scholarly journals Molecular Tuning of an EF-Hand-like Calcium Binding Loop

1997 ◽  
Vol 110 (2) ◽  
pp. 173-184 ◽  
Author(s):  
Steven K. Drake ◽  
Michael A. Zimmer ◽  
Craig Kundrot ◽  
Joseph J. Falke
Keyword(s):  
Calcium Binding ◽  
Ion Selectivity ◽  
Side Chain ◽  
Binding Motif ◽  
Side Chains ◽  
Binding Parameters ◽  
Size Selectivity ◽  
The Third ◽  
Ef Hand ◽  
Binding Loop

Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca2+ binding motif. The ion binding parameters of this motif are controlled, in part, by the structure of its Ca2+ binding loop, termed the EF-loop. The EF-loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca2+ binding parameters for their unique cellular roles. The present study uses a structurally homologous Ca2+ binding loop, that of the Escherichia coli galactose binding protein, as a model for the EF-loop in studies examining the contribution of the third loop position to intramolecular tuning. 10 different side chains are compared at the third position of the model EF-loop with respect to their effects on protein stability, sugar binding, and metal binding equilibria and kinetics. Substitution of an acidic Asp side chain for the native Asn is found to generate a 6,000-fold increase in the ion selectivity for trivalent over divalent cations, providing strong support for the electrostatic repulsion model of divalent cation charge selectivity. Replacement of Asn by neutral side chains differing in size and shape each alter the ionic size selectivity in a similar manner, supporting a model in which large-ion size selectivity is controlled by complex interactions between multiple side chains rather than by the dimensions of a single coordinating side chain. Finally, the pattern of perturbations generated by side chain substitutions helps to explain the prevalence of Asn and Asp at the third position of natural EF-loops and provides further evidence supporting the unique kinetic tuning role of the gateway side chain at the ninth EF-loop position.

scholarly journals Involvement of Both Dockerin Subdomains in Assembly of the Clostridium thermocellum Cellulosome

1998 ◽  
Vol 180 (24) ◽  
pp. 6581-6585 ◽  
Author(s):  
Betsy Lytle ◽  
J. H. David Wu
Keyword(s):  
Calcium Binding ◽  
Clostridium Thermocellum ◽  
Scaffolding Protein ◽  
Stable Complex ◽  
Binding Motif ◽  
Cellulose Binding Domain ◽  
Catalytic Subunits ◽  
The Third ◽  
Ef Hand ◽  
Cellulose Binding

ABSTRACT Clostridium thermocellum produces an extracellular cellulase complex termed the cellulosome. It consists of a scaffolding protein, CipA, containing nine cohesin domains and a cellulose-binding domain, and at least 14 different enzymatic subunits, each containing a conserved duplicated sequence, or dockerin domain. The cohesin-dockerin interaction is responsible for the assembly of the catalytic subunits into the cellulosome structure. Each duplicated sequence of the dockerin domain contains a region bearing homology to the EF-hand calcium-binding motif. Two subdomains, each containing a putative calcium-binding motif, were constructed from the dockerin domain of CelS, a major cellulosomal catalytic subunit. These subdomains, called DS1 and DS2, were cloned by PCR and expressed in Escherichia coli. The binding of DS1 and DS2 to R3, the third cohesin domain of CipA, was analyzed by nondenaturing gel electrophoresis. A stable complex was formed only when R3 was combined with both DS1 and DS2, indicating that the two halves of the dockerin domain interact with each other and such interaction is required for effective binding of the dockerin domain to the cohesin domain.

A change-in-hand mechanism for S100 signalling

1998 ◽  
Vol 76 (2-3) ◽  
pp. 324-333 ◽  
Author(s):  
Steven P Smith ◽  
Gary S Shaw
Keyword(s):  
Conformational Change ◽  
Calcium Ion ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Three Dimensional ◽  
Calcium Binding Proteins ◽  
C Terminus ◽  
Ef Hand ◽  
Sensor Proteins ◽  
Binding Loop

S100 proteins are a group of small dimeric calcium-binding proteins making up a large subclass of the EF-hand family of calcium-binding proteins. Members of this family of proteins have been proposed to act as intracellular calcium modulatory proteins in a fashion analogous to that of the EF-hand sensor proteins troponin-C and calmodulin. Recently, NMR spectroscopy has provided the three-dimensional structures of the S100 family members S100A6 and S100B in both the apo- and calcium-bound forms. These structures have allowed for the identification of a novel calcium-induced conformational change termed the change-in-hand mechanism. Helix III of the C-terminal calcium-binding loop changes its helix-helix interactions (or handness) with the remainder of the molecule primarily owing to the reorientation of the backbone in an effort to coordinate the calcium ion. This reorientation of helix III exposes several residues in the C-terminus and linker regions of S100B resulting in the formation of a hydrophobic patch surrounded be a number of acidic residues. This site is the proposed region for protein-protein recognition.Key words: S100, calcium-binding protein, EF-hand, conformational change.

Tuning the Equilibrium Ion Affinity and Selectivity of the EF-Hand Calcium Binding Motif:  Substitutions at the Gateway Position†

Biochemistry ◽  
1996 ◽  
Vol 35 (21) ◽  
pp. 6697-6705 ◽  
Author(s):  
Steven K. Drake ◽  
Keith L. Lee ◽  
Joseph J. Falke
Keyword(s):  
Calcium Binding ◽  
Binding Motif ◽  
Ef Hand

scholarly journals Ca 2+ Regulation of Trypanosoma brucei Phosphoinositide Phospholipase C

2015 ◽  
Vol 14 (5) ◽  
pp. 486-494 ◽  
Author(s):  
Sharon King-Keller ◽  
Christina A. Moore ◽  
Roberto Docampo ◽  
Silvia N. J. Moreno
Keyword(s):  
Enzymatic Activity ◽  
Phospholipase C ◽  
Trypanosoma Brucei ◽  
Calcium Binding ◽  
Fluorescent Protein ◽  
Consensus Sequence ◽  
Single Amino Acid ◽  
Binding Motif ◽  
Content Type ◽  
Ef Hand

ABSTRACT We characterized a phosphoinositide phospholipase C (PI-PLC) from the procyclic form (PCF) of Trypanosoma brucei . The protein contains a domain organization characteristic of typical PI-PLCs, such as X and Y catalytic domains, an EF-hand calcium-binding motif, and a C2 domain, but it lacks a pleckstrin homology (PH) domain. In addition, the T. brucei PI-PLC (TbPI-PLC) contains an N-terminal myristoylation consensus sequence found only in trypanosomatid PI-PLCs. A peptide containing this N-terminal domain fused to green fluorescent protein (GFP) was targeted to the plasma membrane. TbPI-PLC enzymatic activity was stimulated by Ca 2+ concentrations below the cytosolic levels in the parasite, suggesting that the enzyme is constitutively active. TbPI-PLC hydrolyzes both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP 2 ), with a higher affinity for PIP 2 . We found that modification of a single amino acid in the EF-hand motif greatly affected the protein's Ca 2+ sensitivity and substrate preference, demonstrating the role of this motif in Ca 2+ regulation of TbPI-PLC. Endogenous TbPI-PLC localizes to intracellular vesicles and might be using an intracellular source of PIP 2 . Knockdown of TbPI-PLC expression by RNA interference (RNAi) did not result in growth inhibition, although enzymatic activity was still present in parasites, resulting in hydrolysis of PIP 2 and a contribution to the inositol 1,4,5-trisphosphate (IP 3 )/diacylglycerol (DAG) pathway.

scholarly journals The Calcium-Dependent Interaction between S100B and the Mitochondrial AAA ATPase ATAD3A and the Role of This Complex in the Cytoplasmic Processing of ATAD3A

2010 ◽  
Vol 30 (11) ◽  
pp. 2724-2736 ◽  
Author(s):  
Benoît Gilquin ◽  
Brian R. Cannon ◽  
Arnaud Hubstenberger ◽  
Boualem Moulouel ◽  
Elin Falk ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Nuclear Translocation ◽  
P53 Protein ◽  
Binding Domain ◽  
Binding Motif ◽  
Temperature Sensitive ◽  
Aaa Atpase ◽  
Calcium Dependent ◽  
Ef Hand ◽  
Cell Growth And Differentiation

ABSTRACT S100 proteins comprise a multigene family of EF-hand calcium binding proteins that engage in multiple functions in response to cellular stress. In one case, the S100B protein has been implicated in oligodendrocyte progenitor cell (OPC) regeneration in response to demyelinating insult. In this example, we report that the mitochondrial ATAD3A protein is a major, high-affinity, and calcium-dependent S100B target protein in OPC. In OPC, ATAD3A is required for cell growth and differentiation. Molecular characterization of the S100B binding domain on ATAD3A by nuclear magnetic resonance (NMR) spectroscopy techniques defined a consensus calcium-dependent S100B binding motif. This S100B binding motif is conserved in several other S100B target proteins, including the p53 protein. Cellular studies using a truncated ATAD3A mutant that is deficient for mitochondrial import revealed that S100B prevents cytoplasmic ATAD3A mutant aggregation and restored its mitochondrial localization. With these results in mind, we propose that S100B could assist the newly synthesized ATAD3A protein, which harbors the consensus S100B binding domain for proper folding and subcellular localization. Such a function for S100B might also help to explain the rescue of nuclear translocation and activation of the temperature-sensitive p53val135 mutant by S100B at nonpermissive temperatures.

scholarly journals Plant Seed Peroxygenase Is an Original Heme-oxygenase with an EF-hand Calcium Binding Motif

2006 ◽  
Vol 281 (44) ◽  
pp. 33140-33151 ◽  
Author(s):  
Abdulsamie Hanano ◽  
Michel Burcklen ◽  
Martine Flenet ◽  
Anabella Ivancich ◽  
Mathilde Louwagie ◽  
...  
Keyword(s):  
Heme Oxygenase ◽  
Calcium Binding ◽  
Binding Motif ◽  
Plant Seed ◽  
Ef Hand

scholarly journals Experimental Insight into the Structural and Functional Roles of the ‘Black’ and ‘Gray’ Clusters in Recoverin, a Calcium Binding Protein with Four EF-Hand Motifs

Molecules ◽  
2019 ◽  
Vol 24 (13) ◽  
pp. 2494
Author(s):  
Sergey E. Permyakov ◽  
Alisa S. Vologzhannikova ◽  
Ekaterina L. Nemashkalova ◽  
Alexei S. Kazakov ◽  
Alexander I. Denesyuk ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Amino Acids ◽  
Calcium Binding ◽  
Binding Protein ◽  
Critical Role ◽  
Binding Motif ◽  
Terminal Domain ◽  
Ef Hand ◽  
Helical Content ◽  
Thermal Stability Of

Recently, we have found that calcium binding proteins of the EF-hand superfamily (i.e., a large family of proteins containing helix-loop-helix calcium binding motif or EF-hand) contain two types of conserved clusters called cluster I (‘black’ cluster) and cluster II (‘grey’ cluster), which provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domains. Cluster I is more conservative and mostly incorporates aromatic amino acids, whereas cluster II includes a mix of aromatic, hydrophobic, and polar amino acids of different sizes. Recoverin is EF-hand Ca2+-binding protein containing two ‘black’ clusters comprised of F35, F83, Y86 (N-terminal domain) and F106, E169, F172 (C-terminal domain) as well as two ‘gray’ clusters comprised of F70, Q46, F49 (N-terminal domain) and W156, K119, V122 (C-terminal domain). To understand a role of these residues in structure and function of human recoverin, we sequentially substituted them for alanine and studied the resulting mutants by a set of biophysical methods. Under metal-free conditions, the ‘black’ clusters mutants (except for F35A and E169A) were characterized by an increase in the α-helical content, whereas the ‘gray’ cluster mutants (except for K119A) exhibited the opposite behavior. By contrast, in Ca2+-loaded mutants the α-helical content was always elevated. In the absence of calcium, the substitutions only slightly affected multimerization of recoverin regardless of their localization (except for K119A). Meanwhile, in the presence of calcium mutations in N-terminal domain of the protein significantly suppressed this process, indicating that surface properties of Ca2+-bound recoverin are highly affected by N-terminal cluster residues. The substitutions in C-terminal clusters generally reduced thermal stability of recoverin with F172A (‘black’ cluster) as well as W156A and K119A (‘gray’ cluster) being the most efficacious in this respect. In contrast, the mutations in the N-terminal clusters caused less pronounced differently directed changes in thermal stability of the protein. The substitutions of F172, W156, and K119 in C-terminal domain of recoverin together with substitution of Q46 in its N-terminal domain provoked significant but diverse changes in free energy associated with Ca2+ binding to the protein: the mutant K119A demonstrated significantly improved calcium binding, whereas F172A and W156A showed decrease in the calcium affinity and Q46A exhibited no ion coordination in one of the Ca2+-binding sites. The most of the N-terminal clusters mutations suppressed membrane binding of recoverin and its inhibitory activity towards rhodopsin kinase (GRK1). Surprisingly, the mutant W156A aberrantly activated rhodopsin phosphorylation regardless of the presence of calcium. Taken together, these data confirm the scaffolding function of several cluster-forming residues and point to their critical role in supporting physiological activity of recoverin.

scholarly journals The predominant calcimedins from Trypanosoma brucei comprise a family of flagellar EF-hand calcium-binding proteins

1992 ◽  
Vol 287 (1) ◽  
pp. 187-193 ◽  
Author(s):  
Y Wu ◽  
N G Haghighat ◽  
L Ruben
Keyword(s):  
Trypanosoma Brucei ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Immunoblot Analysis ◽  
Cross Reactivity ◽  
Calcium Binding Proteins ◽  
Phospholipid Vesicles ◽  
Ef Hand ◽  
Sequence Similarities ◽  
Binding Loop

The cellular complement of calcimedins was identified in Trypanosoma brucei by Ca(2+)-dependent association with phenyl-Sepharose. Predominant calcimedins with molecular mass of 23-26 kDa and 44 kDa, along with minor calcimedins of 96, 120 and 230 kDa, were obtained. The trypanosome calcimedins were unrelated to vertebrate annexins, based upon antibody cross-reactivity and an inability to associate in a Ca(2+)-dependent way with phospholipid vesicles comprised of phosphatidylserine or phosphatidylethanolamine/phosphatidylcholine (1:1, w/w). Partial sequence analysis demonstrated that 44 kDa calcimedin (Tb-44) contained an EF-hand calcium-binding loop. Five CNBr/tryptic fragments exhibited a total of 93% similarity with Tb-17, a 23 kDa EF-hand protein in T. brucei. The trypanosome calcimedins appeared to comprise a family of proteins, based on sequence similarities and antibody cross-reactivity of affinity-purified anti-Tb44 with the 23-26 kDa cluster. No evidence was found for Tb-44 in the related species T. cruzi, Leishmania taraentolae or Crithidia fasciculata. Antibodies against Tb-44 were localized by immunofluorescence along the flagellum of T. brucei. Immunoblot analysis of flagella-enriched preparations demonstrated that Tb-44 and the 23-26 kDa cluster were present in this structure. We conclude that annexin family members are not among the predominant trypanosome proteins that associate with phenyl-Sepharose in a Ca(2+)-dependent way. Instead, the major trypanosome calcimedins comprise a family of flagellar EF-hand calcium-binding proteins.

Functional asymmetry in cohesin binding belies inherent symmetry of the dockerin module: insight into cellulosome assembly revealed by systematic mutagenesis

2008 ◽  
Vol 410 (2) ◽  
pp. 331-338 ◽  
Author(s):  
Alon Karpol ◽  
Yoav Barak ◽  
Raphael Lamed ◽  
Yuval Shoham ◽  
Edward A. Bayer
Keyword(s):  
Calcium Binding ◽  
Striking Similarity ◽  
Type I ◽  
Single Mutation ◽  
High Affinity ◽  
Ef Hand ◽  
Repeated Motifs ◽  
Insight Into ◽  
Binding Loop

The cellulosome is an intricate multi-enzyme complex, known for its efficient degradation of recalcitrant cellulosic substrates. Its supramolecular architecture is determined by the high-affinity intermodular cohesin–dockerin interaction. The dockerin module comprises a calcium-binding, duplicated ‘F-hand’ loop–helix motif that bears striking similarity to the EF-hand loop–helix–loop motif of eukaryotic calcium-binding proteins. In the present study, we demonstrate by progressive truncation and alanine scanning of a representative type-I dockerin module from Clostridium thermocellum, that only one of the repeated motifs is critical for high-affinity cohesin binding. The results suggest that the near-symmetry in sequence and structure of the repeated elements of the dockerin is not essential to cohesin binding. The first calcium-binding loop can be deleted entirely, with almost full retention of binding. Likewise, significant deletion of the second repeated segment can be achieved, provided that its calcium-binding loop remains intact. Essentially the same conclusion was verified by systematically mutating the highly conserved residues in the calcium-binding loop. Mutations in one of the calcium-binding loops failed to disrupt cohesin recognition and binding, whereas a single mutation in both loops served to reduce the affinity significantly. The results are mutually compatible with recent crystal structures of the type-I cohesin–dockerin heterodimer, which demonstrate that the dockerin can bind in an equivalent manner to its cohesin counterpart through either its first or second repeated motif. The observed plasticity in cohesin–dockerin binding may facilitate cellulosome assembly in vivo or, alternatively, provide a conformational switch that promotes access of the tethered cellulosomal enzymes to their polysaccharide substrates.

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