Ca-Dependent Folding of Human Calumenin (original) (raw)
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Biophysical characterisation of calumenin as a charged F508del-CFTR folding modulator
PloS one, 2014
The cystic fibrosis transmembrane regulator (CFTR) is a cyclic-AMP dependent chloride channel expressed at the apical surface of epithelial cells lining various organs such as the respiratory tract. Defective processing and functioning of this protein caused by mutations in the CFTR gene results in loss of ionic balance, defective mucus clearance, increased proliferation of biofilms and inflammation of human airways observed in cystic fibrosis (CF) patients. The process by which CFTR folds and matures under the influence of various chaperones in the secretory pathway remains incompletely understood. Recently, calumenin, a secretory protein, belonging to the CREC family of low affinity calcium binding proteins has been identified as a putative CFTR chaperone whose biophysical properties and functions remain uncharacterized. We compared hydropathy, instability, charge, unfoldability, disorder and aggregation propensity of calumenin and other CREC family members with CFTR associated ch...
Binding-induced folding transitions in calpastatin subdomains A and C
Protein Science, 2009
Calpastatin, the endogenous inhibitor of calpain, is an intrinsically unstructured protein proposed to undergo folding transitions upon binding to the enzyme. As this feature has never been experimentally tested, we have set out to characterize the conformation of two peptides corresponding to its conserved subdomains, A and C, known to interact with calpain in a Ca 2+ -dependent manner. The peptides are disordered in water but show a high propensity for ␣-helical conformation in the presence of trifluoroethanol. The conformational transition is sensitive to Ca 2+ , and is clearly seen upon binding of the peptides to the enzyme. Secondary-structure prediction of all calpastatin sequences shows that the helix-forming potential within these regions is a conserved feature of the inhibitor. Furthermore, quantitative data on the binding strength of calpastatin fragments reveal that binding of the inhibitor is accompanied by a large decrease in its configurational entropy. Taken together, these observations point to significant binding-induced local folding transitions in calpastatin, in a way that ensures highly specific, yet reversible, action of the inhibitor.
Calbindin D28k Exhibits Properties Characteristic of a Ca2+ Sensor
Journal of Biological Chemistry, 2002
Calbindin D 28k is a member of the calmodulin superfamily of Ca 2؉-binding proteins and contains six EFhands. The protein is generally believed to function as a Ca 2؉ buffer, but the studies presented in this work indicate that it may also act as a Ca 2؉ sensor. The results show that Mg 2؉ binds to the same sites as Ca 2؉ with an association constant of ϳ1.4⅐10 3 M ؊1 in 0.15 M KCl. The four high affinity sites in calbindin D 28k bind Ca 2؉ in a non-sequential, parallel manner. In the presence of physiological concentrations of Mg 2؉ , the Ca 2؉ affinity is reduced by a factor of 2, and the cooperativity, which otherwise is modest, increases. Based on the binding constants determined in the presence of physiological salt concentrations, we estimate that at the Ca 2؉ concentration in a resting cell calbindin D 28k is saturated to 40-75% with Mg 2؉ but to less than 9% with Ca 2؉. In contrast, the protein is expected to be nearly fully saturated with Ca 2؉ at the Ca 2؉ level of an activated cell. A substantial conformational change is observed upon Ca 2؉ binding, but only minor structural changes take place upon Mg 2؉ binding. This suggests that calbindin D 28k undergoes Ca 2؉-induced structural changes upon Ca 2؉ activation of a cell. Thus, calbindin D 28k displays several properties that would be expected for a protein involved in Ca 2؉-induced signal transmission and hence may function not only as a Ca 2؉ buffer but also as a Ca 2؉ sensor. Digestion patterns resulting from limited proteolysis of the protein suggest that the loop of EF-hand 2, a variant site that does not bind Ca 2؉ , becomes exposed upon Ca 2؉ binding.
Journal of Biological Chemistry, 2009
Calreticulin is an abundant endoplasmic reticulum resident protein that fulfills at least two basic functions. Firstly, due to its ability to bind monoglucosylated high mannose oligosaccharides, calreticulin is a central component of the folding quality control system of glycoproteins. On the other hand, thanks to its capacity to bind high amounts of calcium, calreticulin is one of the main calcium buffers in the endoplasmic reticulum. This last activity resides on a highly negatively charged domain located at the C terminus. Interestingly, this domain has been proposed to regulate the intracellular localization of calreticulin. Structural information for this domain is currently scarce. Here we address this issue by employing a combination of biophysical techniques and molecular dynamics simulation. We found that calreticulin C-terminal domain at low calcium concentration displays a disordered structure, whereas calcium addition induces a more rigid and compact conformation. Remarkably, this change develops when calcium concentration varies within a range similar to that taking place in the endoplasmic reticulum upon physiological fluctuations. In addition, a much higher calcium concentration is necessary to attain similar responses in a peptide displaying a randomized sequence of calreticulin C-terminal domain, illustrating the sequence specificity of this effect. Molecular dynamics simulation reveals that this ordering effect is a consequence of the ability of calcium to bring into close proximity residues that lie apart in the primary structure. These results place calreticulin in a new setting in which the protein behaves not only as a calcium-binding protein but as a finely tuned calcium sensor. Calreticulin (CRT) 2 is an endoplasmic reticulum (ER) calcium-binding chaperone that has been associated with several cellular functions both inside (1-3) and outside the ER (4-7).
Site-site communication in the EF-hand Ca2+-binding protein calbindin D9k
Nature structural biology, 2000
The cooperative binding of Ca2+ ions is an essential functional property of the EF-hand family of Ca2+-binding proteins. To understand how these proteins function, it is essential to characterize intermediate binding states in addition to the apo- and holo-proteins. The three-dimensional solution structure and fast time scale internal motional dynamics of the backbone have been determined for the half-saturated state of the N56A mutant of calbindin D9k with Ca2+ bound only in the N-terminal site. The extent of conformational reorganization and a loss of flexibility in the C-terminal EF-hand upon binding of an ion in the N-terminal EF-hand provide clear evidence of the importance of site-site interactions in this family of proteins, and demonstrates the strength of long-range effects in the cooperative EF-hand Ca2+-binding domain.
Molecular Dynamics of the Neuronal EF-Hand Ca2+-Sensor Caldendrin
PLoS ONE, 2014
Caldendrin, L-and S-CaBP1 are CaM-like Ca 2+ -sensors with different N-termini that arise from alternative splicing of the Caldendrin/CaBP1 gene and that appear to play an important role in neuronal Ca 2+ -signaling. In this paper we show that Caldendrin is abundantly present in brain while the shorter splice isoforms L-and S-CaBP1 are not detectable at the protein level. Caldendrin binds both Ca 2+ and Mg 2+ with a global K d in the low mM range. Interestingly, the Mg 2+ -binding affinity is clearly higher than in S-CaBP1, suggesting that the extended N-terminus might influence Mg 2+ -binding of the first EF-hand. Further evidence for intra-and intermolecular interactions of Caldendrin came from gel-filtration, surface plasmon resonance, dynamic light scattering and FRET assays. Surprisingly, Caldendrin exhibits very little change in surface hydrophobicity and secondary as well as tertiary structure upon Ca 2+ -binding to Mg 2+ -saturated protein. Complex inter-and intramolecular interactions that are regulated by Ca 2+ -binding, high Mg 2+ -and low Ca 2+ -binding affinity, a rigid first EF-hand domain and little conformational change upon titration with Ca 2+ of Mg 2+ -liganted protein suggest different modes of binding to target interactions as compared to classical neuronal Ca 2+ -sensors.
Biochemistry, 2009
Calbindin-D 28k is a calcium binding protein with six EF hand domains. Calbindin-D 28k is unique in that it functions as both a calcium buffer and sensor protein. It is found in many tissues including the brain, pancreas, kidney and intestine, playing important roles in each. Calbindin-D 28k is known to bind four calcium ions and upon calcium binding undergoes a conformational change. The structure of apo-calbindin-D 28k is in an ordered state, transitioning into a disordered state as calcium is bound. Once fully loaded with four calcium ions, it again takes on an ordered state. The solution structure of disulfide-reduced holo-calbindin-D 28k has been solved by NMR, while the structure of apocalbindin-D 28k has yet to be solved. Differential surface modification of lysine and histidine residues analyzed by mass spectrometry have been used in this study to identify, for the first time, the specific regions of calbindin-D 28k undergoing conformational changes between the holo-and apo-states. Using differential surface modification in combination with mass spectrometry, EF hands 1 and 4 as well as the linkers before EF hand 1 and the linkers between EF hands 4-5 and 5-6 were identified as regions of conformational change between the apo-and holo-calbindin-D 28k . Under the experimental conditions employed, EF hands 2 and 6, which are known to not bind calcium were unaffected in either form. EF hand 2 is highly accessible; however, EF hand 6 was determined to not be surface accessible in either form. Previous research has identified a disulfide bond between cysteines 94 and 100 in the holo-state. Until now it was unknown whether this bond also exists in the apo-form. Our data confirm the presence of the disulfide bond between cysteines 94 and 100 in the holo-form, and indicate that there is predominantly no disulfide bond between these residues in the apo-protein.