scholarly journals Engineering Competitive Magnesium Binding into the First EF-hand of Skeletal Troponin C

2002 ◽  
Vol 277 (51) ◽  
pp. 49716-49726 ◽  
Author(s):  
Jonathan P. Davis ◽  
Jack A. Rall ◽  
Peter J. Reiser ◽  
Lawrence B. Smillie ◽  
Svetlana B. Tikunova
Keyword(s):  
Calcium Binding ◽  
Psoas Muscle ◽  
Calcium Sensitivity ◽  
Troponin C ◽  
Regulatory Domain ◽  
Calcium Dependent ◽  
High Affinity ◽  
Magnesium Binding ◽  
Ef Hand ◽  
Binding Loop

The goal of this study was to examine the mechanism of magnesium binding to the regulatory domain of skeletal troponin C (TnC). The fluorescence of Trp29, immediately preceding the first calcium-binding loop in TnCF29W, was unchanged by addition of magnesium, but increased upon calcium binding with an affinity of 3.3 μm. However, the calcium-dependent increase in TnCF29Wfluorescence could be reversed by addition of magnesium, with a calculated competitive magnesium affinity of 2.2 mm. When a Z acid pair was introduced into the first EF-hand of TnCF29W, the fluorescence of G34DTnCF29Wincreased upon addition of magnesium or calcium with affinities of 295 and 1.9 μm, respectively. Addition of 3 mmmagnesium decreased the calcium sensitivity of TnCF29Wand G34DTnCF29W∼2- and 6-fold, respectively. Exchange of G34DTnCF29Winto skinned psoas muscle fibers decreased fiber calcium sensitivity ∼1.7-fold compared with TnCF29Wat 1 mm[magnesium]freeand ∼3.2-fold at 3 mm[magnesium]free. Thus, incorporation of a Z acid pair into the first EF-hand allows it to bind magnesium with high affinity. Furthermore, the data suggests that the second EF-hand, but not the first, of TnC is responsible for the competitive magnesium binding to the regulatory domain.

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.

scholarly journals Mutation of the high affinity calcium binding sites in cardiac troponin C.

1992 ◽  
Vol 267 (2) ◽  
pp. 825-831 ◽  
Author(s):  
J C Negele ◽  
D G Dotson ◽  
W Liu ◽  
H L Sweeney ◽  
J A Putkey
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Cardiac Troponin ◽  
Troponin C ◽  
High Affinity ◽  
Cardiac Troponin C ◽  
Calcium Binding Sites

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.

scholarly journals Targeting of Protein Kinase Cα to Caveolae

1998 ◽  
Vol 141 (3) ◽  
pp. 601-610 ◽  
Author(s):  
Chieko Mineo ◽  
Yun-Shu Ying ◽  
Christine Chapline ◽  
Susan Jaken ◽  
Richard G.W. Anderson
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Regulatory Domain ◽  
Calcium Dependent ◽  
High Affinity ◽  
Membrane Invagination ◽  
Protein Kinase Cα ◽  
Flanking Sequences ◽  
Modulate Signal ◽  
Regulatory Functions

Previously, we showed caveolae contain a population of protein kinase Cα (PKCα) that appears to regulate membrane invagination. We now report that multiple PKC isoenzymes are enriched in caveolae of unstimulated fibroblasts. To understand the mechanism of PKC targeting, we prepared caveolae lacking PKCα and measured the interaction of recombinant PKCα with these membranes. PKCα bound with high affinity and specificity to caveolae membranes. Binding was calcium dependent, did not require the addition of factors that activate the enzyme, and involved the regulatory domain of the molecule. A 68-kD PKCα-binding protein identified as sdr (serum deprivation response) was isolated by interaction cloning and localized to caveolae. Antibodies against sdr inhibited PKCα binding. A 100–amino acid sequence from the middle of sdr competitively blocked PKCα binding while flanking sequences were inactive. Caveolae appear to be a membrane site where PKC enzymes are organized to carry out essential regulatory functions as well as to modulate signal transduction at the cell surface.

Characterization of Arabidopsis calcium-dependent protein kinases: activated or not by calcium?

2012 ◽  
Vol 447 (2) ◽  
pp. 291-299 ◽  
Author(s):  
Marie Boudsocq ◽  
Marie-Jo Droillard ◽  
Leslie Regad ◽  
Christiane Laurière
Keyword(s):  
Protein Kinases ◽  
Calcium Binding ◽  
Molecular Mechanisms ◽  
Calcium Sensitivity ◽  
In Planta ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Biochemical Level ◽  
Calcium Dependent Protein Kinases ◽  
Ef Hands

CDPKs (calcium-dependent protein kinases), which contain both calmodulin-like calcium binding and serine/threonine protein kinase domains, are only present in plants and some protozoans. Upon activation by a stimulus, they transduce the signal through phosphorylation cascades to induce downstream responses, including transcriptional regulation. To understand the functional specificities of CDPKs, 14 Arabidopsis CPKs (CDPKs in plants) representative of the three main subgroups were characterized at the biochemical level, using HA (haemagglutinin)-tagged CPKs expressed in planta. Most of them were partially or mainly associated with membranes, in agreement with acylation predictions. Importantly, CPKs displayed highly variable calcium-dependences for their kinase activities: seven CPKs from subgroups 1 and 2 were clearly sensitive to calcium with different intensities, whereas six CPKs from subgroup 3 exhibited low or no calcium sensitivity to two generic substrates. Interestingly, this apparent calcium-independence correlated with significant alterations in the predicted EF-hands of these kinases, although they all bound calcium. The noticeable exception, CPK25, was calcium-independent owing to the absence of functional EF-hands. Taken together, the results of the present study suggest that calcium binding differentially affects CDPK isoforms that may be activated by distinct molecular mechanisms.

scholarly journals Role of Calcium Signaling in the Transcriptional Regulation of the Apicoplast Genome ofPlasmodium falciparum

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Sabna Cheemadan ◽  
Ramya Ramadoss ◽  
Zbynek Bozdech
Keyword(s):  
Calcium Binding ◽  
Transcriptional Response ◽  
Mrna Levels ◽  
Transcriptional Responses ◽  
Calcium Dependent ◽  
Regulatory Processes ◽  
Ofplasmodium Falciparum ◽  
Ef Hand ◽  
Apicoplast Genome

Calcium is a universal second messenger that plays an important role in regulatory processes in eukaryotic cells. To understand calcium-dependent signaling in malaria parasites, we analyzed transcriptional responses ofPlasmodium falciparumto two calcium ionophores (A23187 and ionomycin) that cause redistribution of intracellular calcium within the cytoplasm. While ionomycin induced a specific transcriptional response defined by up- or downregulation of a narrow set of genes, A23187 caused a developmental arrest in the schizont stage. In addition, we observed a dramatic decrease of mRNA levels of the transcripts encoded by the apicoplast genome during the exposure ofP. falciparumto both calcium ionophores. Neither of the ionophores caused any disruptions to the DNA replication or the overall apicoplast morphology. This suggests that the mRNA downregulation reflects direct inhibition of the apicoplast gene transcription. Next, we identify a nuclear encoded protein with a calcium binding domain (EF-hand) that is localized to the apicoplast. Overexpression of this protein (termed PfACBP1) inP. falciparumcells mediates an increased resistance to the ionophores which suggests its role in calcium-dependent signaling within the apicoplast. Our data indicate that theP. falciparumapicoplast requires calcium-dependent signaling that involves a novel protein PfACBP1.

Calcium binding to the regulatory domain of skeletal muscle troponin C induces a highly constrained open conformation

1998 ◽  
Vol 281 (3) ◽  
pp. 445-452 ◽  
Author(s):  
Mingda She ◽  
Jun Xing ◽  
Wen-Ji Dong ◽  
Patrick K Umeda ◽  
Herbert C Cheung
Keyword(s):  
Skeletal Muscle ◽  
Calcium Binding ◽  
Troponin C ◽  
Regulatory Domain ◽  
Open Conformation
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