Dynamics of myo1c (myosin-ibeta ) lipid binding and dissociation

Myosin-I is the single-headed member of the myosin superfamily that associates with lipid membranes. Biochemical experiments have shown that myosin-I membrane binding is the result of electrostatic interactions between the basic tail domain and acidic phospholipids. To better understand the dynamics of myosin-I membrane association, we measured the rates of association and dissociation of a recombinant myo1c tail domain (which includes three IQ domains and bound calmodulins) to and from large unilamellar vesicles using fluorescence resonance energy transfer. The apparent second-order rate constant for lipid-tail association in the absence of calcium is fast with nearly every lipid-tail collision resulting in binding. The rate of binding is decreased in the presence of calcium. Time courses of myo1c-tail dissociation are best fit by two exponential rates: a fast component that has a rate that depends on the ratio of acidic phospholipid to myo1c-tail (phosphatidylserine (PS)/tail) and a slow component that predominates at high PS/tail ratios. The dissociation rate of the slow component is slower than the myo1c ATPase rate, suggesting that myo1c is able to stay associated with the lipid membrane during multiple catalytic cycles of the motor. Calcium significantly increases the lifetimes of the membrane-bound state, resulting in dissociation rates 0.001 s(-1).     

Calcium binding to complexes of calmodulin and calmodulin binding proteins

The free energy of coupling for binding of Ca2+ and the calmodulin-sensitive phosphodiesterase to calmodulin was determined and compared to coupling energies for two other calmodulin binding proteins, troponin I and myosin light chain kinase. Free energies of coupling were determined by quantitating binding of Ca2+ to calmodulin complexed to calmodulin binding proteins with Quin 2 to monitor free Ca2+ concentrations. The geometric means of the dissociation constants (-Kd) for Ca2+ binding to calmodulin in the presence of equimolar rabbit skeletal muscle troponin I, rabbit skeletal muscle myosin light chain kinase, and bovine heart calmodulin sensitive phosphodiesterase were 2.1, 1.1, and 0.55 microM. The free-energy couplings for the binding of four Ca2+ and these proteins to calmodulin were -4.48, -6.00, and -7.64 kcal, respectively. The Ca2+-independent Kd for binding of the phosphodiesterase to calmodulin was estimated at 80 mM, indicating that complexes between calmodulin and this enzyme would not exist within the cell under low Ca2+ conditions. The large free-energy coupling values reflect the increase in Ca2+ affinity of calmodulin when it is complexed to calmodulin binding proteins and define the apparent positive cooperativity for Ca2+ binding expected for each system. These data suggest that in vitro differences in free-energy coupling for various calmodulin-regulated enzymes may lead to differing Ca2+ sensitivities of the enzymes.     

Calcium-dependent and -independent binding of soybean calmodulin isoforms to the calmodulin binding domain of tobacco MAPK phosphatase-1

The recent finding of an interaction between calmodulin (CaM) and the tobacco mitogen-activated protein kinase phosphatase-1 (NtMKP1) establishes an important connection between Ca(2+) signaling and the MAPK cascade, two of the most important signaling pathways in plant cells. Here we have used different biophysical techniques, including fluorescence and NMR spectroscopy as well as microcalorimetry, to characterize the binding of soybean CaM isoforms, SCaM-1 and -4, to synthetic peptides derived from the CaM binding domain of NtMKP1. We find that the actual CaM binding region is shorter than what had previously been suggested. Moreover, the peptide binds to the SCaM C-terminal domain even in the absence of free Ca(2+) with the single Trp residue of the NtMKP1 peptides buried in a solvent-inaccessible hydrophobic region. In the presence of Ca(2+), the peptides bind first to the C-terminal lobe of the SCaMs with a nanomolar affinity, and at higher peptide concentrations, a second peptide binds to the N-terminal domain with lower affinity. Thermodynamic analysis demonstrates that the formation of the peptide-bound complex with the Ca(2+)-loaded SCaMs is driven by favorable binding enthalpy due to a combination of hydrophobic and electrostatic interactions. Experiments with CaM proteolytic fragments showed that the two domains bind the peptide in an independent manner. To our knowledge, this is the first report providing direct evidence for sequential binding of two identical peptides of a target protein to CaM. Discussion of the potential biological role of this interaction motif is also provided.     

Electrostatic control of calcineurin's intrinsically-disordered regulatory domain binding to calmodulin

Calcineurin (CaN) is a serine/threonine phosphatase that regulates a variety of physiological and pathophysiological processes in mammalian tissue. The calcineurin (CaN) regulatory domain (RD) is responsible for regulating the enzyme's phosphatase activity, and is believed to be highly-disordered when inhibiting CaN, but undergoes a disorder-to-order transition upon diffusion-limited binding with the regulatory protein calmodulin (CaM). The prevalence of polar and charged amino acids in the regulatory domain (RD) suggests electrostatic interactions are involved in mediating calmodulin (CaM) binding, yet the lack of atomistic-resolution data for the bound complex has stymied efforts to probe how the RD sequence controls its conformational ensemble and long-range attractions contribute to target protein binding. In the present study, we investigated via computational modeling the extent to which electrostatics and structural disorder facilitate CaM/CaN association kinetics. Specifically, we examined several RD constructs that contain the CaM binding region (CAMBR) to characterize the roles of electrostatics versus conformational diversity in controlling diffusion-limited association rates, via microsecond-scale molecular dynamics (MD) and Brownian dynamic (BD) simulations. Our results indicate that the RD amino acid composition and sequence length influence both the dynamic availability of conformations amenable to CaM binding, as well as long-range electrostatic interactions to steer association. These findings provide intriguing insight into the interplay between conformational diversity and electrostatically-driven protein-protein association involving CaN, which are likely to extend to wide-ranging diffusion-limited processes regulated by intrinsically-disordered proteins.     

Calcium binding to tryptic fragments of calmodulin

Fragments of scallop testis calmodulin were prepared by tryptic digestion. One peptide consisted of 75 amino acid residues from N-acetylalanine to lysine at position 75 (F12) and the other of 71 residues from aspartic acid at position 78 to C-terminal lysine (F34). Flow dialysis and equilibrium dialysis experiments revealed the existence of two Ca2+ binding sites in each fragment. Half-saturating concentrations of the Ca2+ titration curves were 11 microM for F12 and 3.2 microM for F34, and Hill coefficients were obtained as 1.14 and 1.84, respectively. The results indicate that the high-affinity sites for Ca2+ are located on the C-terminal region of the calmodulin. The sum of the two Ca2+ titration curves of F12 and F34 fits well to the curves of Ca2+ binding to intact calmodulin. This shows that the characteristic of Ca2+ bindings in intact calmodulin did not change after separation of the whole molecule into two domains, F12 and F34. The domains corresponding to F12 and F34 may exist independently from each other in the intact calmodulin molecule.     

Calcium binding to calmodulin mutants having domain-specific effects on the regulation of ion channels

Calmodulin (CaM) is an essential, eukaryotic protein comprised of two highly homologous domains (N and C). CaM binds four calcium ions cooperatively, regulating a wide array of target proteins. A genetic screen of Paramecia by Kung [Kung, C. et al. (1992) Cell Calcium 13, 413-425] demonstrated that the domains of CaM have separable physiological roles: "under-reactive" mutations affecting calcium-dependent sodium currents mapped to the N-domain, while "over-reactive" mutations affecting calcium-dependent potassium currents localized to the C-domain of CaM. To determine whether and how these mutations affected intrinsic calcium-binding properties of CaM domains, phenylalanine fluorescence was used to monitor calcium binding to sites I and II (N-domain) and tyrosine fluorescence was used to monitor sites III and IV (C-domain). To explore interdomain interactions, binding properties of each full-length mutant were compared to those of its corresponding domain fragments. The calcium-binding properties of six under-reactive mutants (V35I/D50N, G40E, G40E/D50N, D50G, E54K, and G59S) and one over-reactive mutant (M145V) were indistinguishable from those of wild-type CaM, despite their deleterious physiological effects on ion-channel regulation. Four over-reactive mutants (D95G, S101F, E104K, and H135R) significantly decreased the calcium affinity of the C-domain. Of these, one (E104K) also increased the calcium affinity of the N-domain, demonstrating that the magnitude and direction of wild-type interdomain coupling had been perturbed. This suggests that, while some of these mutations alter calcium-binding directly, others probably alter CaM-channel association or calcium-triggered conformational change in the context of a ternary complex with the affected ion channel.     

Calcium binding to calmodulin and its globular domains

The macroscopic Ca(2+)-binding constants of bovine calmodulin have been determined from titrations with Ca2+ in the presence of the chromophoric chelator 5,5'-Br2BAPTA in 0, 10, 25, 50, 100, and 150 mM KCl. Identical experiments have also been performed for tryptic fragments comprising the N-terminal and C-terminal domains of calmodulin. These measurements indicate that the separated globular domains retain the Ca2+ binding properties that they have in the intact molecule. The Ca2+ affinity is 6-fold higher for the C-terminal domain than for the N-terminal domain. The salt effect on the free energy of binding two Ca2+ ions is 20 and 21 kJ. mol-1 for the N- and C-terminal domain, respectively, comparing 0 and 150 mM KCl. Positive cooperativity of Ca2+ binding is observed within each globular domain at all ionic strengths. No interaction is observed between the globular domains. In the N-terminal domain, the cooperativity amounts to 3 kJ.mol-1 at low ionic strength and greater than or equal to 10 kJ.mol-1 at 0.15 M KCl. For the C-terminal domain, the corresponding figures are 9 +/- 2 kJ.mol-1 and greater than or equal to 10 kJ.mol-1. Two-dimensional 1H NMR studies of the fragments show that potassium binding does not alter the protein conformation.     

Energetics of calmodulin domain interactions with the calmodulin binding domain of CaMKII

Calmodulin (CaM) is an essential eukaryotic calcium receptor that regulates many kinases, including CaMKII. Calcium‐depleted CaM does not bind to CaMKII under physiological conditions. However, binding of (Ca²⁺)₄‐CaM to a basic amphipathic helix in CaMKII releases auto‐inhibition of the kinase. The crystal structure of CaM bound to CaMKIIp, a peptide representing the CaM‐binding domain (CaMBD) of CaMKII, shows an antiparallel interface: the C‐domain of CaM primarily contacts the N‐terminal half of the CaMBD. The two domains of calcium‐saturated CaM are believed to play distinct roles in releasing auto‐inhibition. To investigate the underlying mechanism of activation, calcium‐dependent titrations of isolated domains of CaM binding to CaMKIIp were monitored using fluorescence anisotropy. The binding affinity of CaMKIIp for the domains of CaM increased upon saturation with calcium, with the C‐domain having a 35‐fold greater affinity than the N‐domain. Because the interdomain linker of CaM regulates calcium‐binding affinity and contribute to conformational change, the role of each CaM domain was explored further by investigating effects of CaMKIIp on site‐knockout mutants affecting the calcium‐binding sites of a single domain. Investigation of the thermodynamic linkage between saturation of individual calcium‐binding sites and CaM‐domain binding to CaMKIIp showed that calcium binding to Sites III and IV was sufficient to recapitulate the behavior of (Ca²⁺)₄‐CaM. The magnitude of favorable interdomain cooperativity varied depending on which of the four calcium‐binding sites were mutated, emphasizing differential regulatory roles for the domains of CaM, despite the high degree of homology among the four EF‐hands of CaM. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Calcium binding to calmodulin. Cooperativity of the calcium-binding sites

The effects of Mg2+ ion, pH, and KCl concentration on Ca2+ binding to calmodulin were studied by using a Ca2+ ion-sensitive electrode. The Ca2+ ion affinity of calmodulin increased with increasing pH or decreasing KCl concentration. Cooperativity between the Ca2+-binding sites was observed, and increased with decreasing pH or increasing KCl concentration. Free Ca2+ ion concentration was decreased by adding MgCl2 ion at low Mg2+ concentration and increased at higher concentrations in the presence of small amounts of Ca2+ ion. The decrease of free Ca2+ ion concentration by Mg2+ ion strongly suggests cooperativity between the Ca2+-binding sites, and it is difficult to explain the decrease in terms of the ordered binding models previously proposed. These results can be explained by a simple model which has four equivalent binding sites that bind Ca2+ and Mg2+ competitively, and showing cooperativity when either Ca2+ or Mg2+ is bound. Mg2+ ion binding to calmodulin was measured in the presence or absence of Ca2+ to confirm the validity of this model, and no Mg2+-specific site was observed.     

Calcium and lanthanide affinity of the EF-loops from the C-terminal domain of calmodulin

To obtain site-specific information about individual EF-hand motifs, the EF-hand Ca(2+)-binding loops from site III and site IV of calmodulin (CaM) were inserted separately into a non-Ca(2+)-binding cell adhesion protein, domain 1 of CD2 (denoted as CaM-CD2-III-5G-52 and CaM-CD2-IV-5G-52). Structural analyses using various spectroscopic methods have shown that the host protein CD2 retains its native structure after the insertion of the 12-residue loops. The Tb(3+) fluorescence enhancement upon formation of a Tb(3+)-protein complex and the direct competition by La(3+) and Ca(2+) suggest that native Ca(2+)-binding pockets are formed in both engineered proteins. Moreover, as revealed by NMR, both Ca(2+) and La(3+) specifically interact with the residues at the grafted EF-loop. The CaM-CD2-III-5G-52 has stronger affinities to Ca(2+), Tb(3+) and La(3+) than CaM-CD2-IV-5G-52, indicating differential intrinsic metal-binding affinities of the EF-loops.     

Kinetics of the Interaction of myo1c with Phosphoinositides

myo1c is a single-headed myosin that dynamically links membranes to the actin cytoskeleton. A putative pleckstrin homology domain has been identified in the myo1c tail that binds phosphoinositides and soluble inositol phosphates with high affinity. However, the kinetics of association and dissociation and the influence of phospholipid composition on the kinetics have not been determined. Stopped-flow spectroscopy was used to measure the binding and dissociation of a recombinant myo1c construct containing the tail and regulatory domains (myo1c^IQ-tail) to and from 100-nm diameter large unilamellar vesicles (LUVs). We found the time course of association of myo1c^IQ-tail with LUVs containing 2% phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) followed a two-exponential time course, and the rate of the predominant fast phase depended linearly upon the total lipid concentration. The apparent second-order rate constant was approximately diffusion-limited. Increasing the molar ratio of anionic phospholipid by adding phosphatidylserine, additional PtdIns(4,5)P₂, or by situating PtdIns(4,5)P₂ in a more physiologically relevant lipid background increased the apparent association rate constant less than 2-fold. myo1c^IQ-tail dissociated from PtdIns(4,5)P₂ at a slower rate (2.0 s⁻¹) than the pleckstrin homology domain of phospholipase C-δ (13 s⁻¹). The presence of additional anionic phospholipid reduced the myo1c^IQ-tail dissociation rate constant >50-fold but marginally changed the dissociation rate of phospholipase C-δ, suggesting that additional electrostatic interactions in myo1c^IQ-tail help to stabilize binding. Remarkably, high concentrations of soluble inositol phosphates induce dissociation of myo1c^IQ-tail from LUVs, suggesting that phosphoinositides are able to bind to and dissociate from myo1c^IQ-tail as it remains bound to the membrane.Myosin-I isoforms are low molecular weight members of the myosin superfamily that link cell membranes with the actin cytoskeleton and play crucial roles driving a diverse array of dynamic membrane processes (1–5). Cell biological studies have shown that myosin-I isoforms localize and fractionate with cell membranes (2, 6), and biochemical experiments have shown myosin-I isoforms bind directly to lipid membranes (7–10). Thus, a key property of some myosin-I isoforms is their ability to bind membranes.myo1c is a widely expressed vertebrate myosin-I isoform that has roles in a variety of important membrane events, including insulin-stimulated fusion of vesicles containing glucose transporter-4 with the plasma membrane (2, 11), compensatory endocytosis following regulated exocytosis (12), and tensioning of mechano-sensitive ion channels (3). The mechanisms of myo1c targeting and anchoring to specific regions on the membrane to support these functions are not well understood. However, evidence is building that myo1c targeting requires direct binding of myo1c to phosphoinositides in cell membranes (13–16).We have shown that binding of myo1c to membranes is mediated by a putative pleckstrin homology (PH)³ domain in its tail that binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) and other phosphoinositides with high affinity (K_d <0.5 μm in terms of accessible phosphoinositide concentration) (13). myo1c also binds soluble inositol phosphates (e.g. inositol 1,4,5-trisphosphate (InsP₃)) with similar affinity. Point mutations of amino acids known to be essential for phosphoinositide binding in other PH domains inhibit myo1c binding to PtdIns(4,5)P₂ in vitro, and these mutations disrupt membrane localization in vivo (13). The affinity of myo1c for PtdIns(4,5)P₂-containing membranes is increased by the presence of additional anionic phospholipids in the membrane. This increased affinity may be due to nonspecific electrostatic interactions between the anionic phospholipids and positively charged regions within the myo1c tail or regulatory domain (13, 17), which is similar to what has been found for the guanine nucleotide exchange factor, ARNO (18). However, high affinity membrane binding via these nonspecific electrostatic interactions (i.e. binding in the absence of PtdIns(4,5)P₂) requires the membrane composition to contain a nonphysiological mole fraction (e.g. >40% phosphatidylserine) of anionic phospholipids (13, 14).Because phosphoinositide binding is important for the cellular localization and function of myo1c (13), it is important to determine the physical constants that define this interaction. Determining the kinetics of membrane attachment will provide insight into the relationship between membrane attachment and actin attachment lifetimes and will also provide details about the role of anionic lipids in regulating membrane attachment. Therefore, we used stopped-flow kinetics to measure the in vitro association and dissociation kinetics of myo1c from LUVs as a function of phosphoinositide composition and anionic charge.     

Identification of the calmodulin binding domain of connexin 43

Calmodulin (CaM) has been implicated in mediating the Ca(2+)-dependent regulation of gap junctions. This report identifies a CaM-binding motif comprising residues 136-158 in the intracellular loop of Cx43. A 23-mer peptide encompassing this CaM-binding motif was shown to bind Ca(2+)-CaM with 1:1 stoichiometry by using various biophysical approaches, including surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and NMR. Far UV circular dichroism studies indicated that the Cx43-derived peptide increased its alpha-helical contents on CaM binding. Fluorescence and NMR studies revealed conformational changes of both the peptide and CaM following formation of the CaM-peptide complex. The apparent dissociation constant of the peptide binding to CaM in physiologic K(+) is in the range of 0.7-1 microM. Upon binding of the peptide to CaM, the apparent K(d) of Ca(2+) for CaM decreased from 2.9 +/- 0.1 to 1.6 +/- 0.1 microM, and the Hill coefficient n(H) increased from 2.1 +/- 0.1 to 3.3 +/- 0.5. Transient expression in HeLa cells of two different mutant Cx43-EYFP constructs without the putative Cx43 CaM-binding site eliminated the Ca(2+)-dependent inhibition of Cx43 gap junction permeability, confirming that residues 136-158 in the intracellular loop of Cx43 contain the CaM-binding site that mediates the Ca(2+)-dependent regulation of Cx43 gap junctions. Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca(2+)-dependent manner, providing a molecular basis for the well characterized Ca(2+)-dependent inhibition of Cx43-containing gap junctions.     

Calcium/calmodulin inhibits direct binding of spectrin to synaptosomal membranes

Brain spectrin, through its beta subunit, binds with high affinity to protein-binding sites on brain membranes quantitatively depleted of ankyrin (Steiner, J., and Bennett, V. (1988) J. Biol. Chem. 263, 14417-14425). In this study, calmodulin is demonstrated to inhibit binding of brain spectrin to synaptosomal membranes. Submicromolar concentrations of calcium are required for inhibition of binding, with half-maximal effects at pCa = 6.5. Calmodulin competitively inhibits binding of spectrin to protein(s) in stripped synaptosomal membranes, with Ki = 1.3 microM in the presence of 10 microM calcium. A reversible receptor-mediated process, and not proteolysis, is responsible for inhibition since the effect of calcium/calmodulin is reversed by the calmodulin antagonist trifluoperazine and by chelation of calcium with sodium [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The target of calmodulin is most likely the spectrin attachment protein(s) rather than spectrin itself since: (a) membrane binding of the brain spectrin beta subunit, which does not associate with calmodulin, is inhibited by calcium/calmodulin, and (b) red cell spectrin which binds calmodulin very weakly, is inhibited from interacting with membrane receptors in the presence of calcium/calmodulin. Ca2+/calmodulin inhibited association of erythrocyte spectrin with synaptosomal membranes but had no effect on binding of erythrocyte or brain spectrin to ankyrin in erythrocyte membranes. These experiments demonstrate the potential for differential regulation of spectrin-membrane protein interactions, with the consequence that Ca2+/calmodulin can dissociate direct spectrin-membrane interactions locally or regionally without disassembly of the areas of the membrane skeleton stabilized by linkage of spectrin to ankyrin. A membrane protein of Mr = 88,000 has been identified that is dissociated from spectrin affinity columns by calcium/calmodulin and is a candidate for the calmodulin-sensitive spectrin-binding site in brain.     

The rate of MgADP binding to and dissociation from acto-S1

The rate of binding and dissociation of MgADP from its ternary complex with actin and S1 was measured by following the extent to which fixed concentrations of MgADP slow down MgATP-induced dissociation of acto-S1. The solution of the equations describing this process shows that at any MgADP concentration the apparent rate of acto-S1 dissociation should be proportional to a square root of the equilibrium constant for MgADP dissociation and to MgATP concentration. By measuring the apparent rate of acto-S1 dissociation as a function of MgATP concentration, the rate of MgADP binding and dissociation were determined as 5 X 10(6) M-1 X s-1 and 1400 s-1, respectively. These rates were unchanged by modification of SH1 thiol of S1 by a variety of fluorescence and spin-labels, but dissociation rate was drastically reduced when SH1 was labelled with 5-iodoacetamidofluorescein.     

Calcium binding to calmodulin leads to an N-terminal shift in its binding site on the ryanodine Receptor

The skeletal muscle calcium release channel, ryanodine receptor, is activated by calcium-free calmodulin and inhibited by calcium-bound calmodulin. Previous biochemical studies from our laboratory have shown that calcium-free calmodulin and calcium bound calmodulin protect sites at amino acids 3630 and 3637 from trypsin cleavage (Moore, C. P., Rodney, G., Zhang, J. Z., Santacruz-Toloza, L., Strasburg, G., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). We now demonstrate that both calcium-free calmodulin and calcium-bound calmodulin bind with nanomolar affinity to a synthetic peptide matching amino acids 3614-3643 of the ryanodine receptor. Deletion of the last nine amino acids (3635-3643) destroys the ability of the peptide to bind calcium-free calmodulin, but not calcium-bound calmodulin. We propose a novel mechanism for calmodulin's interaction with a target protein. Our data suggest that the binding sites for calcium-free calmodulin and calcium-bound calmodulin are overlapping and, when calcium binds to calmodulin, the calmodulin molecule shifts to a more N-terminal location on the ryanodine receptor converting it from an activator to an inhibitor of the channel. This region of the ryanodine receptor has previously been identified as a site of intersubunit contact, suggesting the possibility that calmodulin regulates ryanodine receptor activity by regulating subunit-subunit interactions.     

Structural dynamics of the microtubule binding and regulatory elements in the kinesin-like calmodulin binding protein

Kinesins are molecular motors that power cell division and transport of various proteins and organelles. Their motor activity is driven by ATP hydrolysis and depends on interactions with microtubule tracks. Essential steps in kinesin movement rely on controlled alternate binding to and detaching from the microtubules. The conformational changes in the kinesin motors induced by nucleotide and microtubule binding are coordinated by structural elements within their motor domains. Loop L11 of the kinesin motor domain interacts with the microtubule and is implicated in both microtubule binding and sensing nucleotide bound to the active site of kinesin. Consistent with its proposed role as a microtubule sensor, loop L11 is rarely seen in crystal structures of unattached kinesins. Here, we report four structures of a regulated plant kinesin, the kinesin-like calmodulin binding protein (KCBP), determined by X-ray crystallography. Although all structures reveal the kinesin motor in the ATP-like conformation, its loop L11 is observed in different conformational states, both ordered and disordered. When structured, loop L11 adds three additional helical turns to the N-terminal part of the following helix α4. Although interactions with protein neighbors in the crystal support the ordering of loop L11, its observed conformation suggests the conformation for loop L11 in the microtubule-bound kinesin. Variations in the positions of other features of these kinesins were observed. A critical regulatory element of this kinesin, the calmodulin binding helix positioned at the C-terminus of the motor domain, is thought to confer negative regulation of KCBP. Calmodulin binds to this helix and inserts itself between the motor and the microtubule. Comparison of five independent structures of KCBP shows that the positioning of the calmodulin binding helix is not decided by crystal packing forces but is determined by the conformational state of the motor. The observed variations in the position of the calmodulin binding helix fit the regulatory mechanism previously proposed for this kinesin motor.     

Allostery in Orai1 binding to calmodulin revealed from conformational thermodynamics

Here, we study microscopic mechanism of complex formation between Ca²⁺-bound calmodulin (holoCaM) and Orai1 that regulates Ca²⁺-dependent inactivation process in eukaryotic cells. We compute conformational thermodynamic changes in holoCaM with respect to complex of Orai1 bound to C-terminal domain of holoCaM using histograms of dihedral angles of the proteins over trajectories from molecular dynamics simulations. Our analysis shows that the N-terminal domain residues L4, T5, Q41, N42, T44 and E67 of holoCaM get destabilized and disordered due to Orai1 binding to C-terminal domain of calmodulin affect the N-terminal domain residues. Among these residues, polar T44, having maximum destabilization and disorder via backbone fluctuations, shows the largest change in solvent exposure. This suggests that N-terminal domain is allosterically regulated via T44 by the binding of Orai1 to the C-terminal domain.     

Calcium binding proteins and cellular regulation

An important feature of cellular regulation is the precise control of intracellular calcium levels. This is accomplished both by dynamic organelle release and sequestration of calcium and by specific calcium active transport mechanisms located in the plasma membrane. The actual calcium signal for mediation of a cellular response is carried out by specific intracellular proteins, the most widely studied examples are calmodulin and troponin C. The recent discovery of phospholipid protein kinase and calcimedins suggests receptor mediation via several independent proteins. The physiological importance of a particular protein as a calcium messenger rests on several features: 1) calcium binding is of the order of 1–10 μm, 2) the protein is known to be localized at the site of proposed action, 3) if translocation occurs upon activation, the time required is consistent with the time course of the physiologic response and 4) substrates or effectors at the next level of action when isolated can be demonstrated to have similar activation kinetics as $\text{in situ}$.     

Investigating the disorder–order transition of calmodulin binding domain upon binding calmodulin using molecular dynamics simulation

We have studied the conformational transition of the calmodulin binding domains (CBD) in several calmodulin‐binding kinases, in which CBD changes from the disordered state to the ordered state when binding with calmodulin (CaM). Targeted molecular dynamics simulation was used to investigate the binding process of CaM and CBD of CaM‐dependent kinase I (CaMKI–CBD). The results show that CaMKI–CBD began to form an α‐helix and the interaction free energy between CaM and CaMKI–CBD increased once CaM fully encompassed CaMKI–CBD. Two series of CaM/CBD complex systems, including the complexes of CaM with the initially disordered and the final ordered CBD, were constructed to study the interaction using molecular dynamics simulations. Our analyses suggest that the VDW interaction plays a dominant role in CaM/CBD binding and is a key factor in the disorder–order transition of CBD. Additionally, the entropy effect is not in favor of the formation of the CaM/CBD complex, which is consistent with the experimental evidence. Based on the results, it appears that the CBD conformational change from a non‐compact extended structure to compact α‐helix is critical in gaining a favorable VDW interaction and interaction free energy. Copyright © 2009 John Wiley & Sons, Ltd.     

Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence

Cooperative calcium binding to the two homologous domains of calmodulin (CaM) induces conformational changes that regulate its association with and activation of numerous cellular target proteins. Calcium binding to the pair of high-affinity sites (III and IV in the C-domain) can be monitored by observing calcium-dependent changes in intrinsic tyrosine fluorescence intensity (lambda(ex)/lambda(em) of 277/320 nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is more difficult to measure with optical spectroscopy because that domain of CaM does not contain tryptophan or tyrosine. We recently demonstrated that calcium-dependent changes in intrinsic phenylalanine fluorescence (lambda(ex)/lambda(em) of 250/280 nm) of an N-domain fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci. 10:1758-1768). Using steady-state and time-resolved fluorescence methods, we now show that these excitation and emission wavelength pairs for phenylalanine and tyrosine fluorescence can be used to monitor equilibrium calcium titrations of the individual domains in full-length CaM. Calcium-dependent changes in phenylalanine fluorescence specifically indicate ion occupancy of sites I and II in the N-domain because phenylalanine residues in the C-domain are nonemissive. Tyrosine emission from the C-domain does not interfere with phenylalanine fluorescence signals from the N-domain. This is the first demonstration that intrinsic fluorescence may be used to monitor calcium binding to each domain of CaM. In this way, we also evaluated how mutations of two residues (Arg74 and Arg90) located between sites II and III can alter the calcium-binding properties of each of the domains. The mutation R74A caused an increase in the calcium affinity of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G caused an increase in calcium affinity of sites in both domains. This approach holds promise for exploring the linked energetics of calcium binding and target recognition.