O objetivo deste trabalho é estudar a afinidade e especificidade de sítios EF-hand, e correlacionar estas propriedades com a estrutura primária do sítio, com as interações entre aminoácidos nas posições de coordenação, e com prováveis características da estrutura terciária da proteína. Os efeitos de três mutações no sítio EF-hand da cadeia leve regulatória de miosina (RLC) foram estudados: D5S, em que o aspartato presente na posição 5 do sítio foi substituído por uma serina; D9E, substituindo o aspartato da posição 9 por um glutamato, e D12E, substituindo o aspartato da posição 12 por um glutamato. Todas as combinações destas três mutações foram produzidas. Os mutantes simples D5S e D9E e o duplo mutante D5S/D9E têm baixa afinidade por cálcio. Todos os mutantes contendo a mutação D12E são específicos para cálcio, com afinidades maiores que RLC tipo selvagem. Todos os mutantes estudados possuem menor afinidade por magnésio que RLC tipo selvagem. As mudanças na energia livre de ligação e as energias de acoplamento sugerem que há interações inespecíficas entre todas as posições, e uma interação específica entre uma serina na posição 5 e um glutamato na posição 9. Esta interação ocorre somente na presença de magnésio, e quando há um aspartato na posição 12. O glutamato na posição 9 pode ser capaz de coordenar a ligação de magnésio diretamente no duplo mutante D5S/D9E. Embora um aminoácido ou um certo arranjo deles possa determinar características específicas do sítio EF-hand, o conjunto de propriedades depende da estrutura terciária, uma vez que sítios homólogos podem possuir afinidades e especificidades bastante diferentes. / The aim of this thesis was to study affinity and specificity in EF-hand sites, and how these properties are related to the site primary structure, interactions between amino acids in coordinating positions, and probable tertiary structure properties. The effects of three mutations on the EF-hand Ca2+/Mg2+ binding site of smooth muscle myosin regulatory light chain (RLC) were studied: D5S, in which an aspartate is replaced by a serine in position 5 of the loop; D9E, in which an aspartate is replaced by a glutamate in position 9, and D12E, in which the aspartate in position 12 is replaced by a glutamate. All possible combinations of the three mutations were produced. The single mutants D5S and D9E and the double mutant D5S/D9E have low affinity for Ca2+. All the mutants containing mutation D12E are Ca2+-specific and have higher affinities than wild type, even when containing mutations D5S or D9E. All the mutants studied have lower affinity for Mg2+ than wild type RLC. Coupling energies and changes in binding free energy suggest that all positions interact in a non-specific way, and a specific interaction occurs between a serine in position 5 and a glutamate in position 9. This interaction can be seen only in the presence of magnesium, and with an apartate in position 12. Glutamate in position 9 may be able to coordinate Mg2+ directly in the double mutant D5S/D9E. Even though an amino acid or a few amino acids in certain positions can determine specific characteristics for an EF-hand site, the site properties depend on the tertiary structure, since homologue sites can have very different affinities and specificities.

O objetivo deste trabalho é estudar a afinidade e especificidade de sítios EF-hand, e correlacionar estas propriedades com a estrutura primária do sítio, com as interações entre aminoácidos nas posições de coordenação, e com prováveis características da estrutura terciária da proteína. Os efeitos de três mutações no sítio EF-hand da cadeia leve regulatória de miosina (RLC) foram estudados: D5S, em que o aspartato presente na posição 5 do sítio foi substituído por uma serina; D9E, substituindo o aspartato da posição 9 por um glutamato, e D12E, substituindo o aspartato da posição 12 por um glutamato. Todas as combinações destas três mutações foram produzidas. Os mutantes simples D5S e D9E e o duplo mutante D5S/D9E têm baixa afinidade por cálcio. Todos os mutantes contendo a mutação D12E são específicos para cálcio, com afinidades maiores que RLC tipo selvagem. Todos os mutantes estudados possuem menor afinidade por magnésio que RLC tipo selvagem. As mudanças na energia livre de ligação e as energias de acoplamento sugerem que há interações inespecíficas entre todas as posições, e uma interação específica entre uma serina na posição 5 e um glutamato na posição 9. Esta interação ocorre somente na presença de magnésio, e quando há um aspartato na posição 12. O glutamato na posição 9 pode ser capaz de coordenar a ligação de magnésio diretamente no duplo mutante D5S/D9E. Embora um aminoácido ou um certo arranjo deles possa determinar características específicas do sítio EF-hand, o conjunto de propriedades depende da estrutura terciária, uma vez que sítios homólogos podem possuir afinidades e especificidades bastante diferentes. / The aim of this thesis was to study affinity and specificity in EF-hand sites, and how these properties are related to the site primary structure, interactions between amino acids in coordinating positions, and probable tertiary structure properties. The effects of three mutations on the EF-hand Ca2+/Mg2+ binding site of smooth muscle myosin regulatory light chain (RLC) were studied: D5S, in which an aspartate is replaced by a serine in position 5 of the loop; D9E, in which an aspartate is replaced by a glutamate in position 9, and D12E, in which the aspartate in position 12 is replaced by a glutamate. All possible combinations of the three mutations were produced. The single mutants D5S and D9E and the double mutant D5S/D9E have low affinity for Ca2+. All the mutants containing mutation D12E are Ca2+-specific and have higher affinities than wild type, even when containing mutations D5S or D9E. All the mutants studied have lower affinity for Mg2+ than wild type RLC. Coupling energies and changes in binding free energy suggest that all positions interact in a non-specific way, and a specific interaction occurs between a serine in position 5 and a glutamate in position 9. This interaction can be seen only in the presence of magnesium, and with an apartate in position 12. Glutamate in position 9 may be able to coordinate Mg2+ directly in the double mutant D5S/D9E. Even though an amino acid or a few amino acids in certain positions can determine specific characteristics for an EF-hand site, the site properties depend on the tertiary structure, since homologue sites can have very different affinities and specificities.

Essential metals like Ca2+ and Zn2+ play critical roles in biological processes through protein interactions. Conversely, non-essential metals (e.g., Gd3+ and Pb2+) also interact with proteins, often with toxic effects. Molecular metal toxicity is assumed to be due to ionic displacement, and studies have demonstrated that Pb2+ replaces Zn2+, Ca2+ and other essential metals in proteins. The focus of this work was to compare protein Ca2+ and Pb2+ -binding sites and to investigate a mechanism of Pb2+ toxicity in Ca2+-binding proteins, particularly the intracellular trigger protein calmodulin (CaM) which binds four Ca2+ ions and interacts with numerous molecular targets via Ca2+-induced conformational change. A statistical analysis of PDB structural data for Pb2+ and Ca2+-binding (EF-hand and non-EF-hand) proteins revealed fewer binding ligands in Pb2+ sites (4 ± 2), than non-EF-Hand (6 ± 2) and EF-Hand (7 ± 1) Ca2+-binding sites. Pb2+ binds predominantly with sidechain Glu (38.4%), which is less prevalent in both non-EF-Hand (10.4%) and EF-Hand (26.6%) sites. Interestingly, analyses of proteins where Pb2+ replaces Ca2+ (calmodulin) or Zn2+ (5-aminolaevulinic acid dehydratase) revealed structural changes presumably unrelated to ionic displacement. These results suggested that Pb2+ adopts diverse binding geometries and that opportunistic binding outside of known Ca2+-binding sites may play a role in molecular metal toxicity. Ca2+-binding affinities (Kd) using phenylalanine and tyrosine fluorescence were found to be 1.15 ± 0.68 X 10-5 M and 2.04 ± 0.02 X 10-6 M for the N- and C-terminal domains, respectively. The Kd for Pb2+-binding in the N-terminal domain, 1.40 ± 0.30 X 10-6 M, was 8-fold higher than Ca2+. Binding of Pb2+ in the C-terminal domain produced a biphasic response with Kd values 7.34 ± 0.95 X 10-7 M and 1.93 ± 0.32 X 10-6 M, suggesting a single higher affinity Pb2+-binding site in the C-terminal domain with nearly equivalent affinity for the remaining sites. Competitive effects of Pb2+ added to Ca2+-loaded CaM were examined using multiple NMR techniques. Pb2+ was found to displace Ca2+ only in the N-terminal domain, however structural/dynamic changes were observed in the central helix apparently due to Pb2+-binding in secondary sites. These data supported our hypothesis that CaM structure and function is altered by opportunistic Pb2+-binding.

The Wnt signaling network plays critical roles in development and is implicated in human disease. Wnts comprise a complex signaling network that, upon ligand binding, activates the phosphoprotein Dishevelled (Dvl), leading to distinct outputs including polarized cell movement (known as planar cell polarity, Wnt/PCP) and stabilization of the transcription factor β-catenin (Wnt/β-catenin). The mechanisms that determine a specific output are not completely understood, especially because they share receptors and cellular effectors, such as Naked-cuticle 1 (Nkd), a Dvl-interacting protein. The Nkd protein contains a myristoylation domain and an EF-hand, a putative calcium binding domain. Genetic evidence in Drosophila demonstrates that Nkd acts as a Wnt/β-catenin antagonist, while in contrast, Nkd modulates both branches of Wnt signaling in vertebrates. We hypothesize that the specialized role of Nkd in Drosophila is due to a disrupted EF-hand that cannot not bind calcium. Indeed, this change is unique to Drosophila and is not present in closely related insects all the way up to vertebrates. To test the role of the Nkd EF-hand in Wnt signal integration, we created two different mutations in the zebrafish Nkd: one with a neutralized EF-hand, as well as a Drosophila-like EF-hand, and manipulate Nkd activity in the zebrafish. Using a combination of biochemical and functional assays, we identified a requirement for the Nkd EF-hand in Wnt/PCP but not in Wnt/β-catenin transcriptional outputs. We demonstrate that the Drosophila-like antagonizes Wnt/β-catenin more robustly than zebrafish Nkd. The EF-hand of Nkd is similar to the EF-hand of a known calcium binding protein, Recoverin, a myristoyl-swtich protein that shuttles between the membrane and the cytoplasm depending on its calcium bound state. Consistently, we observe that NkdWT, but not the two mutant forms, shows localization changes in the calcium fluxing cells that also host converging Wnt signals versus calcium quiescent cells. Our functional data suggests that the Nkd EF-hand is important for Wnt signal integration. Interestingly, Nkd only contains one EF-hand, and proteins that bind calcium tend to have multiple. Calcium binding can also be influenced by binding partners. Because of this, we investigate the role of the Nkd binding protein Dvl and their possible calcium affinity. Dvl is a pivotal point in the Wnt signaling network, leading to the output decision of a cell. EF-hand of Nkd binds to the PDZ domain of Dvl. Interestingly, the Dvl PDZ domain contains a region rich in negatively charged amino acids that could aid in binding calcium. In the same manner as Nkd, we generated a Dvl with neutralized putative EF-hand and tested its function and localization relative to wildtype Dvl. This work elucidates the elegant mechanism by which a cell receiving multiple Wnt signals integrates the information into a specific response. The Nkd EF-hand may serve to interpret the physiology of a cell receiving multiple cues and provides mechanistic insight into Wnt signal integration in vivo.

The Wnt signaling network has critical roles in development and disease. Simplified, this complex network has two distinct outputs: the Wnt/β-catenin module activates the phosphoprotein Dishevelled (Dvl) and leads to transcriptional activation while the Wnt/Planar Cell Polarity (PCP) module activates Dvl and leads to calcium release and directed cell movement. Wnt/β-catenin and Wnt/PCP share signaling components like Frizzled receptors, Dvl, and Naked (Nkd). It is an open question how converging Wnt signals diverge into separate outcomes. In this thesis, I used molecular techniques, functional studies in the zebrafish, and biochemical approaches to determine the role of Nkd in Wnt signaling. Nkd contains and EF-hand, a putative calcium binding domain, and is known to antagonize Wnt/β-catenin and disrupt Wnt/PCP signaling. We utilized a tissue that requires both Wnt/β-catenin and Wnt/PCP signaling to properly pattern the left/right axes of the embryo; the dorsal forerunner cells (DFCs). The DFCs exhibit aperiodic calcium release as they migrate to form the Kupffer's Vesicle (KV), the organ of asymmetry. Calcium inhibition in the DFCs disrupts their migration, alters KV formation, and disrupts left/right patterning. Nkd is enriched in the DFCs during migration and KV formation and endogenous Nkd knockdown in the DFCs produces the same phenotypes as calcium inhibition, making Nkd a candidate molecule for directing converging Wnt signals to distinct outcomes. To assess the role of the EF-hand in Nkd function, I created point mutations predicted to disrupt EF-hand affinity for calcium. Through functional studies in zebrafish embryos, I determined that Nkd EF-hand is necessary for Nkd function in Wnt/PCP signaling, but dispensable for Wnt/β-catenin signaling. Although Nkd has not been shown to bind calcium, our functional data with the Nkd EF-hand point mutant provides compelling evidence for a role for calcium in Nkd function in directing Wnt signaling output. EF-hand affinity for calcium is influenced by binding partners, and since Nkd binds to Dvl in the Dvl PDZ domain, we screened the domain for a region rich in amino acids that facilitate ion binding. We identified a 12-amino acid sequence in the Dvl PDZ domain with potential to create a negatively charged pocket to help coordinate calcium binding. We expressed the Nkd EF-hand (EFX) Dvl basic domain and PDZ domain (bPDZ). The purified EFX and bPDZ constructs were used to investigate the interaction between Nkd, Dvl, and calcium. I show, by circular dichroism, that the Nkd/Dvl complex undergoes a calcium-induced change in secondary structure. This reveals the mechanism by which Nkd directs Dvl from the default Wnt/β-catenin signaling module to the Wnt/PCP module in response to calcium.

In our efforts to engineer a DNA binding and cleaving protein with greater sequence discrimination, we have designed dimeric proteins derived from engrailed homeodomain and calmodulin. Previous research by our group has shown that a hydrolytically active lanthanide binding site can be incorporated into a DNA binding motif. To understand protein-DNA interaction and improve the sequence selectivity of the chimeric complex, two lanthanide-binding homodimers were designed and expressed. One of the dimers, F2, is coupled together by a flexible polypeptide linker and the other, R7C, is a disulfide cross-linked cysteine mutant at the N-terminus. Studies of fluorescence of tryptophan residues document that the overall affinity for lanthanide and calcium is similar to traditional EF-hand peptides (1-10 μM). Metal titrations monitored by circular dichroism (CD) revealed that the secondary structures of the dimers contained a lower degree of -helicity than the designed monomeric protein due to additional modifications, but because of their flexibility and their two active-site domain, hydrolytic activity was several folds faster than our previously designed proteins and peptides. Unlike earlier reports on our chimeras, F2 also demonstrated the capability to hydrolyze DNA in the presence of some biological relevant metal ions suggesting different cleavage mechanisms were carried out. Extensive DNA sequencing studies on cleavage patterns with oligonucleotide duplexes confirmed the unique sequence selectivity and kinetic properties of F2. Two engrailed homeodomain target sites, TAATTA, were favored for hydrolytic activity corresponding to one domain acting as a DNA anchor on the first target site while the other was an "opportunist" at recognizing the second site. Nonetheless, the hydrolytic behavior at the phosphodiester bond on a specific dsDNA sequence is in good agreement with the behavior of restriction endonucleases. Unlike restriction enzymes, metallated F2 has not only demonstrated the ability to cleave DNA plasmid, but it also excises the entire nucleotide on a selected sequence. This homodimer is the first example of an active and selective hydrolytic artificial nuclease based on the modular turn substitution design approach that can be a potential template for genomic modification.

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.

Calpains are intracellular enzymes that merge cysteine protease and calcium sensing activities together in one molecule. They respond to Ca2+ signals and modify the activity of their targets by selective proteolysis. Calpains are involved in normal cellular process like cell migration and apoptosis. The over-activation of calpain due to disturbances in Ca2+ homeostasis or inactivation due to mutations, contribute to diseases like ischemic injury and muscular dystrophy. The classical calpains 1 and 2 are heterodimeric enzymes containing a large (80 kDa) subunit and a small subunit (28 kDa). Dimerization occurs through the 5th EF-hand of penta-EF-hand (PEF) domains present in both large and small subunits. In this study, I have used structural genomics approaches to explore the PEF and C2-like regulatory domains of some of the other 12 human calpain isoforms. I have shown that recombinant PEF domain of skeletal muscle-specific calpain 3 exists as a stable homodimer when produced alone. Modelling studies suggest that there would be no barriers for dimerization of the full-length enzyme through the PEF domains which would place the protease cores at opposite ends of the dimer. Co-expression studies using small subunit were performed with PEF domains of calpains 1, 3, 8, 9, 11, 12 and 13. A differential tagging system was devised to differentiate heterodimers from homodimers. The PEF domains of calpains 1, 3, 9 and 13 co-expressed with the small subunit, while the others failed to express. The PEF domains of calpains 1 and 9 formed heterodimers. Conversely, the PEF domain of calpain 3 formed a homodimer and that of calpain 13 predominantly formed a homodimer with a small amount of heterodimer. Homodimerization of calpains implies they are less-likely to be inhibited by the endogenous calpain inhibitor, calpastatin. C2-like regulatory domains of calpains 5-13 were also studied. The structure of the distal C2-like domain of calpain 7 was solved. It is markedly different from canonical C2 domains and may not bind Ca2+. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-02-11 12:30:29.18

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No / The human epidermis holds an autocrine acetylcholine production and degradation including functioning membrane integrated and cytosolic butyrylcholinesterase (BuchE). Here we show that BuchE activities increase 9-fold in the presence of calcium (0.5 × 10-3 M) via a specific EF-hand calcium binding site, whereas acetylcholinesterase (AchE) is not affected. 45Calcium labelling and computer simulation confirmed the presence of one EF-hand binding site per subunit which is disrupted by H2O2-mediated oxidation. Moreover, we confirmed the faster hydrolysis by calcium-activated BuchE using the neurotoxic organophosphate O-ethyl-O-(4-nitrophenyl)-phenylphosphonothioate (EPN). Considering the large size of the human skin with 1.8 m2 surface area with its calcium gradient in the 10¿3 M range, our results implicate calcium-activated BuchE as a major protective mechanism against suicide inhibition of AchE by organophosphates in this non-neuronal tissue