Structure–Function Relationships in Ca2+ Cycling Proteins

Shi

Proc. Natl. Acad. Sci. U.S.A.

et al. 2005

Although it is known that calcium is a very important messenger involved in many eukaryotic cellular processes, much less is known about calcium's role in bacteria. CcbP, a Ca 2؉ -binding protein, was isolated from the heterocystous cyanobacterium Anabaena sp. PCC 7120, and the ccbP gene was cloned and inactivated. In the absence of combined nitrogen, inactivation of ccbP resulted in multiple contiguous heterocysts, whereas overexpression of ccbP suppressed heterocyst formation. Calmodulin, which is not present in Anabaena species, could also suppress heterocyst formation in both Anabaena sp. PCC 7120 and Anabaena variabilis. HetR induction upon nitrogen step-down was slow in the strain overexpressing ccbP. It has been demonstrated that [Ca 2ϩ ] i is tightly regulated in some bacteria (6), and there is evidence indicating that calcium is critical to some bacterial cellular processes such as sporulation of Bacillus (7), chemotaxis of Escherichia coli (8), and heterocyst differentiation of cyanobacteria (9). Heterocyst development may represent one of the earliest examples of pattern formation in evolution (10). Heterocysts are specialized cells for nitrogen fixation of some filamentous cyanobacteria (11)(12)(13)(14). When heterocystous cyanobacteria are subjected to nitrogen step-down, some vegetative cells differentiate and become heterocysts. Many genes are specifically involved in heterocyst differentiation. The genes that are required for initiation of cell differentiation include ntcA (15, 16) and hetR (17). HetR is a serine-type protease that plays an important role in heterocyst formation (18).Evidence obtained by manipulating extracellular calcium concentration and by using inhibitors of Ca 2ϩ -binding proteins suggested that Ca 2ϩ could be involved in heterocyst differentiation (9). Here we describe a Ca 2ϩ -binding protein, CcbP, from Anabaena sp. PCC 7120. Our study shows that free calcium accumulates in differentiating cells and mature heterocysts, correlated with a drop in the level of the Ca 2ϩ -binding protein.The free Ca 2ϩ concentration appears to be critical for the differentiation process. Materials and MethodsThe following protocols can be found in Supporting Text, which is published as supporting information on the PNAS web site: growth of the strains of Anabaena; isolation of the Ca 2ϩ -binding protein CcbP; isolation of the ccbP gene from total genomic DNA; expression of recombinant ccbP in Escherichia coli; construction of plasmids for inactivation of the ccbP gene, for copper-regulated expression of ccbP, for copper-regulated expression of rat calmodulin (CaM) gene, for ccbP promoter regulation of GFP expression, and for expression of obelin, a reporter for the level of free Ca 2ϩ .Assays for Ca 2؉ -Binding Proteins. 45 Ca 2ϩ overlay assay was performed as follows: Proteins were first separated by SDS͞PAGE and then transferred to a poly(vinylidene difluoride) membrane. It was then washed three times with buffer C (20 mM Tris⅐HCl, pH 7.2͞5 mM MgCl 2 ͞60 mM KCl͞10 mM imidazole). The memb...

Section: Discussionmentioning

confidence: 99%

CcbP, a calcium-binding protein from Anabaena sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation

Shi

Proc. Natl. Acad. Sci. U.S.A.

et al. 2005

“…Ryanodine and inositol 1,4,5-trisphosphate (IP 3 ) receptor subtypes on the sarco/endoplasmic reticulum (SR/ER) comprise a unique family of intracellular Ca 2+ release channels that mediate Ca 2+ mobilisation in response to various stimuli (Meissner, 1994;Bezprozvanny and Ehrlich, 1995;MacLennan et al, 2002;Patterson et al, 2004). Release of Ca 2+ from the SR/ER generates a negative potential on the luminal side, which is likely to inhibit subsequent Ca 2+ release processes.…”

Section: Introductionmentioning

confidence: 99%

Essential role of the TRIC-B channel in Ca2+ handling of alveolar epithelial cells and in perinatal lung maturation

et al. 2009

Journal of Biological Chemistry

“…Ca 2ϩ binding sites in CSQ are supposed to be very different from those in the Ca 2ϩ pump (sarco(endo)plasmic reticulum calcium ATPase (SERCA)), calmodulin, and troponin C. CSQ sites need to be made and broken but not over the low cytosolic Ca 2ϩ concentration range or with the same stoichiometry and precision as those formed and subsequently disrupted in the Ca 2ϩ pump or those which are intrinsic to the EF-hand structure (5). Therefore, the high capacity and low affinity Ca 2ϩ binding by CSQ is likely nonspecific, although the first few ions that bind at low Ca 2ϩ concentrations may have some specificity.…”

mentioning

confidence: 99%

Comparing Skeletal and Cardiac Calsequestrin Structures and Their Calcium Binding

Park

Kim

et al. 2004

Self Cite

131

Calsequestrin, the major calcium storage protein of both cardiac and skeletal muscle, binds and releases large numbers of Ca 2؉ ions for each contraction and relaxation cycle. Here we show that two crystal structures for skeletal and cardiac calsequestrin are nearly superimposable not only for their subunits but also their front-to-front-type dimers. Ca 2؉ binding curves were measured using atomic absorption spectroscopy. This method enables highly accurate measurements even for Ca 2؉ bound to polymerized protein. The binding curves for both skeletal and cardiac calsequestrin were complex, with binding increases that correlated with protein dimerization, tetramerization, and oligomerization. The Ca 2؉ binding capacities of skeletal and cardiac calsequestrin are directly compared for the first time, with ϳ80 Ca 2؉ ions bound per skeletal calsequestrin and ϳ60 Ca 2؉ ions per cardiac calsequestrin, as compared with net charges for these molecules of ؊80 and ؊69, respectively. Deleting the negatively charged and disordered C-terminal 27 amino acids of cardiac calsequestrin results in a 50% reduction of its calcium binding capacity and a loss of Ca 2؉ -dependent tetramer formation. Based on the crystal structures of rabbit skeletal muscle calsequestrin and canine cardiac calsequestrin, Ca 2؉ binding capacity data, and previous lightscattering data, a mechanism of Ca 2؉ binding coupled with polymerization is proposed.Calsequestrin (CSQ) 1 binds and releases large quantities of Ca 2ϩ through its high capacity (40 -50 mol of Ca 2ϩ ion per molecule) and relatively low affinity interactions with Ca 2ϩ (K d ϭ 1 mM) (1). Because of this Ca 2ϩ -buffering capacity of CSQ in the lumenal space, the concentration of free Ca 2ϩ in the sarcoplasmic reticulum (SR) can be maintained below the inhibitory level of the Ca 2ϩ pump (1 mM), and simultaneously, the SR can maintain the ability to rapidly deliver a high capacity Ca 2ϩ signal to the cytoplasm. Even though the lumenal space is minuscule compared with the extracellular space, the high concentrations (ϳ100 mg/ml) of CSQ make the SR an efficient storage compartment for Ca 2ϩ (2).CSQ is associated physically with the RyR protein by a nucleation event that involves CSQ binding to the basic lumenal domains of triadin (3) or junctin (4). These two proteins interact with RyR in the junctional face region of the SR, and this network of interacting proteins assures that high concentrations of Ca 2ϩ are stored very near to the site of Ca 2ϩ release. Ca 2ϩ release from CSQ through the Ca 2ϩ release channel is regulatory but not limiting.The Ca 2ϩ binding and dissociation mechanisms of CSQ are not yet clearly understood. Ca 2ϩ binding sites in CSQ are supposed to be very different from those in the Ca 2ϩ pump (sarco(endo)plasmic reticulum calcium ATPase (SERCA)), calmodulin, and troponin C. CSQ sites need to be made and broken but not over the low cytosolic Ca 2ϩ concentration range or with the same stoichiometry and precision as those formed and subsequently disrupted in the Ca 2ϩ pump or...