Calcium-induced sensitization of the central helix of calmodulin to proteolysis

Mackall, Julia C.; Klee, Claude B.

doi:10.1021/bi00243a028

Cited by 33 publications

(35 citation statements)

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“…The membrane-embedded sequence of site 2 protease (a zinc metalloprotease) is believed to unwind an ␣-helix of the membrane-bound sterol regulatory element binding protein into a less-ordered structure to facilitate proteolytic cleavage (41). Calcium binding in calmodulin is also known to induce flexibility in the central helix of calmodine for proteolytic attack (24).…”

Section: Discussionmentioning

confidence: 99%

Crystal Structures of the BlaI Repressor from Staphylococcus aureus and Its Complex with DNA: Insights into Transcriptional Regulation of the bla and mec Operons

Safo

Zhao

et al. 2005

J Bacteriol

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The 14-kDa BlaI protein represses the transcription of blaZ, the gene encoding ␤-lactamase. It is homologous to MecI, which regulates the expression of mecA, the gene encoding the penicillin binding protein PBP2a. These genes mediate resistance to ␤-lactam antibiotics in staphylococci. Both repressors can bind either bla or mec DNA promoter-operator sequences. Regulated resistance genes are activated via receptor-mediated cleavage of the repressors. Cleavage is induced when ␤-lactam antibiotics bind the extramembrane sensor of the sensor-transducer signaling molecules, BlaR1 or MecR1. The crystal structures of BlaI from Staphylococcus aureus, both in free form and in complex with 32 bp of DNA of the mec operator, have been determined to 2.0-and 2.7-Å resolutions, respectively. The structure of MecI, also in free form and in complex with the bla operator, has been previously reported. Both repressors form homodimers, with each monomer composed of an N-terminal DNA binding domain of winged helix-turn-helix topology and a C-terminal dimerization domain. The structure of BlaI in complex with the mec operator shows a protein-DNA interface that is conserved between both mec and bla targets. The recognition helix ␣3 interacts specifically with the conserved TACA/TGTA DNA binding motif. BlaI and, probably, MecI dimers bind to opposite faces of the mec DNA double helix in an up-and-down arrangement, whereas MecI and, probably, BlaI dimers bind to the same DNA face of bla promoter-operator DNA. This is due to the different spacing of mec and bla DNA binding sites. Furthermore, the flexibility of the dimeric proteins may make the C-terminal proteolytic cleavage site more accessible when the repressors are bound to DNA than when they are in solution, suggesting that the induction cascade involves bound rather than free repressor.␤-Lactam antibiotics are becoming less effective therapy for treating staphylococcal infections as resistance to them increases. Resistance is mediated by a ␤-lactamase (encoded by blaZ) that hydrolyzes penicillins (4) and an alternate penicillin binding protein target (PBP2a, encoded by mecA) to which ␤-lactam antibiotics bind poorly (17). The transcription of blaZ and mecA is corepressed by related regulators (encoded by blaI and mecI). Both BlaI and MecI bind to palindromic promoteroperator sequences, presumably as dimers, and can interchangeably repress the transcription of either target gene (8,26,36). Signal transduction through either of two transmembrane inducers, BlaR1 or MecR1, leads to proteolytic autocleavage of the cytoplasmic domains of these molecules. Autocleavage of the signal transducer is followed by cleavage of the cognate repressor (BlaI or MecI, respectively) and induction of the transcription of blaZ or mecA (42). The exact mechanism of cleavage is unclear at present. The current hypothesis (1, 42) is that ␤-lactam binding to the extramembrane sensor of BlaR1 or MecR1 triggers autocleavage of its cytoplasmic domain at a single site, leaving the putative cytoplasmic protease te...

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Section: Discussionmentioning

confidence: 99%

Crystal Structures of the BlaI Repressor from Staphylococcus aureus and Its Complex with DNA: Insights into Transcriptional Regulation of the bla and mec Operons

Safo

Zhao

et al. 2005

J Bacteriol

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“…Apo-CaM is also known to be significantly~and differentially! more sensitive to proteolytic attack than Ca-CaM~Walsh et al, 1977;Kawasaki et al, 1986;Mackall & Klee, 1991!. Unless mitigated in some way, the instability of apo-CaM could therefore enhance its susceptibility to proteolytic cleavage in vivo.…”

Section: Discussionmentioning

confidence: 99%

Ligand binding and thermodynamic stability of a multidomain protein, calmodulin

Masino¹,

Martin²,

Bayley³

2000

Protein Science

124

196

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Chemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca 2ϩ or Mg 2ϩ . The reversible urea unfolding of either isolated apo-domain follows a two-state mechanism with relatively low DG8 20 values of ;2.7~N-domain! and ;1.9 kcal0mol~C-domain!. The apo-C-domain is significantly unfolded at normal temperatures~20-25 8C!. The greater affinity of the C-domain for Ca 2ϩ causes it to be more stable than the N-domain at @Ca 2ϩ # Ն0.3 mM. By contrast, Mg 2ϩ causes a greater stabilization of the N-rather than the C-domain, consistent with measured Mg 2ϩ affinities. For the intact protein~6Ca 2ϩ !, the bimodal denaturation profiles can be analyzed to give two DG8 20 values, which differ significantly from those of the isolated domains, with one domain being less stable and one domain more stable. The observed stability of the domains is strongly dependent on solution conditions such as ionic strength, as well as specific effects due to metal ion binding. In the intact protein, different folding intermediates are observed, depending on the ionic composition. The results illustrate that a protein of low intrinsic stability is liable to major perturbation of its unfolding properties by environmental conditions and liganding processes and, by extension, mutation. Hence, the observed stability of an isolated domain may differ significantly from the stability of the same structure in a multidomain protein. These results address questions involved in manipulating the stability of a protein or its domains by site directed mutagenesis and protein engineering.

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“…Only in this way can we explain why only the complex of two peptide molecules and one CaM molecule is observed in our experiments. Evidence for communication between both lobes of CaM has recently been reported by several groups (42)(43)(44)(45)(46). In this way, it would not be necessary for the two bound PGD peptides to interact with each other in the complex.…”

mentioning

confidence: 86%

Calcium-Calmodulin-induced Dimerization of the Carboxyl-terminal Domain from Petunia Glutamate Decarboxylase

Yuan

Vogel

1998

Journal of Biological Chemistry

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The acidic, bilobed protein calmodulin (CaM; molecular mass of 16.7 kDa) can activate some 40 distinct proteins in a calcium-dependent manner. The majority of the CaM-binding domain regions of the target proteins are basic and hydrophobic in nature, are devoid of multiple negatively charged residues, and have a propensity to form an ␣-helix. The CaM-binding domain in the Cterminal region of petunia glutamate decarboxylase (PGD) is atypical because it contains five negatively charged residues. Therefore, we chose to study the binding of calcium-CaM to a 26-residue synthetic peptide encompassing the C-terminal region of PGD. Gel band shift assays, fluorescence spectroscopy, and NMR titration studies showed that a single unique complex of calcium-CaM with two PGD peptides is formed. The formation of a 1:2 protein-peptide complex is unusual; normally, calcium-CaM forms 1:1 complexes with the majority of its target proteins. Circular dichroism spectroscopy showed that the bound PGD peptides have an ␣-helical structure. NMR studies of biosynthetically [methyl-13 C]methionine-labeled CaM revealed that all the Met side chains in CaM are involved in the binding of the PGD peptides. Analysis of fluorescence spectra showed that the single Trp residue of the two peptides becomes bound to the N-and C-terminal lobes of CaM. These results predict that binding of calcium-CaM to PGD will give rise to dimerization of the protein, which may be necessary for activation. Possible models for the structure of the protein-peptide complex, such as a dimeric peptide structure, are discussed. Calmodulin (CaM)1 is a 148-amino acid acidic protein that acts as a ubiquitous calcium regulatory protein in eukaryotic cells. It can bind and activate Ͼ40 different target proteins (for recent reviews, see Refs. 1-4). These proteins include several protein kinases, phosphatase 2B (calcineurin), multiple components in smooth muscle contraction, ion channels, etc. In the x-ray structure, Ca 2ϩ -CaM adopts a dumbbell-shaped structure, with the two lobes connected by a 26-residue-long ␣-helix (5). Each lobe contains two helix-loop-helix Ca 2ϩ -binding motifs that are interconnected by a small ␤-sheet between the two Ca 2ϩ -binding loops. In solution, the middle portion of the central ␣-helical linker region is flexible, as demonstrated by NMR spectroscopy (6). Methionine-rich hydrophobic patches become exposed on each lobe of CaM following the binding of four Ca 2ϩ ions (5), whereas these two hydrophobic patches are absent or not fully exposed in apo-CaM structures (7,8). When the concentration of the secondary messenger Ca 2ϩ increases inside cells from ϳ10 Ϫ7 to ϳ10 Ϫ5 M, four Ca 2ϩ ions will bind to CaM, and CaM will expose its two hydrophobic patches for target protein binding. This sequence of events is confirmed by high resolution x-ray and NMR structures of the complexes between Ca 2ϩ -CaM and peptides derived from CaM-binding domains of target proteins (9 -11).CaM-binding domains in typical target proteins do not have any amino acid sequence hom...

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Calcium-induced sensitization of the central helix of calmodulin to proteolysis

Cited by 33 publications

References 45 publications

Crystal Structures of the BlaI Repressor from Staphylococcus aureus and Its Complex with DNA: Insights into Transcriptional Regulation of the bla and mec Operons

Crystal Structures of the BlaI Repressor from Staphylococcus aureus and Its Complex with DNA: Insights into Transcriptional Regulation of the bla and mec Operons

Ligand binding and thermodynamic stability of a multidomain protein, calmodulin

Calcium-Calmodulin-induced Dimerization of the Carboxyl-terminal Domain from Petunia Glutamate Decarboxylase

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