The effect of macromolecular crowding, ionic strength and calcium binding on calmodulin dynamics

The flexibility in the structure of calmodulin (CaM) allows its binding to over 300 target proteins in the cell. To investigate the structure-function relationship of CaM, we combined methods of computer simulation and experiments based on circular dichroism (CD) to investigate the structural characteristics of CaM that influence its target recognition in crowded cell-like conditions. We developed a unique multiscale solution of charges computed from quantum chemistry, together with protein reconstruction, coarse-grained molecular simulations, and statistical physics, to represent the charge distribution in the transition from apoCaM to holoCaM upon calcium binding. Computationally, we found that increased levels of macromolecular crowding, in addition to calcium binding and ionic strength typical of that found inside cells, can impact the conformation, helicity and the EF hand orientation of CaM. Because EF hand orientation impacts the affinity of calcium binding and the specificity of CaM's target selection, our results may provide unique insight into understanding the promiscuous behavior of calmodulin in target selection inside cells.     

Conformational frustration in calmodulin–target recognition

Calmodulin (CaM) is a primary calcium (Ca²⁺)‐signaling protein that specifically recognizes and activates highly diverse target proteins. We explored the molecular basis of target recognition of CaM with peptides representing the CaM‐binding domains from two Ca²⁺‐CaM‐dependent kinases, CaMKI and CaMKII, by employing experimentally constrained molecular simulations. Detailed binding route analysis revealed that the two CaM target peptides, although similar in length and net charge, follow distinct routes that lead to a higher binding frustration in the CaM–CaMKII complex than in the CaM–CaMKI complex. We discovered that the molecular origin of the binding frustration is caused by intermolecular contacts formed with the C‐domain of CaM that need to be broken before the formation of intermolecular contacts with the N‐domain of CaM. We argue that the binding frustration is important for determining the kinetics of the recognition process of proteins involving large structural fluctuations. Copyright © 2015 John Wiley & Sons, Ltd.     

Analysis of the functional coupling between calmodulin's calcium binding and peptide recognition properties

The enhancement of calmodulin's (CaM) calcium binding activity by an enzyme or a recognition site peptide and its diminution by key point mutations at the protein recognition interface (e.g., E84K-CaM), which is more than 20 A away from the nearest calcium ligation structure, can be described by an expanded version of the Adair-Klotz equation for multiligand binding. The expanded equation can accurately describe the calcium binding events and their variable linkage to protein recognition events can be extended to other CaM-regulated enzymes and can potentially be applied to a diverse array of ligand binding systems with allosteric regulation of ligand binding, whether by other ligands or protein interaction. The 1.9 A resolution X-ray crystallographic structure of the complex between E84K-CaM and RS20 peptide, the CaM recognition site peptide from vertebrate smooth muscle and nonmuscle forms of myosin light chain kinase, provides insight into the structural basis of the functional communication between CaM's calcium ligation structures and protein recognition surfaces. The structure reveals that the complex adapts to the effect of the functional mutation by discrete adjustments in the helix that contains E84. This helix is on the amino-terminal side of the helix-loop-helix structural motif that is the first to be occupied in CaM's calcium binding mechanism. The results reported here are consistent with a sequential and cooperative model of CaM's calcium binding activity in which the two globular and flexible central helix domains are functionally linked, and provide insight into how CaM's calcium binding activity and peptide recognition properties are functionally coupled.     

Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity

The first primary structure for a nonmuscle myosin light chain kinase (nmMLCK) has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, and a possible effect on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences (residues 1,068-1,080 and 1,082-1,101, respectively). Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrate a direct link between nmMLCK and non-muscle cell function, and provide a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation.     

Identification of the critical structural determinants of the EF-hand domain arrangements in calcium binding proteins

EF-hand calcium binding proteins (CaBPs) share strong sequence homology, but exhibit great diversity in structure and function. Thus although calmodulin (CaM) and calcineurin B (CNB) both consist of four EF hands, their domain arrangements are quite distinct. CaM and the CaM-like proteins are characterized by an extended architecture, whereas CNB and the CNB-like proteins have a more compact form. In this study, we performed structural alignments and molecular dynamics (MD) simulations on 3 CaM-like proteins and 6 CNB-like proteins, and quantified their distinct structural and dynamical features in an effort to establish how their sequences specify their structures and dynamics. Alignments of the EF2-EF3 region of these proteins revealed that several residues (not restricted to the linker between the EF2 and EF3 motifs) differed between the two groups of proteins. A customized inverse folding approach followed by structural assessments and MD simulations established the critical role of these residues in determining the structure of the proteins. Identification of the critical determinants of the two different EF-hand domain arrangements and the distinct dynamical features relevant to their respective functions provides insight into the relationships between sequence, structure, dynamics and function among these EF-hand CaBPs.     

Prediction of the conformation and geometry of loops in globular proteins: testing ArchDB, a structural classification of loops

In protein structure prediction, a central problem is defining the structure of a loop connecting 2 secondary structures. This problem frequently occurs in homology modeling, fold recognition, and in several strategies in ab initio structure prediction. In our previous work, we developed a classification database of structural motifs, ArchDB. The database contains 12,665 clustered loops in 451 structural classes with information about phi-psi angles in the loops and 1492 structural subclasses with the relative locations of the bracing secondary structures. Here we evaluate the extent to which sequence information in the loop database can be used to predict loop structure. Two sequence profiles were used, a HMM profile and a PSSM derived from PSI-BLAST. A jack-knife test was made removing homologous loops using SCOP superfamily definition and predicting afterwards against recalculated profiles that only take into account the sequence information. Two scenarios were considered: (1) prediction of structural class with application in comparative modeling and (2) prediction of structural subclass with application in fold recognition and ab initio. For the first scenario, structural class prediction was made directly over loops with X-ray secondary structure assignment, and if we consider the top 20 classes out of 451 possible classes, the best accuracy of prediction is 78.5%. In the second scenario, structural subclass prediction was made over loops using PSI-PRED (Jones, J Mol Biol 1999;292:195-202) secondary structure prediction to define loop boundaries, and if we take into account the top 20 subclasses out of 1492, the best accuracy is 46.7%. Accuracy of loop prediction was also evaluated by means of RMSD calculations.     

Identification of the critical structural determinants of the EF-hand domain arrangements in calcium binding proteins

EF-hand calcium binding proteins (CaBPs) share strong sequence homology, but exhibit great diversity in structure and function. Thus although calmodulin (CaM) and calcineurin B (CNB) both consist of four EF hands, their domain arrangements are quite distinct. CaM and the CaM-like proteins are characterized by an extended architecture, whereas CNB and the CNB-like proteins have a more compact form. In this study, we performed structural alignments and molecular dynamics (MD) simulations on 3 CaM-like proteins and 6 CNB-like proteins, and quantified their distinct structural and dynamical features in an effort to establish how their sequences specify their structures and dynamics. Alignments of the EF2-EF3 region of these proteins revealed that several residues (not restricted to the linker between the EF2 and EF3 motifs) differed between the two groups of proteins. A customized inverse folding approach followed by structural assessments and MD simulations established the critical role of these residues in determining the structure of the proteins. Identification of the critical determinants of the two different EF-hand domain arrangements and the distinct dynamical features relevant to their respective functions provides insight into the relationships between sequence, structure, dynamics and function among these EF-hand CaBPs.     

Comparison of Ca++-regulated events in the intestinal brush border

The intestinal epithelial cell and specifically the cytoskeleton of the brush border are thought to be controlled by micromolar levels of free calcium. Calcium-binding proteins of this system include intestinal calcium binding protein (CaBP), calmodulin (CaM), villin, and a 36,000-mol-wt protein substrate of tyrosine kinases. To assess the sequence of events as the intracellular Ca++ level rises, we determined the amount of CaM and CaBP in the intestinal epithelium by western blotting and tested the Ca++ binding of CaM and CaBP by equilibrium dialysis. The Ca++-dependent actin severing activity of villin was analyzed in the presence of physiological CaM levels and increasing calcium concentrations. In addition, we analyzed the Ca++ levels required for interaction between CaM and the microvillus 110,000-mol-wt protein as well as fodrin and the interaction between a polypeptide of 36,000 mol wt (P-36) and actin. The results suggest that CaBP serves as the predominant Ca++ buffer in the cell, but CaM can effectively buffer ionic calcium in the microvillus and thus protect actin from the severing activity of villin. CaM binds to its cytoskeletal receptors, 110,000-mol-wt protein and fodrin differently, governed by the free Ca++ and pH. The interaction between P-36 and actin, however, appears to require an unphysiologically high calcium concentration (10(-4) to 10(-3) M) to be meaningful. The results provide a coherent picture of the different Ca++ regulated events occurring when the free calcium rises into the micromolar level in this unique system. This study would suggest that as the Ca++ rises in the intestinal epithelial cell an ordered sequence of Ca++ saturation of intracellular receptors occurs with the order from the lowest to highest Ca++ requirements being CaBP less than CaM less than villin less than P-36.     

Closer proximity between opposing domains of vertebrate calmodulin following deletion of Met(145)-Lys(148)

To investigate the structural linkage between the opposing globular domains in vertebrate calmodulin (CaM), we have constructed a CaM mutant (CaMX(145)) deficient in the last four amino acids between Met(145) and Lys(148) at the carboxyl terminal. Circular dichroism and fluorescence spectroscopic measurements were used to detect changes in the average secondary and tertiary structure of CaMX(145) in comparison to full-length CaM. Complementary measurements of the maximal calcium-binding stoichiometry and ability to activate the plasma membrane (PM) Ca-ATPase permit an assessment of the functional significance of observed structural changes. In comparison with native CaM, we find that CaMX(145) exhibits (i) a large reduction in alpha-helical content, (ii) a dramatic decrease in the average spatial separation between the opposing globular domains, (iii) the loss of one high-affinity calcium-binding site, and (iv) a diminished binding affinity for the PM-Ca-ATPase. Thus, the sequence near the carboxyl terminus functions to stabilize high-affinity calcium binding at one site and facilitates important intramolecular interactions that maintain CaM in an extended conformation. However, despite the large conformational changes resulting from deletion of the last four amino acids at the carboxyl terminal, CaMX(145) can fully activate the PM-Ca-ATPase. These results indicate that target protein binding can restore the nativelike structure critical to function, emphasizing that the structure of the central helix is not critical to CaM function under equilibrium conditions. Rather, the central helix functions to maintain the spatial separation between the opposing domains in CaM that may be critical to high-affinity binding and the rapid activation of the PM-Ca-ATPase, which are necessary for optimal calcium signaling. Thus, following initial association between CaM and target proteins, structural changes involving the carboxyl-terminal sequence have the potential to play an important role in triggering the structural collapse of CaM that facilitates the rapid and cooperative binding of the opposing globular domains with target proteins, which is important to high-affinity binding and rapid enzyme activation.     

Calmodulin, conformational states, and calcium signaling. A single-molecule perspective

Single-molecule fluorescence measurements can provide a new perspective on the conformations, dynamics, and interactions of proteins. Recent examples are described illustrating the application of single-molecule fluorescence spectroscopy to calcium signaling proteins with an emphasis on the new information available in single-molecule fluorescence burst measurements, resonance energy transfer, and polarization modulation methods. Calcium signaling pathways are crucial in many cellular processes. The calcium binding protein calmodulin (CaM) serves as a molecular switch to regulate a network of calcium signaling pathways. Single-molecule spectroscopic methods can yield insights into conformations and dynamics of CaM and CaM-regulated proteins. Examples include studies of the conformations and dynamics of CaM, binding of target peptides, and interaction with the plasma-membrane Ca2+ pump. Single-molecule resonance energy transfer measurements revealed conformational substates of CaM, and single-molecule polarization modulation spectroscopy was used to probe interactions between CaM and the plasma-membrane Ca2+-ATPase.     

Structural uncoupling between opposing domains of oxidized calmodulin underlies the enhanced binding affinity and inhibition of the plasma membrane Ca-ATPase

Stabilization of the plasma membrane Ca-ATPase (PMCA) in an inactive conformation upon oxidation of multiple methionines in the calcium regulatory protein calmodulin (CaM) is part of an adaptive cellular response to minimize ATP utilization and the generation of reactive oxygen species (ROS) under conditions of oxidative stress. To differentiate oxidant-induced structural changes that selectively modify the amino-terminal domain of CaM from those that modulate the conformational coupling between the opposing domains, we have engineered a tetracysteine binding motif within helix A in the amino-terminal domain of calmodulin (CaM) that permits the selective and rigid attachment of the conformationally sensitive fluorescent probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)(2) (FlAsH-EDT(2)). The position of the FlAsH label in the amino-terminal domain provides a signal for monitoring its binding to the CaM-binding sequence of the PMCA. Following methionine oxidation, there is an enhanced binding affinity between the amino-terminal domain and the CaM-binding sequence of the PMCA. To identify oxidant-induced structural changes, we used frequency domain fluorescence anisotropy measurements to assess the structural coupling between helix A and the amino- and carboxyl-terminal domains of CaM. Helix A undergoes large amplitude motions in apo-CaM; following calcium activation, helix A is immobilized as part of a conformational switch that couples the opposing domains of CaM to stabilize the high-affinity binding cleft associated with target protein binding. Methionine oxidation disrupts the structural coupling between opposing globular domains of CaM, without affecting the calcium-dependent immobilization of helix A associated with activation of the amino-terminal domain to promote high-affinity binding to target proteins. We suggest that this selective disruption of the structural linkage between the opposing globular domains of CaM relieves steric constraints associated with high-affinity target binding, permitting the formation of new contact interactions between the amino-terminal domain and the CaM-binding sequence that stabilizes the PMCA in an inhibited conformation.     

Distinguishing unfolding and functional conformational transitions of calmodulin using ultraviolet resonance Raman spectroscopy

Calmodulin (CaM) is a ubiquitous moderator protein for calcium signaling in all eukaryotic cells. This small calcium‐binding protein exhibits a broad range of structural transitions, including domain opening and folding–unfolding, that allow it to recognize a wide variety of binding partners in vivo. While the static structures of CaM associated with its various binding activities are fairly well‐known, it has been challenging to examine the dynamics of transition between these structures in real‐time, due to a lack of suitable spectroscopic probes of CaM structure. In this article, we examine the potential of ultraviolet resonance Raman (UVRR) spectroscopy for clarifying the nature of structural transitions in CaM. We find that the UVRR spectral change (with 229 nm excitation) due to thermal unfolding of CaM is qualitatively different from that associated with opening of the C‐terminal domain in response to Ca²⁺ binding. This spectral difference is entirely due to differences in tertiary contacts at the interdomain tyrosine residue Tyr138, toward which other spectroscopic methods are not sensitive. We conclude that UVRR is ideally suited to identifying the different types of structural transitions in CaM and other proteins with conformation‐sensitive tyrosine residues, opening a path to time‐resolved studies of CaM dynamics using Raman spectroscopy.     

Calmodulin’s flexibility allows for promiscuity in its interactions with target proteins and peptides

The small bilobal calcium regulatory protein calmodulin (CaM) activates numerous target enzymes in response to transient changes in intracellular calcium concentrations. Binding of calcium to the two helix-loop-helix calcium-binding motifs in each of the globular domains induces conformational changes that expose a methionine-rich hydrophobic patch on the surface of each domain of the protein, which it uses to bind to peptide sequences in its target enzymes. Although these CaM-binding domains typically have little sequence identity, the positions of several bulky hydrophobic residues are often conserved, allowing for classification of CaM-binding domains into recognition motifs, such as the 1–14 and 1–10 motifs. For calcium-independent binding of CaM, a third motif known as the IQ motif is also common. Many CaM-peptide complexes have globular conformations, where CaM’s central linker connecting the two domains unwinds, allowing the protein to wrap around a single predominantly α-helical target peptide sequence. However, novel structures have recently been reported where the conformation of CaM is highly dissimilar to these globular complexes, in some instances with less than a full compliment of bound calcium ions, as well as novel stoichiometries. Furthermore, many divergent CaM isoforms from yeast and plant species have been discovered with unique calcium-binding and enzymatic activation characteristics compared to the single CaM isoform found in mammals.     

A citrate-binding site in calmodulin

Calmodulin (CaM) is a major Ca2+ messenger which, upon Ca2+ activation, binds and activates a number of target enzymes involved in crucial cellular processes. The dependence on Ca2+ ion concentration suggests that CaM activation may be modulated by low-affinity Ca2+ chelators. The effect on CaM structure and function of citrate ion, a Ca2+ chelator commonly found in the cytosol and the mitochondria, was therefore investigated. A series of structural and biochemical methods, including tryptic mapping, immunological recognition by specific monoclonal antibodies, CIDNP-NMR, binding to specific ligands and association with radiolabeled citrate, showed that citrate induces conformational modifications in CaM which affect the shape and activity of the protein. These changes were shown to be associated with the C-terminal lobe of the molecule and involve actual binding of citrate to CaM. Analyzing X-ray structures of several citrate-binding proteins by computerized molecular graphics enabled us to identify a putative citrate-binding site (CBS) on the CaM molecule around residues Arg106-His107. Owing to the tight proximity of this site to the third Ca(2+)-binding loop of CaM, binding of citrate is presumably translated into changes in Ca2+ binding to site III (and indirectly to site IV). These changes apparently affect the structural and biochemical properties of the conformation-sensitive protein.     

Between promiscuity and specificity: novel roles of EF-hand calcium sensors in neuronal Ca2+ signalling

In recent years, substantial progress has been made towards an understanding of the physiological function of EF-hand calcium sensor proteins of the Calmodulin (CaM) superfamily in neurons. This deeper appreciation is based on the identification of novel target interactions, structural studies and the discovery of novel signalling mechanisms in protein trafficking and synaptic plasticity, in which CaM-like sensor proteins appear to play a role. However, not all interactions are of plausible physiological relevance and in many cases it is not yet clear how the CaM signaling network relates to the proposed function of other EF-hand sensors. In this review, we will summarize these findings and address some of the open questions on the functional role of EF-hand calcium binding proteins in neurons.     

Helix A stabilization precedes amino-terminal lobe activation upon calcium binding to calmodulin

The structural coupling between opposing domains of CaM was investigated using the conformationally sensitive biarsenical probe 4,5-bis(1,3,2-dithioarsolan-2-yl)resorufin (ReAsH), which upon binding to an engineered tetracysteine motif near the end of helix A (Thr-5 to Phe-19) becomes highly fluorescent. Changes in conformation and dynamics are reflective of the native CaM structure, as there is no change in the (1)H- (15)N HSQC NMR spectrum in comparison to wild-type CaM. We find evidence of a conformational intermediate associated with CaM activation, where calcium occupancy of sites in the amino-terminal and carboxyl-terminal lobes of CaM differentially affect the fluorescence intensity of bound ReAsH. Insight into the structure of the conformational intermediate is possible from a consideration of calcium-dependent changes in rates of ReAsH binding and helix A mobility, which respectively distinguish secondary structural changes associated with helix A stabilization from the tertiary structural reorganization of the amino-terminal lobe of CaM necessary for high-affinity binding to target proteins. Helix A stabilization is associated with calcium occupancy of sites in the carboxyl-terminal lobe ( K d = 0.36 +/- 0.04 microM), which results in a reduction in the rate of ReAsH binding from 4900 M (-1) s (-1) to 370 M (-1) s (-1). In comparison, tertiary structural changes involving helix A and other structural elements in the amino-terminal lobe require calcium occupancy of amino-terminal sites (K d = 18 +/- 3 microM). Observed secondary and tertiary structural changes involving helix A in response to the sequential calcium occupancy of carboxyl- and amino-terminal lobe calcium binding sites suggest an important involvement of helix A in mediating the structural coupling between the opposing domains of CaM. These results are discussed in terms of a model in which carboxyl-terminal lobe calcium activation induces secondary structural changes within the interdomain linker that release helix A, thereby facilitating the formation of calcium binding sites in the amino-terminal lobe and linked tertiary structural rearrangements to form a high-affinity binding cleft that can associate with target proteins.     

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

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

SSToSS--sequence-structural templates of single-member superfamilies

The presence of sequence homologues and the availability of structural information of proteins enable better understanding of the biological function of a protein family. A majority of entries in protein structural databank are single member superfamilies for which it is hard to derive motifs due to the paucity of structural homologues. Important conserved segments for these superfamilies have been identified and compiled into a database, SSToSS (Sequence Structural Templates of Single member Superfamily). Conserved regions, recognized by permitted amino acid exchanges, are mapped on the structure and various structural features (solvent accessibility, secondary structure content, hydrogen bonding and residue packing) are examined. These conserved segments with high structural feature content are projected as sequence-structural templates for the particular superfamily member. Interactive three-dimensional displays of the templates in three-dimensional structure (in Chime and RASMOL) are provided for better understanding and visualization. In SSToSS database, we also provide the application of sequence-structural templates in three different areas: multiple-motif based sequence search, multiple sequence alignment and homology modeling. In each case, the inclusion of the sequence-structural templates can give rise to sensitive and accurate results. This enables the inclusion of singletons to provide added value to the recognition of additional members, comparative modeling and in designing experiments.