Concerted Action of the High Affinity Calcium Binding Sites in Skeletal Muscle Troponin C

Abstract

Mutants of each of the four divalent cation binding sites of chicken skeletal muscle troponin C (TnC) were constructed using site-directed mutagenesis to convert Asp to Ala at the first coordinating position in each site. With a view to evaluating the importance of site-site interactions both within and between the N- and C-terminal domains, in this study the mutants are examined for their ability to associate with other components of the troponin-tropomyosin regulatory complex and to regulate thin filaments. The functional effects of each mutation in reconstitution assays are largely confined to the domain in which it occurs, where the unmutated site is unable to compensate for the defect. Thus the mutants of sites I and II bind to the regulatory complex but are impaired in ability to regulate tension and actomyosin ATPase activity, whereas the mutants of sites III and IV regulate activity but are unable to remain bound to thin filaments unless Ca2+ is present. When all four sites are intact, free Mg2+ causes a 50-60-fold increase in TnC's affinity for the other components of the regulatory complex, allowing it to attach firmly to thin filaments. Calcium can replace Mg2+ at a concentration ratio of 1:5000, and at this ratio the Ca2.TnC complex is more tightly bound to the filaments than the Mg2.TnC form. In the C-terminal mutants, higher concentrations of Ca2+ (above tension threshold) are required to effect this transformation than in the recombinant wild-type protein, suggesting that the mutants reveal an attachment mediated by Ca2+ in the N-domain sites.

Biological Function and Site II Ca2+-induced Opening of the Regulatory Domain of Skeletal Troponin C Are Impaired by Invariant Site I or II Glu Mutations

“…As demonstrated by Gagné et al (26) and in the present 2 Gagné et al (26) on the other hand have concluded, based on their NMR structure, that Val 65 , positioned at the C-terminal end of helix C and immediately adjunct to loop II, is the pivot point associated with site II. Houdusse et al (14), based on their structural analyses of the rabbit crystals, have identified small and larger hinge regions for each of EF-hands I and II The small regions involve residues at the Cterminal ends of helices A and C. study, the substitution of Glu 41 by Ala also impairs the closed to open structural transition as well as functionality (31) as assessed by tension measurements in the skinned muscle fiber system.…”

“…The effects of mutating other liganding residues in sites I and II of sTnC on its Ca 2ϩ binding and functional properties have been previously reported (31,(63)(64)(65)(66)(67)(68). These have been largely concerned with loop position 1 and/or 3 and include in loop I the mutations D30V and D30A (position 1) and D32G and D32A (position 3).…”

The Role of Troponins in Muscle Contraction

“…The two C-terminal high-affinity Ca 2+ -binding domains (EF3 and EF4) are functional in all cardiac and skeletal TnC isoforms. These two high-affinity Ca 2+ -binding sites are believed to be fully occupied by either Ca 2+ or Mg 2+ , depending on the physiological conditions of the muscle, and are likely to play a structural role providing stability to the Tn complex (25)(26)(27). The TnC-based Ca 2+ regulation of contraction appears to have separate mechanisms in skeletal vs. cardiac muscles.…”

Immunological and Biological Properties of Bet v 4, a Novel Birch Pollen Allergen with Two EF-hand Calcium-binding Domains

“…, as demonstrated by 45 Atopic allergies, which comprise a wide range of IgE-mediated disorders such as hay fever, asthma, atopic dermatitis, and food allergies, affect 12-20% of the population. Pollen originating from trees, grasses, and weeds are among the most important sources of airborne allergens and are associated with seasonal patterns of allergic diseases.…”