Sarcoplasmic Calcium-binding Protein from Nereis diversicolor
The calmodulin superfamily of proteins also includes' sarcoplasmic Ca2+-binding proteins (SCPs) that can be found in both vertebrate and invertebrate muscle.129 The function of SCPs is not yet known, but their sequence homology with Ca2+-binding proteins of known tertiary structure suggests that they originally contained four helix-loop-helix Ca2+-binding domains. Ca2+ binding has been preserved in the first and third domains of all known SCPs, but only one, if any, of domains II and IV is functional. The three-dimensional crystal structure of an SCP from the sandworm Nereis diversicolor analyzed at 3.0 Å resolution130 can be seen in Figure 3.26. (See color plate section, page C-11.) The C-terminal half (domains III and IV) of the molecule contains two Ca2+- binding EF-hands (green and red in Figure 3.26) similar to calbindin D9k and the globular domains of troponin C and calmodulin. The N-terminal half is, on the contrary, markedly different from the normal helix-loop-helix geometry. Domain I binds Ca2+ with a novel helix-loop-helix conformation, whereas domain H lacks calcium-binding capacity. The two halves are packed closely together, and are not, as in troponin C or calmodulin, connected by a solvent-exposed \(\alpha\)-helix.
Membrane Cytoskeleton and Phospholipid Binding Proteins
It has long been suspected that Ca2+ ions are somehow involved in exocytosis. Recently several groups131 have isolated intracellular proteins that associate with membranes, and/or membrane cytoskeleton proteins, in a Ca2+-dependent manner, and that seem able to mediate vesicle fusion or aggregation at Ca2+ concentrations above 200 \(\mu\)M. These proteins—endonexin, calelectrin, p36, and pII—have stretches of consensus amino-acid sequences that are also found in a phospholipase A2 inhibitor protein, lipocortin.132 It appears that further studies of this new class of proteins, known as annexins, will lead to new insights into cell-signaling pathways. Multiple functions have been proposed for the annexins, but no cellular role has yet been defined.133 The first crystal structure of an annexin, human annexin V—which in vitro will form voltage-gated Ca2+ channels—has been determined recently.172 In annexin, the three Ca2+-binding sites are located on the side of the molecule that is involved in membrane binding.
An interesting Ca2+-activated intracellular protease, sometimes called calpain, was discovered during the last decade.134 The ending -pain refers to its relation with other proteolytic enzymes like papain. It may seem dangerous to have a proteolytic enzyme loose inside a cell, and it must have rather specialized functions and be under strict control. The complete primary structure of the calcium protease (Mr ≈ 80,000) in chicken tissues has recently been deduced from the nucleotide sequence of cloned DNA.135,136 The findings are quite unexpected.
The protein contains four distinct domains. The first and third domains have no clear sequence homologies with known protein sequences, but the second domain has a high homology with the proteolytic enzyme papain, and the fourth domain is highly homologous to calmodulin. This fourth domain thus has four EF-hand-type Ca2+-binding sites, although the third site has a somewhat unusual loop sequence. Here we apparently are faced with an unusual invention by Nature: by fusing the gene for a protease with that of the canonical Ca2+ receptor, she has created a molecule in which a regulatory protein is covalently linked to its target enzyme!
Protein Kinase C
Before we leave our brief survey of intracellular Ca2+-binding proteins, we must write a few lines about an important Ca2+-regulated kinase (a phosphorylating enzyme), i.e., protein kinase C (PKC). The activity of this enzyme, or rather family of enzymes,137 appears to be regulated by three factors: phospholipids, in particular phosphatidylserine; diacyl-glycerols, one of the products of inositol lipid breakdown; and Ca2+ ions. The high-activity form of PKC, which appears responsible for much of the phosphorylation activity of many cells, is presumably membrane-bound, whereas the low-activity form may be partly cytosolic (Figure 3.27). The schematic structure of rabbit PKC (Mr ≈ 77 kDa) according to Ohno et al.138 is shown in Figure 3.28. The Ca2+ site(s) are presumably in the regulatory domain. No typical "EF-hand" pattern has been found in the amino-acid sequence. A protein kinase that requires Ca2+ but not phospholipids nor calmodulin for activity has been purified from soybean. From the amino-acid sequence the protein appears to have a calmodulin-like Ca2+-binding domain, very much as in calpain.139