput.Biol. 5:e1000544.

Calcium-Induced Structural Transitions of the Calmodulin−Melittin System Studied by Electrospray Mass Spectrometry: Conformational Subpopulations and Metal-Unsaturated Intermediates

Calmodulin (CaM) is a calcium-sensing protein that can bind to and activate various target enzymes. Here, electrospray ionization mass spectrometry (ESI-MS) was used to investigate calcium-induced structural changes of CaM, as well as binding to the model target melittin (Mel). Nonspecific metalation artifacts were eliminated by conducting the experiments in negative ion mode and with calcium tartrate as metal source [Pan et al. (2009) Anal. Chem. 81, 5008]. Two coexisting CaM subpopulations can be distinguished on the basis of their ESI charge state distributions, namely, relatively disordered conformers (CaM(D), high charge states) and more tightly folded proteins (CaM(F), low charge states). Calcium titration experiments on isolated CaM reveal that the transition from apo-CaM(D) to Ca(4).CaM(F) proceeds with apparent K(d) values of 10, 14, 30, and 12 microM. In the presence of Mel, a gradual [Ca(2+)] increase results in an overall population shift from apo-CaM(D) to Ca(4).CaM(F).Mel. This transition involves various intermediates, Ca(n).CaM(F).Mel with n = 0, ..., 3, as well as apo-CaM(D).Mel. Thus, neither the binding of four Ca(2+) nor the existence of a tightly folded CaM conformation is a prerequisite for target binding. Millisecond time-resolved ESI-MS experiments were conducted to monitor the response of a premixed CaM-Mel solution to a calcium concentration jump, thereby mimicking the conditions encountered in a cellular signaling context. The resulting data suggest that the formation of Ca(4).CaM(F).Mel proceeds along three parallel kinetic pathways: (1) metal binding to CaM(D) followed by formation of a compact protein-target complex, (2) folding of the apoprotein, then target binding, followed by metal complexation, (3) target binding to apo-CaM(D) followed by sequential metal binding. The exact structural properties of the various metal-unsaturated CaM species, as well as their physiological roles, remain to be elucidated.

Time-Resolved FRET Detection of Structural Distributions Involving FKBP12.6 and Calmodulin Bound Within Macromolecular RyR Channels

We used multi-exponential analysis of time-resolved (TR) fluorescence decays to investigate the array of distances underlying fluorescence resonance energy transfer (FRET) between donors and acceptors bound within functional RyR channels in sarcoplasmic reticulum membranes. Previously, we have used steady-state (SS) FRET to demonstrate that CaM is oriented with the N-lobe proximal to the FKBP12.6 subunit on each lateral face of the RyR tetrameric complex (Cornea et al. 2009). Here, we used cysteine mutagenesis and sulfhydryl-specific fluorescent labeling to attach Alexa Fluor (AF) donor probes to single-cysteines at FKBP12.6 sites 49 or 85 (denoted D49 and D85), and acceptor probes to calmodulin (CaM) N-lobe sites 26 or 34 (denoted A26 and A34). The fluorescence of RyR-bound donor-labeled FKBP12.6 was decreased in the presence of saturating acceptor-labeled CaM, indicating FRET. Forster analysis of TR- and SS-FRET data of donor/acceptor pairs AF350/AF488 and AF488/AF568 (with different ranges of sensitivity) yielded similarly-ranked energy transfer efficiencies: D85/A26 ≥ D49/A26 > D49/D34 > D85/34. Distances calculated from these FRET efficiencies are remarkably similar for the two different donor/acceptor probe pairs. However, this kind of analysis can extract only averaged distances from an RyR sample in which distinct biophysical states are known to co-exist. Further multi-exponential analysis of the donor-only (AF350-FKBP12.6) and donor+acceptor (AF350-FKBP12.6/AF488-CaM) TR-FRET data sets resolved at least two simultaneous distance populations for each pair of labeled sites. These distances and their distribution responded to changes in [Ca2+]. We conclude that TR-FRET provides a powerful approach for resolving dynamic transitions between structural states within functional, multimeric RyR channels. Further studies will aim to determine whether these findings reflect transitions between different conformations of CaM itself, or of the underlying RyR channel.

Investigations of Calmodulin Conformations Resolved by Single Molecule Microscopy

Measurements of the distance between two dye molecules covalently linked to the calcium signaling protein calmodulin (CaM) have been previously performed by our group to investigate the conformations of CaM in solution. It was shown that calmodulin exists in a wide range of distinct conformations whose amplitudes depend upon free calcium concentrations (1). Currently, we are investigating affects that the choice of dye pair or labeling site has on measured conformations. This is done using an alternating laser excitation (ALEX) single molecule microscope system that has been custom built in our laboratory. Time correlated single photon counting in bulk samples is used to determine the time resolved anisotropy of the dye pair and the orientational mobility of each dye. Analysis of burst measurements using interphoton time burst selection criteria and the probability distribution analysis reveal a wide range of CaM conformations. Conformational analysis is performed using both discrete states and the maximum entropy method. The maximum entropy method reveals the most probable underlying conformational distribution that fits our data. Finally, we are investigating fluorescence fluctuations within CaM conformations using conformationally sorted fluorescence correlation spectroscopy. 1. Slaughter et al., J. Phys. Chem. B, 2004, 108, 10388-10397

FRET Detection of Calmodulin Binding and Structural Rearrangements Within the Cardiac RyR2 Calcium Release Channel

Calmodulin (CaM) binds to a conserved domain of the ryanodine receptor isoforms expressed in skeletal muscle (RyR1) and cardiac muscle (RyR2) to evoke isoform-specific changes in channel gating. To better understand CaM's interactions with the RyR2 isoform, we are using fluorescence resonance energy transfer (FRET) to define the orientation and kinetics of CaM binding, and to resolve structural rearrangements linked to channel regulation. A FRET donor was targeted to the RyR2 cytoplasmic assembly by preincubating cardiac sarcoplasmic reticulum membranes with a fluorescent-labeled FKBP12.6 (F-FKBP). An acceptor fluorophore was attached within the N-lobe of CaM (F-CaM). A decrease in F-FKBP fluorescence upon addition of F-CaM provided a specific, real-time readout of CaM binding to the RyR2, despite the presence of additional non-RyR CaM targets in the cardiac membranes. FRET demonstrated that the affinity of F-CaM binding to RyR2 was greater in 100 μM than in 30 nM Ca2+. The maximal FRET observed in the presence of saturating [F-CaM] increased as a function of [Ca2+] (30 nM to 1 mM). The Ca2+ dependence of this increase in FRET was similar to the Ca2+ dependence of [3H]ryanodine binding to RyR2 assayed in equivalent media (KCa ∼5 μM). A marked decrease in FRET between FKBP12.6 and CaM was observed when the acceptor was shifted from CaM's N-lobe to CaM's C-lobe. We conclude that CaM binds to the RyR2 in an extended conformation, with its N-lobe oriented nearest to the FKBP12.6 subunit. CaM's conformation and orientation when bound to the RyR2 are therefore similar to what has been demonstrated previously for the RyR1 isoform (Cornea et al., 2009). Ca2+ dependent changes in FRET between FKBP12.6 and CaM may reflect structural changes within the RyR2 linked to channel activation by Ca2+.

The Effect of Calcium Ions and Peptide Ligands on the Relative Stabilities of the Calmodulin Dumbbell and Compact Structures

A combination of ion mobility and mass spectrometry methods was used to characterize the molecular shape of the protein calmodulin (CaM) and its complexes with calcium and a number of peptide ligands. CaM, a calcium-binding protein composed of 148 amino acid residues, was found by X-ray crystallography to occur both in a globular shape and in the shape of an extended dumbbell. Here, it was found, as solutions of CaM and CaM complexes were sprayed into the solvent-free environment of the mass spectrometer, that major structural features of the molecule and the stoichiometry of the units constituting a complex in solution were preserved in the desolvation process. Two types of CaM structures were observed in our experiments: a compact and an extended form of CaM with measured cross sections in near-perfect agreement with those calculated for the known globular and extended dumbbell X-ray geometries. Calcium-free solutions yielded predominantly an extended CaM conformation. Ca(n)(2+)-CaM complexes were observed in calcium-containing solutions, n = 0-4, with the population of the compact conformation increasing relative to the elongated conformation as n increases. For n = 4, a predominantly compact globular conformation was observed. Solutions containing the peptide CaMKII(290-309), the CaM target domain of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) enzyme, yielded predominantly globular Ca(4)(2+)-CaM-CaMKII(290-309) complexes. Similar results were obtained with the 26-residue peptide melittin. For the 14-residue C-terminal melittin fragment, on the other hand, formation of both a 1:1 and a 1:2 CaM-peptide complex was detected. On the basis of the entirety of our results, we conclude that the collapse of extended (dumbbell-like) CaM structures into more compact globular structures occurs upon specific binding of four calcium ions. Furthermore, this calcium-induced structural collapse of CaM appears to be a prerequisite for formation of a particularly stable CaM-peptide complex involving peptides long enough to be engaged in interactions with both lobes of CaM.

Modulation of Calmodulin Plasticity by the Effect of Macromolecular Crowding

In vitro biochemical reactions are most often studied in dilute solution, a poor mimic of the intracellular space of eukaryotic cells, which are crowded with mobile and immobile macromolecules. Such crowded conditions exert volume exclusion and other entropic forces that have the potential to impact chemical equilibria and reaction rates. In this article, we used the well-characterized and ubiquitous molecule calmodulin (CaM) and a combination of theoretical and experimental approaches to address how crowding impacts CaM's conformational plasticity. CaM is a dumbbell-shaped molecule that contains four EF hands (two in the N-lobe and two in the C-lobe) that each could bind Ca 2+, leading to stabilization of certain substates that favor interactions with other target proteins. Using coarse-grained molecular simulations, we explored the distribution of CaM conformations in the presence of crowding agents. These predictions, in which crowding effects enhance the population of compact structures, were then confirmed in experimental measurements using fluorescence resonance energy transfer techniques of donor- and acceptor-labeled CaM under normal and crowded conditions. Using protein reconstruction methods, we further explored the folding-energy landscape and examined the structural characteristics of CaM at free-energy basins. We discovered that crowding stabilizes several different compact conformations, which reflects the inherent plasticity in CaM's structure. From these results, we suggest that the EF hands in the C-lobe are flexible and can be thought of as a switch, while those in the N-lobe are stiff, analogous to a rheostat. New combinatorial signaling properties may arise from the product of the differential plasticity of the two distinct lobes of CaM in the presence of crowding. We discuss the implications of these results for modulating CaM's ability to bind Ca 2+ and target proteins.

Interactions of the Anti-Psychotic Drug Trifluoperazine with Calmodulin

Calmodulin (CaM) is a Ca2+-sensing protein essential to eukaryotic signal transduction pathways. It has two homologous domains (N and C), each binding two Ca2+ ions. The anti-psychotic drug trifluoperazine (TFP; Stelazine) is a CaM antagonist known to bind hydrophobic clefts of CaM that are exposed upon Ca2+ binding. Equilibrium Ca2+ titrations monitored by changes in steady-state fluorescence of intrinsic Phe and Tyr residues were used to evaluate the effect of TFP on the Ca2+ affinity of full length CaM (CaM1–148), N-domain (CaM1–80) and C-domain (CaM76–148) over a range of TFP:CaM ratios. Low levels of TFP (1:1, 2:1 ratios) decreased the Ca2+ affinity of CaM. TFP had the greatest effect on Ca2+ binding to sites III and IV, in the C-domain of CaM1–148, but affected both domains. At an 8:1 ratio of TFP:CaM, the effect reversed and the Ca2+ affinity of CaM increased. 1H-15N-HSQC NMR showed that resonances assigned to apo and Ca2+-saturated C-domain were the most perturbed during TFP titration, while a smaller subset of N-domain resonances were affected. The stoichiometry of TFP binding to apo-CaM1–148 was determined to be 2:1, and 4:1 for (Ca2+)4-CaM. Crystallographic structures of TFP bound to (Ca2+)4-CaM1–148 indicate two possible orientations of TFP when bound in 1:1 vs 2:1 and 4:1 TFP:CaM ratios. A new structure of a (Ca2+)2-CaM76–148 -TFP complex showed the trifluoromethyl group of TFP in both positions seen previously; distinct conformation of Met 144 correlated with orientation of TFP. NMR of apo-CaM76–148 will be used to determine whether apo CaM-TFP complex adopts the semi-open conformation of apo CaM bound to a myosin peptide.

Effect of Trifluoperazine on Ca2+ -Bound Calmodulin binding to Fas Death Domain for DISC Formation

Fas death receptor-activated signaling pathway is one important regulating mechanism of apoptosis in a variety of cells. The formation of the death inducing signaling complex (DISC) is a critical step for Fas-mediated signaling of apoptosis. Recent experimental studies showed that calmodulin (CaM) binds to Fas and regulates Fas-mediated DISC formation and the binding of CaM to Fas is inhibited by CaM antagonist, trifluoperazine (TFP). However, the exact molecular mechanisms for the effect of TFP on Fas-mediated DISC formation are still unknown. Knowledge about these is important for identifying new drug candidate to regulate Fas-mediated signaling pathway for apoptosis. In this study, we investigated the effect of TFP on CaM/Fas binding with molecular dynamics simulations. Conformation and binding free energy analyses were performed to examine the connections between the conformational changes of CaM by TFP and CaM/Fas binding affinity. Conformational characteristics of Fas by TFP were also examined for the further determining TFP effects on Fas recruiting FADD to form DISC. Binding free energy analyses showed that CaM antagonist, TFP inhibited CaM binding to Fas. The results are consistent with experimental results. The further conformational analyses showed that TFP significantly changed the CaM conformation, resulted in the increased Fas conformational fluctuations and the degree of correlation between motions of the residues in Fas, which provides structural insight for Fas further binding to FADD for DISC formation. Understanding the molecular mechanisms of CaM antagonist TFP in CaM/Fas binding for Fas-mediated DISC formation should provide important insight into the function of CaM antagonists in regulating Fas-mediated apoptosis.Keywords: CaM antagonist TFP; CaM/Fas binding; DISC; binding free energy, conformational analysis

Conformation and Free Energy Analyses of the Complex of Calcium-Bound Calmodulin and the Fas Death Domain

Previous studies have demonstrated a calcium-dependent interaction of calmodulin (CaM) and Fas that is regulated during Fas-induced apoptosis in several cell lines, including cholangiocarcinoma, Jurkat cells, and osteoclasts. The binding of CaM and Fas has been identified on residues 231–254 of Fas; the V254N point mutation decreases the CaM/Fas binding, and the C-terminal deletion mutation increases the CaM/Fas binding. Recent studies have shown that CaM is recruited into the Fas-mediated death-inducing signaling complex (DISC) in a calcium-dependent manner. However, the molecular mechanisms whereby Fas mutations and CaM/Fas binding might regulate Fas-mediated DISC formation are unknown. In this study we investigated the binding thermodynamics and conformation of the CaM/Fas complexes with combined explicit solvent molecular-dynamics simulations and implicit solvent binding free-energy calculations. The binding free-energy analysis demonstrated that the Fas V254N point mutation reduced its binding affinity with CaM. In contrast, the Fas mutant with the deletion of the 15 amino acid at the C-terminus increased its binding to CaM. These observations are consistent with previous findings from biochemical studies. Conformational analyses further showed that the Fas V254N mutation resulted in an unstable conformation, whereas the C-terminal deletion mutation stabilized the Fas conformation, and both mutations resulted in changes of the degree of correlation between the motions of the residues in Fas. Analysis of the CaM/Fas complex revealed that CaM/Fas binding stabilized the conformation of both CaM and Fas and changed the degree of correlated motion of the residues of CaM and Fas. The results presented here provide structural evidence for the roles of Fas mutations and CaM/Fas binding in Fas-induced DISC formation. Understanding the molecular mechanisms of CaM/Fas binding in Fas-mediated DISC formation should provide important insights into the function of Fas mutations and CaM in regulating Fas-mediated apoptosis.

Calmodulin Association with Connexin32-derived Peptides Suggests trans-Domain Interaction in Chemical Gating of Gap Junction Channels

Calmodulin plays a key role in the chemical gating of gap junction channels. Two calmodulin-binding regions have previously been identified in connexin32 gap junction protein, one in the N-terminal and another in the C-terminal cytoplasmic tail of the molecule. The aim of this study was to better understand how calmodulin interacts with the connexin32-binding domains. Lobe-specific interactions of calmodulin with connexin32 peptides were studied by stopped flow kinetics, using Ca2+ binding-deficient mutants. Peptides corresponding to the N-terminal tail (residues 1–22) of connexin32 engaged both the N- and C-terminal lobes (N- and C-lobes) of calmodulin, binding with higher affinity to the C-lobe of calmodulin (Ca2+ dissociation rate constants k3,4, 1.7 ± 0.5 s–1) than to the N-lobe (k1,2, 10.8 ± 1.3 s–1). In contrast, peptides representing the C-terminal tail domain (residues 208–227) of connexin32 bound either the C- or the N-lobe but only one calmodulin lobe at a time (k3,4, 2.6 ± 0.1 s–1 or k1, 13.8 ± 0.5 s–1 and k2, 1000 s–1). The calmodulin-binding domains of the N- and C-terminal tails of connexin32 were best defined as residues 1–21 and 216–227, respectively. Our data, showing separate functions of the N- and C-lobes of calmodulin in the interactions with connexin32, suggest trans-domain or trans-subunit bridging by calmodulin as a possible mechanism of gap junction gating.

Ca 2+ dissociation from the C-terminal EF-hand pair in calmodulin: A steered molecular dynamics study

We used steered molecular dynamics (SMD) to simulate the process of Ca 2+ dissociation from the EF-hand motifs of the C-terminal lobe of calmodulin. Based on an analysis of the pulling forces, the dissociation sequences and the structural changes, we show that the Ca 2+-coordinating residues lose their binding to Ca 2+ in a stepwise fashion. The two Ca 2+ ions dissociate from the two EF-hands simultaneously, with two distinct groups among the five Ca 2+-coordinating residues affecting the EF-hand conformational changes differently. These results provide new insights into the effects of Ca 2+ on calmodulin conformation, from which a novel sequential mechanism of Ca 2+-calmodulin dissociation is proposed.

Dynamic Conformational Changes of Calmodulin when Bound to Nitric Oxide Synthase using FRET

Calmodulin (CaM) is a ubiquitous Ca2+‐sensor protein that binds and activates the Nitric Oxide Synthase (NOS) enzymes (E.C. ). Although the structure of CaM has been determined when bound to a peptide derived from the CaM‐binding domain of endothelial NOS, the conformation of CaM when bound to the three mammalian NOS holoenzymes has yet to be determined. The objective of this study is to determine the dynamic conformational changes of CaM when bound to all three NOS isoforms through the use of Förster Resonance Energy Transfer (FRET). We have produced a double cysteine CaM mutant (T34C/T110C) using site‐directed mutagenesis for the performance of FRET measurements. Far‐red shifted fluorescent dyes were employed so that they could be selectively excited in the presence of the NOS cofactors that absorb light strongly between 250 and 500 nm. FRET studies were performed in the presence of a range of physiologically relevant free Ca2+ concentrations for CaM binding to the constitutive NOS enzymes. These studies will provide a better understanding of the differences between the Ca2+‐dependent and Ca2+‐independent binding of CaM to the NOS isozymes. This work is supported by NSERC (J.G.G.).