No abstract

cMyBP-C [cardiac (MyBP-C) myosin-binding protein-C)] is a sarcomeric protein involved both in thick filament structure and in the regulation of contractility. It is composed of eight IgI-like and three fibronectin-3-like domains (termed C0-C10). Mutations in the gene encoding cMyBP-C are a principal cause of HCM (hypertrophic cardiomyopathy). cMyBP-C binds to the LMM (light meromyosin) portion of the myosin rod via its C-terminal domain, C10. We investigated this interaction in detail to determine whether HCM mutations in beta myosin heavy chain located within the LMM portion alter the binding of cMyBP-C, and to define the precise region of LMM that binds C10 to aid in developing models of the arrangement of MyBP-C on the thick filament. In co-sedimentation experiments recombinant C10 bound full-length LMM with a K(d) of 3.52 microM and at a stoichiometry of 1.14 C10 per LMM. C10 was also shown to bind with similar affinity to LMM containing either the HCM mutations A1379T or S1776G, suggesting that these HCM mutations do not perturb C10 binding. Using a range of N-terminally truncated LMM fragments, the cMyBP-C-binding site on LMM was shown to lie between residues 1554 and 1581. Since it had been reported previously that acidic residues on myosin mediate the C10 interaction, three clusters of acidic amino acids (Glu1554/Glu1555, Glu1571/Glu1573 and Glu1578/Asp1580/Glu1581/Glu1582) were mutated in full-length LMM and the proteins tested for C10 binding. No effect of these mutations on C10 binding was however detected. We interpret our results with respect to the localization of the proposed trimeric collar on the thick filament.

Regulation of the hypoxic response in humans is regulated by the post-translational hydroxylation of hypoxia inducible transcription factor; a recombinant form of a human prolyl-4-hydroxylase (PHD2) was characterised and shown to have an unexpectedly high affinity for, and to copurify with endogenous levels of, its Fe(ii) cofactor and 2-oxoglutarate cosubstrate.

6 -dependent dioxygenase family (2OG oxygenases). Crystallographic and spectroscopic studies imply that the enzyme⅐Fe⅐2OG intermediate has an octahedral metal coordination, with three of the coordination sites occupied by the 2-oxo-acid of 2OG and a water molecule. In some cases, binding of substrate to this complex has been shown to induce loss of the iron bound water so enabling dioxygen binding. Oxidative decarboxylation of 2OG results in the formation of a ferryl species (Fe(VI)ϭO) that effects oxidation of the substrate/cosubstrate with the regeneration of Fe(II) at the active site (for review, see Refs. 1-3). In addition, spectroscopic studies have demonstrated that interaction of the non-iron-ligated oxygen atom of the carboxylate side chain of the metal binding Asp/Glu residue with the iron bound water molecule has a role in oxygen activation (4).Hydroxylation is the most common reaction catalyzed by the 2OG oxygenases, but family members also catalyze other oxidative reactions including demethylations, desaturations, epoxidations, and rearrangements. Related enzymes, which do not employ 2OG as a co-substrate, have been shown to catalyze oxidative fragmentations and cyclization reactions (for review, see Refs. 2 and 5). Recently, two 2OG oxygenases have been reported to catalyze oxidative halogenations (6, 7); the structure of one of these halogenases, SyrB2, revealed that the iron was coordinated by only two enzyme-derived His residues (8). The carboxylate-bearing residue found in all prior 2OG oxygenase structures was substituted by an Ala in SyrB2 apparently leaving sufficient space for a chloride ion to fill the vacant iron coordination site.Four 2OG oxygenases are known to be involved in regulation of the hypoxic response in humans via the post-translational hydroxylation of specific residues in the ␣-subunit of hypoxiainducible factor (HIF): three prolyl hydroxylases (9, 10) and an asparagine hydroxylase (FIH, factor inhibiting HIF (11-13)). Hydroxylation of specific proline residues signals for the proteolytic destruction of HIF-␣ (9, 10), whereas hydroxylation of an asparagine residue (Asn-803 in human HIF-1␣) (Fig. 1) in the C-terminal transactivation domain blocks recruitment of *