3.12: Molecular Aspects of Calcium Ion-regulated Intracellular Processes (Part 2)

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 D_9k 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.

Ca²⁺-dependent Proteases

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 (M_r ≈ 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 (M_r ≈ 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

Figure 3.27 - Outline of the cellular events that result in the activation of protein kinase C (PKC). The enzyme apparently exists in at least two states. Recent sequence work indicates that it has a Ca²⁺-binding site of the EF-hand type. When no Ca²⁺ ion is bound, and when the "concentration" of diacylglycerol (DG) in the inner layer of the plasma membrane is low, the kinase exists in a low-activity form, possibly dissociated from the membrane. When a hormone binds to a plasmamembrane receptor (R), cleavage of phosphoinositol into 1,4,5-IP₃ and DG is induced. The latter lipid may bind to and activate the calcium-loaded form of PKC. The active form of protein kinase C will now phosphorylate other cytoplasmic proteins, and in this way modify their biochemical properties. R = receptor; PL-C = phospholipase C; G = a GTP-binding protein that is assumed to act as an intermediary between the receptor and the membrane bound PL-C.

Figure 3.28 - Schematic representation of the structure of rabbit protein kinase C.¹³⁸ Three highly homologous protein kinases C were actually identified with M_r ≈ 76,800. The kinase region shows clear similarity with other kinases. The regulatory domain should contain binding sites for Ca²⁺, phosphatidyl serine (PS), and diacylglycerol (DG).