Calmodulin: a versatile calcium mediator protein

1994 ◽  
Vol 72 (9-10) ◽  
pp. 357-376 ◽  
Author(s):  
Hans J. Vogel
Keyword(s):  
Amino Acids ◽  
Intracellular Calcium ◽  
Calcium Binding ◽  
Unnatural Amino Acids ◽  
Metal Ion ◽  
Structural Information ◽  
Limited Proteolysis ◽  
Hydrophobic Surface ◽  
Site Directed Mutagenesis ◽  
Binding Domains

The level of intracellular calcium is strictly regulated in all cells. In a resting cell, the [Ca2+] is ≤ 10−7 M and during activation it rises to approximately 10−6 M. Calmodulin (CaM) is the secondary messenger protein that has to translate this modest rise in intracellular calcium into a physiological response in all eukaryotic cells. CaM can activate almost 30 different target systems, including smooth muscle contraction, protein kinases and phosphatases, nitric oxide synthases, and calcium-extruding pumps. It is an acidic protein of 148 amino acids with four helix–loop–helix calcium-binding domains and it has a characteristic dumbbell shape in the crystal structure. In this review I discuss which features of CaM allow it to be such a universal and versatile calcium regulator. First of all, the positive cooperative calcium binding to all four binding sites of CaM in the presence of a target protein allows the protein to act effectively during a calcium transient. Secondly, the high Met content of two hydrophobic surface patches on the two domains of CaM creates a flexible and pliable, yet sticky, interaction surface that does not place high demands on the specificity of the interaction. Consequently, calcium-CaM can bind effectively to the CaM-binding domains of all its target proteins, despite their lack of amino acid sequence homology; their only common feature is that they are hydrophobic basic peptides that have a propensity to form an α-helix. CaM's capacity to recognize its CaM-binding domains is further enhanced by its third crucial feature, the intrinsic flexibility of the central linker region; this allows the two domains of CaM to slide over the surface of the α-helical bound peptide, to find their most favourable binding orientation. In this review I have also presented selected examples of a variety of experimental techniques that have contributed to our understanding of this unique multitasking protein. These include studies with well-established techniques such as site-directed mutagenesis, chemical modification, limited proteolysis, circular dichroism, and two-dimensional nuclear magnetic resonance (NMR), as well as novel or less common approaches involving the use of unnatural amino acids, metal-ion NMR, lysine pKa determinations, and isotope-edited Fourier transform infrared spectroscopy. In combination with available structural information, these studies have provided considerable detail in our understanding of this versatile calcium regulatory protein.Key words: calmodulin, calcium metabolism, methionine, lysine, NMR spectroscopy.

Molecular basis of the complex formation between the two calcium-binding proteins S100A8 (MRP8) and S100A9 (MRP14)

2005 ◽  
Vol 386 (5) ◽  
pp. 429-434 ◽  
Author(s):  
Nadja Leukert ◽  
Clemens Sorg ◽  
Johannes Roth
Keyword(s):  
Amino Acids ◽  
Complex Formation ◽  
Calcium Binding ◽  
Site Directed Mutagenesis ◽  
Dimer Interface ◽  
Yeast Two Hybrid System ◽  
Hydrophobic Amino Acids ◽  
Functional Relevance ◽  
First Time

Abstract S100 proteins form characteristic homo- and/or heterodimers that play a role in calcium-mediated signaling. We characterized the formation of the human S100A8/S100A9 heterodimer using the yeast two-hybrid system. Employing site-directed mutagenesis we found that distinct hydrophobic amino acids of helix I/I′ are located at a crucial site of the S100A8/S100A9 dimer interface, whereas conserved residues within helix IV/IV′ are not important for heterodimerization. Furthermore, amino acids Y16 and F68 prevent homodimerization of human S100A8. These data demonstrate for the first time the functional relevance of distinct hydrophobic amino acids for human S100A8/S100A9 complex formation in vivo.

scholarly journals Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

2004 ◽  
Vol 379 (3) ◽  
pp. 795-803 ◽  
Author(s):  
Chung-Chun WU ◽  
Min-Che CHEN ◽  
Ya-Ru CHANG ◽  
Tsuey-Ying HSU ◽  
Jen-Yang CHEN
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Thymidine Kinase ◽  
Crucial Role ◽  
Metal Ion ◽  
Epstein Barr Virus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Barr Virus ◽  
Epstein Barr

Thymidine kinase (TK), encoded by EBV (Epstein–Barr virus), is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignancies. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure–function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed significant changes in activity and binding affinity for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. For site 3, only mutants D392E (Asp392→Glu) and R393H retain activity, indicating that a negative charge is important for Asp392 and a positive charge is required for Arg393. The increased binding affinities of these two mutants for 3´-deoxy-2´,3´-didehydrothymidine suggest that the two residues are also important for substrate selection. Interestingly, the changed metal-ion usage pattern of D392E reveals that Asp392 plays multiple roles in this region. His394 cannot be compensated by other amino acids, also indicating a crucial role. In site 4, the F402Y mutant retains full activity; however, F402S retains only 60% relative activity. Strikingly, when Phe402 is substituted with serine residue, the original preferred pyrimidine substrates, such as 3´-azido-3´-deoxythymidine, iododeoxyuridine and β-l-5-iododioxolane uracil (l-form substrate), have decreased competitiveness with thymidine, suggesting that Phe402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.

Characterization of active-site residues in diadenosine tetraphosphate hydrolase from Lupinus angustifolius

2001 ◽  
Vol 357 (2) ◽  
pp. 399-405 ◽  
Author(s):  
Danuta MAKSEL ◽  
Paul R. GOOLEY ◽  
James D. SWARBRICK ◽  
Andrzej GURANOWSKI ◽  
Christine GANGE ◽  
...  
Keyword(s):  
Amino Acids ◽  
Metal Ion ◽  
Solution Structure ◽  
Ph Dependence ◽  
Three Dimensional ◽  
Lupinus Angustifolius ◽  
Site Directed Mutagenesis ◽  
Nudix Hydrolase ◽  
Active Site Residues ◽  
Dimensional Solution

Site-directed mutagenesis has been used to characterize the functions of key amino acid residues in the catalytic site of the ‘nudix’ hydrolase, (asymmetrical) diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) hydrolase (EC 3.6.1.17) from Lupinus angustifolius, the three-dimensional solution structure of which has recently been solved. Residues within the nudix motif, Gly-(Xaa)5-Glu-(Xaa)7-Arg-Glu-Uaa-Xaa-(Glu)2-Xaa-Gly (where Xaa represents unspecified amino acids and Uaa represents the bulky aliphatic amino acids Ile, Leu or Val) conserved in ‘nudix enzymes’, and residues important for catalysis from elsewhere in the molecule, were mutated and the expressed proteins characterized. The results reveal a high degree of functional conservation between lupin asymmetric Ap4A hydrolase and the 8-oxo-dGTP hydrolase from Escherichia coli. Charged residues in positions equivalent to those that ligate an enzyme-bound metal ion in the E. coli 8-oxo-dGTP hydrolase [Harris, Wu, Massiah and Mildvan (2000) Biochemistry 39, 1655–1674] were shown to contribute to catalysis to similar extents in the lupin enzyme. Mutations E55Q, E59Q and E125Q all reduced kcat markedly, whereas mutations R54Q, E58Q and E122Q had smaller effects. None of the mutations produced a substantial change in the Km for Ap4A, but several extensively modified the pH-dependence and fluoride-sensitivities of the hydrolase. It was concluded that the precisely positioned glutamate residues Glu-55, Glu-59 and Glu-125 are conserved as functionally significant components of the hydrolytic mechanism in both of these members of the nudix family of hydrolases.

scholarly journals The Calcium Binding Loops of the Cytosolic Phospholipase A2 C2 Domain Specify Targeting to Golgi and ER in Live Cells

2004 ◽  
Vol 15 (1) ◽  
pp. 371-383 ◽  
Author(s):  
John H. Evans ◽  
Stefan H. Gerber ◽  
Diana Murray ◽  
Christina C. Leslie
Keyword(s):  
Plasma Membrane ◽  
Intracellular Calcium ◽  
Calcium Binding ◽  
Structural Information ◽  
Lipid Binding ◽  
Live Cells ◽  
C2 Domain ◽  
Cytosolic Phospholipase

Translocation of cytosolic phospholipase A2 (cPLA2) to Golgi and ER in response to intracellular calcium mobilization is regulated by its calcium-dependent lipid-binding, or C2, domain. Although well studied in vitro, the biochemical characteristics of the cPLA2C2 domain offer no predictive value in determining its intracellular targeting. To understand the molecular basis for cPLA2C2 targeting in vivo, the intracellular targets of the synaptotagmin 1 C2A (Syt1C2A) and protein kinase Cα C2 (PKCαC2) domains were identified in Madin-Darby canine kidney cells and compared with that of hybrid C2 domains containing the calcium binding loops from cPLA2C2 on Syt1C2A and PKCαC2 domain backbones. In response to an intracellular calcium increase, PKCαC2 targeted plasma membrane regions rich in phosphatidylinositol-4,5-bisphosphate, and Syt1C2A displayed a biphasic targeting pattern, first targeting phosphatidylinositol-4,5-bisphosphate-rich regions in the plasma membrane and then the trans-Golgi network. In contrast, the Syt1C2A/cPLA2C2 and PKCαC2/cPLA2C2 hybrids targeted Golgi/ER and colocalized with cPLA2C2. The electrostatic properties of these hybrids suggested that the membrane binding mechanism was similar to cPLA2C2, but not PKCαC2 or Syt1C2A. These results suggest that primarily calcium binding loops 1 and 3 encode structural information specifying Golgi/ER targeting of cPLA2C2 and the hybrid domains.

scholarly journals Site-Directed Mutagenesis of Myoglobin for Studies of Their Interaction with Iron(III) by Multi-Spectroscopic Techniques

2013 ◽  
Vol 2013 ◽  
pp. 1-11
Author(s):  
Qian Tang ◽  
Xiao-Jun Peng ◽  
Hong-Yu Cao ◽  
Yan-Jie Yang ◽  
Jing Ma ◽  
...  
Keyword(s):  
Amino Acids ◽  
Metal Ion ◽  
Binding Capacity ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Spectroscopic Techniques ◽  
Wild Type ◽  
Bimolecular Quenching ◽  
And Function ◽  
Binding Distance

In order to investigate how the amino acids on the surface of myoglobin molecule influence myoglobin's structure and function, a variety of spectroscopy techniques were applied in the study of the interaction between Fe(III) and myoglobin (wild type and its mutants, D44K, D60K, and K56D). The results demonstrate that Fe(III) can quench the fluorescence of wild type and mutants of myoglobin, and the quenching mechanisms are static quenching. It is found that the binding distance between Fe(III) and myoglobin mutants gets smaller, the binding capacity increases by the values of binding constant and the bimolecular quenching constant as well as the binding distance. Those data also indicate that the metal ion Fe(III) can interact strongly with myoglobin mutants. The three-dimensional conformation change after surface amino acids are replaced is detected by the UV absorption spectroscopy and fluorescence spectroscopy, which make mutants become more dynamic and change its function and interaction with Fe(III) strongly.

Identification of metal ion binding peptides containing unnatural amino acids by phage display

2013 ◽  
Vol 23 (9) ◽  
pp. 2598-2600 ◽  
Author(s):  
Jonathan W. Day ◽  
Chan Hyuk Kim ◽  
Vaughn V. Smider ◽  
Peter G. Schultz
Keyword(s):  
Amino Acids ◽  
Phage Display ◽  
Unnatural Amino Acids ◽  
Metal Ion ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
Binding Peptides

Immunogold labeling of the calcium binding protein synexin in isolated adrenal chromaffin granules and chromaffin cells

1990 ◽  
Vol 48 (3) ◽  
pp. 952-953
Author(s):  
Gemma A.J. Kuijpers ◽  
Harvey B. Pollard
Keyword(s):  
Adrenal Medulla ◽  
Calcium Binding ◽  
Chromaffin Cells ◽  
Cell Activation ◽  
Structural Information ◽  
Dependent Manner ◽  
Chromaffin Granules ◽  
Immuno Electron Microscopy ◽  
Ultrathin Sections ◽  
Adrenal Chromaffin

Exocytotic fusion of granules in the adrenal medulla chromaffin cell is triggered by a rise in the concentration of cytosolic Ca2+ upon cell activation. The protein synexin, annexin VII, was originally found in the adrenal medulla and has been shown to cause aggregation and to support fusion of chromaffin granules in a Ca2+-dependent manner. We have previously suggested that synexin may there fore play a role in the exocytotic fusion process. In order to obtain more structural information on synexin, we performed immuno-electron microscopy on frozen ultrathin sections of both isolated chromaffin granules and chromaffin cells.Chromaffin granules were isolated from bovine adrenal medulla, and synexin was isolated from bovine lung. Granules were incubated in the presence or absence of synexin (24 μg per mg granule protein) and Ca2+ (1 mM), which induces maximal granule aggregation, in 0.3M sucrose-40m MMES buffer(pH 6.0). Granules were pelleted, washed twice in buffer without synexin and fixed with 2% glutaraldehyde- 2% para formaldehyde in 0.1 M phosphate buffer (GA/PFA) for 30 min. Chromaffin cells were isolated and cultured for 3-5 days, and washed and incubated in Krebs solution with or without 20 uM nicotine. Cells were fixed 90 sec after on set of stimulation with GA/PFA for 30 min. Fixed granule or cell pellets were washed, infiltrated with 2.3 M sucrose in PBS, mounted and frozen in liquid N2.

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