The roles of metals in proteins have been discussed. Striated muscle proteins, actin, troponin C and myosin, are used as examples to elucidate the interaction between metal ions and proteins and the functional roles of metal ions play in the particular protein.
Many metals have been found in association with proteins. The relationship between proteins and metals involves two different aspects:
Regarding the active roles of metals in proteins, the following different types can be summarized:
The following table lists some of the variety of metals found in proteins and the roles they play.
|Metal||Example of protein||Role of Metal|
|Cu||Ascorbic acid oxidase||Oxidation-reduction|
|Zn||Alcohol dehydrogenase||Helps bind NAD+|
|Mn||Histidine ammonia-lyase||Aids in catalysis by electron withdrawal|
|Ca||Troponin-C||Triggers muscle contraction|
Muscles are very complex protein assembles. Ca2+ and Mg2+ are heavily involved in muscle structures and functions. The following sections will illustrate how Ca2+ and Mg2+ are involved in striated muscle's structures and functions.
Striated muscles are formed of bundles of muscle fibres, the basic unit of which is called the myofibril. Two majour types of filaments contained in myofibrils are 'thick' filaments and 'thin' filaments.
Thick filaments are made of myosin molecules each of which contains six subunits. The structure of myosin molecule were elucidated in 1960s of several laboratories (H.E. Huxley, 1963; Perry, 1967; Slayter and Lowey, 1967; Lowey et al, 1969) As showed in the figure below obtained from PPS2 course,
two heavy chains (200,000 daltons in size) have their alpha-helical tails wound each other to form a rod with two globular heads sticking out at one end of the rod. Two pairs of light chains (Two of 19,000 daltons, one of 21,000 daltons and one of 17,000 daltons) are attached to the two heads. Rod regions self-associate to form bipolar filaments with the globular heads protruding to interact with thin filaments.
Thin filaments contain actin filament as backbone with regulatory proteins tropomyosin and troponin attached to them. The ratio between actin monomer, tropomyosin and troponin is 7:1:1. Actin filament has the characteristic form of two chains of polymerized globular monomers wound round each other. Tropomyosin, a two-stranded coiled-coil of alpha-helices, is rod-like and forms head-to-tail polymers lying between the two actin chains. Troponin is attached to actin-tropomyosin complex at intervals of 38.5nm, and contains three subunits: troponin-I, which is responsible for preventing myosin head binding to actininteraction; troponin-C, which in the presence of Ca2+ is able to remove the inhibition imposed by troponin I and tropomyosin and start muscle contraction; and troponin-T, which binds the whole troponin complex to tropomyosin (Ebashi et al , 1969). The following figure shows the assembly of thin filament (obtained from PPS2 ourse).
On the basis of X-ray diffraction and electron microscope investigation, a steric blocking model has been proposed as a mechanism for the Ca2+-regulated control of striated muscle contraction ( Huxley, 1972; Wakabayashi, et al, 1975). Muscle contraction is because of the result of anti-parallel movement of thick and thin filaments upon each other, starts with the release of Ca2+ from sarcoplasmic reticulum. Troponin-C binds Ca2+ and initiate a series of conformational changes among thin filament proteins culminating in the release of inhibition of actin and myosin interaction. Myosin binding to actin induces ATPase activity in myosin heads which bend and pull attached actin filaments towards the centre of the bipolar myosin filaments.The following figure (Modified from the figure of Squire, 1981. A: actin; M: myosin.) shows the comparison of the mechanical steps involved in muscle contraction cycle (a) and the corresponding biochemical steps (b). In step 3, upon stimulation, myosin head with ADP and Pi attached forms cross-bridge with adjacent actin filament and the myosin head is still perpendicular to actin filament; in step 4, the interaction between actin and myosin causes the release of ADP and Pi which results in the conformational changes in myosin and the hinge between myosin head and rod bends aproximately 45o. This bending pulls the thin filament about 100A towards the M line in a "rowing" action. In step 1, ATP binds to myosin head causing it to detach from thin filament but still remain the bending conformation. In step 2, bound ATP is hydrolysed and myosin returns to original relaxed conformation.
A movie of this contraction cycle can be seen by click here .
Ca2+ (or Mg2+) in actin has the function of maintaining the structure of actin. Although it is not clear wether Ca2+ or Mg2+ is the structural metal ion in vivo, pure actin can interact with either ions.
Actin monomer in striated muscle is about 41,000 daltons in size and consists of two domains which can be further subdivided into two subdomains. Normally, isolated G-actin from rabbit skeletal muscle contains ATP and Ca2+ binding to a specific site. Ca2+ is responsible in stablizing the actin structure and in association with ATP which is hydrolysed when G-actin polymerises into actin filament. ATP is located in the cleft between the two domains and its beta- and gamma- phosphates interact with Ca2+ (Kabsch, et al, 1990).
Ca2+ sits in a hydrophilic pocket formed by the beta- and gama-phosphates of ATP and the residues Asp11, Gln137 and Asp154. The following figure (Modified from PDB file 1atn.pdb) shows the three-dimentional structure of G-actin with the bound ATP (in red colour) and Ca2+ (in green colour. The three Ca2+ at left-hand side are bound to DNaseI which forms complex with actin in order to produce crystals for x-Ray analysis.). Residues Asp11, Gln137 and Asp154 of actin are highlighted (in black colour).
The following schematic drawing ( From Kabsch, et al, 1990) shows the interaction of ATP with actin and Ca2+. The residues specified near the circular segment in the left form a pocket covering adenosine of ATP.
Troponin C, as mentioned before, is a Ca2+ binding protein in muscle complex and is responsible for Ca2+ -dependant regulation of muscle contraction. Ca2+ here has the triggering role. Troponin C can bind up to four Ca2+ per molecule (Greaser and Gergely, 1973). Two of the binding sites (sites III and IV) have high affinity for Ca2+ (Ka = 107M-1) and are located in the C-terminal domain of the molecule and can also bind Mg2+. The other two sites (sites I and II), located in the N-terminal domain, have low affinity and are Ca2+ specific (Ka = 105M-1) (Potter and Gergely, 1975).
The binding sites for Ca2+ in troponin C are of the typical EF hand structures which are oftenly found in Ca2+ -binding proteins. The basic structure of EF hand motif contains two alpha-helices almost perpendicular to each other with a loop in between. Ca2+ are bound to a specific constellation of seven oxygen atoms from side chains of Asp, Asn, Glu, Thr and Ser residues, backbone carbonyl, or intermidiary water molecules in the 12-residue loop segment . The following figure shows a typical EF hand motif with a Ca2+ (in yellow colour) sitting in the loop segment (modified from PDB file parv.pdb).
The following link shows the sequence of skeletal troponin C from turkey and the secondary segments along the sequence. It is clearly showed that an alpha-helix links the N-terminal and C-terminal domains and each domain contains two EF hand motifs.
This picture is confirmed in a three-dimentional structure of the turkey skeletal troponin C which has two Ca2+ (in yellow colour) bound to the C-terminal high affinity sites III and IV (modified from PDB file 5tnc).
Many studies have measured the conformational changes of troponin C upon binding of Ca2+. Without Ca2+ at sites I and II, the N-terminal part of the molecule has a distinctive configuration of the helices producing a cluster of hydrophobic residues, which stablizes the N-terminal structure. In contrast, C-terminal domain only has two out of four helices (F and G) coiled at the absence of Ca2+. Ca2+ binding to sites III and IV induces the coiling of helices E and H and thus stablizes the C-terminal (Nagy and gergely 1979). Binding of Ca2+ to regulatory sites I and II does not induce changes in secondary structure and thus tertiary structure of troponin C thus the question was raised as what conformational transition of troponin C triggers muscle contraction. Herzberg and James (1985) suggested two possible structural changes. First, when Ca2+ binds, the conformational changes in the middle of the central helix would cause a change in the relative disposition of the two halves of the molecule. This would result in a change in the relative position of troponin I and thus release the inhibition. Alternatively, a Ca2+-induced change in the inter-helical angles in sites I and II might stablize the interaction of the region with troponin I, and/or could be transmitted to C-terminal via a conformational change of the central helix.
Myosin has ATPase activity. This activity is Mg2+-dependant. Here it is more likely that Mg2+ binds to substrate ATP to make it a more suitable substrate. So far no evidence of the direct interaction of Mg2+ with myosin has been indicated.
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