Evolutionary Covariant Positions within Calmodulin EF-hand Sequences Promote Ligand Binding

Intracellular calcium signaling is an essential regulatory mechanism through calcium-mediated signal transduction pathways involved in many cell processes, such as exocytosis, motility, apoptosis, excitability, transcription, and muscle contraction. The calcium-binding, ubiquitous, and highly conserved protein calmodulin (CaM) is an important regulator of hundreds of target proteins involved in cellular calcium signaling. CaM comprises of two pairs of EF-hand calcium-binding domains and these structural regions of the protein are highly conserved. Studying the molecular mechanisms underlying the binding of calcium to the EF-hands of CaM is critical in understanding the calcium-mediated cellular processes and how improper binding of calcium can lead to various human pathologies. Previous site-specific binding measurements indicate that each of the four EF-hands of CaM have distinct affinities for calcium. In this study, we have utilized covariance patterns and site-specific mutagenesis to analyze calcium affinity in the two EF-hands of the N-lobe of CaM in order to determine the specific amino acids that are evolutionarily conserved to coordinate calcium. The specific amino acids in CaM that we studied are theorized to coevolve, which means that in their protein coding genes, when a mutation occurs, a compensatory mutation is likely to follow to conserve structure and function of CaM. Since CaM is a highly conserved protein with a known structure, covariance analyses will help in understanding which amino acid contacts are most important for the coordination of calcium in the EF-hands of CaM and to determine which amino acids are under evolutionary constraint. Covariance algorithms, multiple sequence analyses and accompanied protein structure analyses were used to identify the two high scoring amino acid pairs in the N-lobe EF-hands: positions 22 and 24 in EF-hand site 1 and positions 58 and 60 in EF-hand site 2. The amino acids in these locations were mutated and accompanied calcium binding was measured to better understand the effects of the mutations on calcium binding. We have found that both the D24N mutation in site 1 and the D58N mutation in site 2 disrupt binding likely due to the removal of a necessary aspartate in the binding site. However, the combined D58N and N60D mutations restore binding in site 2 by providing the necessary aspartate in the covariant location. The N60D mutation by itself has little impact on calcium binding in site 2. Therefore, it is evident that evolution conserves at least one aspartate in the covariant positions of the binding site and the presence of two aspartates in the covariant positions of the binding site has little affect on calcium binding. We are currently studying the covariant positions in site 1 and future work includes structurally analyzing the covariant positions in the C-lobe of CaM and studying covariance patterns of other calcium-binding proteins with EF-hand binding domains.Biochemistr

In Silico Prediction and Analysis of Caenorhabditis EF-hand Containing Proteins

Calcium (Ca+2) is a ubiquitous messenger in eukaryotes including Caenorhabditis. Ca+2-mediated signalling processes are usually carried out through well characterized proteins like calmodulin (CaM) and other Ca+2 binding proteins (CaBP). These proteins interact with different targets and activate it by bringing conformational changes. Majority of the EF-hand proteins in Caenorhabditis contain Ca+2 binding motifs. Here, we have performed homology modelling of CaM-like proteins using the crystal structure of Drosophila melanogaster CaM as a template. Molecular docking was applied to explore the binding mechanism of CaM-like proteins and IQ1 motif which is a ∼25 residues and conform to the consensus sequence (I, L, V)QXXXRXXXX(R,K) to serve as a binding site for different EF hand proteins. We made an attempt to identify all the EF-hand (a helix-loop-helix structure characterized by a 12 residues loop sequence involved in metal coordination) containing proteins and their Ca+2 binding affinity in Caenorhabditis by analysing the complete genome sequence. Docking studies revealed that F165, F169, L29, E33, F44, L57, M61, M96, M97, M108, G65, V115, F93, N104, E144 of CaM-like protein is involved in the interaction with IQ1 motif. A maximum of 170 EF-hand proteins and 39 non-EF-hand proteins with Ca+2/metal binding motif were identified. Diverse proteins including enzyme, transcription, translation and large number of unknown proteins have one or more putative EF-hands. Phylogenetic analysis revealed seven major classes/groups that contain some families of proteins. Various domains that we identified in the EF-hand proteins (uncharacterized) would help in elucidating their functions. It is the first report of its kind where calcium binding loop sequences of EF-hand proteins were analyzed to decipher their calcium affinities. Variation in Ca+2-binding affinity of EF-hand CaBP could be further used to study the behaviour of these proteins. Our analyses postulated that Ca+2 is likely to be key player in Caenorhabditis cell signalling

Determining The Site Specific Metal Binding and Structural Properties of EF-Hand Protein Using Grafting Approach

Calmodulin is an essential EF-hand protein with a helix-loop-helix calcium binding motif. Understanding Ca(II) dependent activation of calmodulin and other EF-hand proteins is limited by Ca(II)-induced conformational change, multiple and cooperative binding of Ca(II) ions, and interactions between the paired EF-hand motifs. The goal of this research project is to probe key determinants for calcium binding properties and pairing interactions at the site specific level using a grafting approach and high resolution NMR. An individual Ca(II) binding site of the EF-hand motifs of calmodulin was grafted into a non-calcium dependent protein, CD2, to bypass limitations associated with natural EF-hand proteins and peptide fragments. Using high resolution NMR, we have shown that the grafted EF-loop III of calmodulin in the host protein retains its native conformation with a strong loop and β-conformation preference. Grafted ligand residues in the engineered protein are directly involved in binding of Ca(II) and La(III). The NMR studies support our hypothesis that both ligand arrangement and dynamic properties play essential role in tuning Ca(II) binding affinities. Using pulse-field diffusion NMR and protein engineering, we further demonstrated that grafted EF- loop remains as a monomer. Although the EF-loop with flanking helices dimerizes in the presence of Ca(II). Additionally, removal of conserved hydrophobic residues at the flanking helices of the EF-hand motif leads to be monomer in the absence and presence of metal ions. Our results suggest that conserved hydrophobic residues are essential for the pair-paired interaction in the coupled EF-hand protein. We have shown that our developed grafting approach can be applied to probe intrinsic Ca(II) binding affinities of different Ca(II) binding sites

Dynamik der cGMP-Synthese in Sehzellen - Regulation membranständiger Guanylatzyklasen durch modifizierte Kalzium-Sensor-Proteine

Membrane bound guanylate cyclases GC1 and GC2 are important for the phototransduction in photoreceptors. They regulate, in an interplay with a phosphodiesterase, the concentration of the intracellular messenger cyclic guanosine monophosphate (cGMP). At low calciumconcentrations both GCs are activated by guanylate cyclase activating proteins (GCAP1 and GCAP2). The cGMP-concentration increases and the photoreceptor adapts. If GCs become activated a GC-dimer forms a complex with an unidentified number of GCAPs. The stoichiometry and calcium dependent conformational changes within this complex are unknown. GCAP2 has three cysteine residues, interestingly one in the first and one in the third calcium-binding-motif (EF-hand-motif). In this work cysteine mutants of GCAP2 were generated, heterologously expressed, and purified. All cysteine mutants exhibited EC50- and IC$_{50}$-values comparable to GCAP2-wildtype. However, mutants with no cysteine residue in the first EF-Hand-motif activated GCs weaker compared to GCAP2-wildtype. This indicates an important function of this cysteine residue in GC-regulation. In further experiments I investigated the accessibility of cysteine residues for the thiolreactive substance 5,5'-Dithiobis(2-nitrobenzoic acid). The cysteine residues within the first and third EF-hand-motif were only accessible at low calcium concentrations. This was surprising because the first EF-hand-motif is assumed to bind calcium with only very low affinity. Thus no calcium induced conformational change was expected. By determining the calcium sensitivities of the DTNB-reaction, which could be interpreted as apparent calcium affinities of the EF-hand-motifs, I was able to develop following model: calcium dissociation from the third EF-hand-motif in GCAP2 induces a conformational change that causes GC-activation. Furthermore I demonstrated that magnesium increases the apparent calcium affinities of the first EF-hand-motif in GCAP2 and of the first and third EF-hand-motif in GCAP1. I coupled thiolreactive dyes to cysteine mutants of GCAP2. Thereby I wanted to detect conformational changes in the vicinity of the dye. Furthermore, by recording Förster Resonance Energy Transfer between dye labeled GCAP1- and GCAP2-mutants I wanted to show the simultaneous binding of both GCAP isoforms to GC1. Both experimental approaches gave negative results. Using a soluble, enzymatic active GC1-construct instead of wildtype GC1 produced also not the expected results

Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins

BACKGROUND: A wide range of stimuli evoke rapid and transient increases in [Ca(2+)](cyt )in plant cells which are transmitted by protein sensors that contain EF-hand motifs. Here, a group of Oryza sativa L. genes encoding calmodulin (CaM) and CaM-like (CML) proteins that do not possess functional domains other than the Ca(2+)-binding EF-hand motifs was analyzed. RESULTS: By functional analyses and BLAST searches of the TIGR rice database, a maximum number of 243 proteins that possibly have EF-hand motifs were identified in the rice genome. Using a neighbor-joining tree based on amino acid sequence similarity, five loci were defined as Cam genes and thirty two additional CML genes were identified. Extensive analyses of the gene structures, the chromosome locations, the EF-hand motif organization, expression characteristics including analysis by RT-PCR and a comparative analysis of Cam and CML genes in rice and Arabidopsis are presented. CONCLUSION: Although many proteins have unknown functions, the complexity of this gene family indicates the importance of Ca(2+)-signals in regulating cellular responses to stimuli and this family of proteins likely plays a critical role as their transducers

Fraternal twins: Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1, two homologous EF-hand containing calcium binding adaptor proteins with distinct functions

Changes in the intracellular calcium concentration govern cytoskeletal rearrangement, mitosis, apoptosis, transcriptional regulation or synaptic transmission, thereby, regulating cellular effector and organ functions. Calcium binding proteins respond to changes in the intracellular calcium concentration with structural changes, triggering enzymatic activation and association with downstream proteins. One type of calcium binding proteins are EF-hand super family proteins. Here, we describe two recently discovered homologous EF-hand containing adaptor proteins, Swiprosin-1/EF-hand domain containing 2 (EFhd2) and Swiprosin-2/EF-hand domain containing 1 (EFhd1), which are related to allograft inflammatory factor-1 (AIF-1). For reasons of simplicity and concision we propose to name Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1 from now on EFhd2 and EFhd1, according to their respective gene symbols. AIF-1 and Swiprosin-1/EFhd2 are already present in Bilateria, for instance in Drosophila melanogaster and Caenhorhabditis elegans. Swiprosin-2/EFhd1 arose later from gene duplication in the tetrapodal lineage. Secondary structure prediction of AIF-1 reveals disordered regions and one functional EF-hand. Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1 exhibit a disordered region at the N-terminus, followed by two EF-hands and a coiled-coil domain. Whereas both proteins are similar in their predicted overall structure they differ in a non-homologous stretch of 60 amino acids just in front of the EF-hands. AIF-1 controls calcium-dependent cytoskeletal rearrangement in innate immune cells by means of its functional EF-hand. We propose that Swiprosin-1/EFhd2 as well is a cytoskeleton associated adaptor protein involved in immune and brain cell function. Pro-inflammatory conditions are likely to modulate expression and function of Swiprosin-1/EFhd2. Swiprosin-2/EFhd1, on the other hand, modulates apoptosis and differentiation of neuronal and muscle precursor cells, probably through an association with mitochondria. We suggest furthermore that Swiprosin-2/EFhd1 is part of a cellular response to oxidative stress, which could explain its pro-survival activity in neuronal, muscle and perhaps some malignant tissues

A high-resolution structure of the EF-hand domain of human polycystin-2.

Autosomal dominant polycystic kidney disease (ADPKD) affects over 1:1000 of the worldwide population and is caused by mutations in two genes, PKD1 and PKD2. PKD2 encodes a 968-amino acid membrane spanning protein, Polycystin-2 (PC-2), which is a member of the TRP ion channel family. The C-terminal cytoplasmic tail contains an EF-hand motif followed by a short coiled-coil domain. We have determined the structure of the EF-hand region of PC-2 using NMR spectroscopy. The use of different boundaries, compared with those used in previous studies, have enabled us to determine a high resolution structure and show that the EF hand motif forms a standard calcium-binding pocket. The affinity of this pocket for calcium has been measured and mutants that both decrease and increase its affinity for the metal ion have been created

The myeloid expressed EF-hand proteins display a diverse pattern of lipid raft association

AbstractEF-hand proteins are known to translocate to membranes, suggesting that they are involved in signaling events located in the cell membrane. Many proteins involved in signaling events associate cholesterol rich membrane domains, so called lipid rafts, which serve as platforms for controlled protein–protein interaction. Here, we demonstrate that the myeloid expressed EF-hand proteins can be distinguished into three classes with respect to their membrane association. Grancalcin, a myeloid expressed penta EF-hand protein, is constitutively located in lipid rafts. S100A9 (MRP14) and S100A8 (MRP8) are translocated into detergent resistant lipid structures only after calcium activation of the neutrophils. However, the S100A9/A8 membrane association is cholesterol and sphingolipid independent. On the other hand, the association of S100A12 (EN-RAGE) and S100A6 (calcyclin) with membranes is detergent sensitive. These diverse affinities to lipid structures of the myeloid expressed EF-hand proteins most likely reflect their different functions in neutrophils

Functional characteristics of the calcium modulated proteins seen from an evolutionary perspective

We have constructed dendrograms relating 173 EF-hand proteins of known amino acid sequence. We aligned all of these proteins by their EF-hand domains, omitting interdomain regions. Initial dendrograms were computed by minimum mutation distance methods. Using these as starting points, we determined the best dendrogram by the method of maximum parsimony, scored by minimum mutation distance. We identified 14 distinct subfamilies as well as 6 unique proteins that are perhaps the sole representatives of other subfamilies. This information is given in tabular form. Within subfamilies one can easily align interdomain regions. The resulting dendrograms are very similar to those computed using domains only. Dendrograms constructed using pairs of domains show general congruence. However, there are enough exceptions to caution against an overly simple scheme in which one pair of gene duplications leads from one domain precurser to a four domain prototype from which all other forms evolved. The ability to bind calcium was lost and acquired several times during evolution. The distribution of introns does not conform to the dendrogram based on amino acid sequences. The rates of evolution appear to be much slower within subfamilies, especially within calmodulin, than those prior to the definition of subfamily