Calcium binding site

Parvalbumin is a muscle protein with a single polypeptide chain of 109 amino acids. Its function is uncertain, but calcium binding to this protein probably plays a role in muscle relaxation. The helix-loop-helix motif appears three times in this structure, in two of the cases there is a calcium-binding site. Figure 2.13 shows this motif which is called an EF hand because the fifth and sixth helices from the amino terminus in the structure of parvalbumin, which were labeled E and F, are the parts of the structure that were originally used to illustrate calcium binding by this motif. Despite this trivial origin, the name has remained in the literature. 

The three-dimensional structure of HRP C is largely a-helical, although there is also a small region of (3-sheet. There are two domains, the distal and proximal, between which the heme group is located. These domains probably originated as a result of gene duplication, a proposal supported by their common calcium binding sites and other structural elements (Veitch 2004). 

Numbers of Calcium-Binding Sites in Selected Metalloproteins... 

M.-C. Kilhoffer, J. G. Demaille, and D. Gerard, Terbium as luminescent probe of calmodulin calcium-binding sites. Domains I and II contain the high-affinity sites, FEBS Lett. 116, 269-272 (1980). 

P. Kanellis, J. Yang, H. C. Cheung, and R. E. Lenkinski, Synthetic peptide analogs of skeletal troponin C Fluorescence studies of analogs of the low-affinity calcium-binding site II, Arch. Biochem. Biophys. 220, 530-540 (1983). 

N. A. Malik, G. M. Anatharamaiah, A. Gawish, and H. C. Cheung, Structural and biological studies on synthetic peptide analogues of a low-affinity calcium-binding site of skeletal troponin C, Biochim. Biophys. Acta 911, 221-230 (1987). 

During the last ten years, it has become apparent that calcium-dependent papain-like peptidases called calpains (EC 3.4.22.17) represent an important intracellular nonlysosomal enzyme system [35][36], These enzymes show limited proteolytic activity at neutral pH and are present in virtually every eukaryotic cell type. They have been found to function in specific proteolytic events that alter intracellular metabolism and structure, rather than in general turnover of intracellular proteins. Calpains are composed of two nonidentical subunits, each of which contains functional calcium-binding sites. Two types of calpains, i.e., /i-calpain and m-calpain (formerly calpain I and calpain II, respectively), have been identified that differ in their Ca2+ requirement for activation. The activity of calpains is regulated by intracellular Ca2+ levels. At elevated cytoplasmic calcium concentrations, the precursor procal-pain associates with the inner surface of the cell membrane. This interaction seems to trigger autoproteolysis of procalpain, and active calpain is released into the cytoplasm [37]. 

Fig. 56. The calcium-binding sites from carp muscle calcium-binding protein (a) backbone of the entire E-F hand (b) detailed view of the E-F calcium-binding site, including those side chains which are Ca ligands (c) detailed view of the C-D calcium-binding site, rotated to match part b. Oxygens are shown as open circles and a-carbons as solid dots.

Fig. 7. The calcium binding sites of HRP C (detail). Ligands to the distal (A) and proximal (B) calcium atoms are shown. The O-donor ligand sets are hsted in Section IV,D.

The presence of calcium in horseradish peroxidase was demonstrated originally by Haschke and Friedhoff, working with the C and A (imspec-ified, but likely to have been predominantly A2) isoenzymes (209). HRP C and HRP A contain 2.0 0.13 and 1.4 0.19 moles calcium per mole enzyme, respectively, as determined by atomic absorption spectroscopy. Incubation in 6 M guanidinium hydrochloride and 10 mM EDTA for 4 hours at neutral pH and room temperature gave calcium-depleted enzymes with specific activities decreased by 40% and 15%, respectively. The thermal stability of calcium-depleted HRP C was also reduced compared to native enzyme. Reconstitution was successful only with calcium-depleted HRP C (209). It remains to be established whether this reflects true structural differences between the calcium binding sites of the two isoenz5unes, or is a consequence of the relatively harsh... 

The proximal calcium binding site is coupled to the heme group by virtue of the fact that one of its ligands, Thrl71, is adjacent to the proximal histidine residue, Hisl70 (Fig. 4). The results of site-directed mutagenesis studies at this position are awaited with interest. An illustration of the importance of both calcium sites to the structure and function of HRP C is afforded by the need to incorporate calcium as a component of in vitro folding mixtures to obtain active recombinant enzyme from solubilized inclusion bodies (64). 

Fig. 9. The calcium binding site in P. aeruginosa CCP. One of the propionates of the high-potential (HP) heme is hydrogen bonded to two bound water (OW) molecules.

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