The X-ray structure of the homotetrameric lysosomal acid hydrolase, human beta-glucuronidase (332,000 Mr), has been determined at 2.6 A resolution. The tetramer has approximate dihedral symmetry and each promoter consists of three structural domains with topologies similar to a jelly roll barrel, an immunoglobulin constant domain and a TIM barrel respectively. Residues 179-204 form a beta-hairpin motif similar to the putative lysosomal targeting motif of cathepsin D, supporting the view that lysosomal targeting has a structural basis. The active site of the enzyme is formed from a large cleft at the interface of two monomers. Residues Glu 451 and Glu 540 are proposed to be important for catalysis. The structure establishes a framework for understanding mutations that lead to the human genetic disease mucopolysaccharidosis VII, and for using the enzyme in anti-cancer therapy.
The unusual dumbbell shape of troponin C is due to the presence of a long a-helix of nine turns that connects the amino-and carboxyl-terminal calcium-binding domains. The center of the long helix appears to be stabilized by several salt bridges. The long helix is also bent about 160 at glycine-92. Calmodulin, which lacks the central glycine, also is predicted to be stabilized by salt bridges in the central helix. The presence of a proline residue in the center of the long helix of ascidian troponin C and the myosin regulatory light chains suggests that a sharper bend may occur in these molecules. The conservation of the bend and salt bridges in the related calcium-binding proteins suggests they may have an important biological function. The structure of troponin C suggests that intrahelix salt bridges between neighboring charged residues may be involved in the stabilization ofprotein secondary structure. The preponderance of potential salt bridges in other muscle proteins as well may be related to their elongated structures and their participation in the contractile process. Recent x-ray diffraction results on troponin C and calmodulin crystals have revealed that these molecules are dumbbell shaped with a long central helix extending between the amino and carboxyl domains (1-4). The nine-turn helix in troponin C has about three turns buried (either wholly or partially) in each ofthe flanking domains, but this leaves three turns in the center exposed fully to the solvent. It is well known that single a helices are highly unstable in an aqueous environment (5) and, thus, the exposed helix in these calcium-binding proteins was totally unexpected. In fact, there is no precedent for such an arrangement among the large number of proteins whose x-ray structures are known. An examination of the troponin C amino acid sequence in the DE-linker helix, however, shows that the residues have a high potential for helix formation. A proposed structure of troponin C (6) had predicted this region to be a random coil as would have been expected by the corresponding BC-linking region of parvalbumin on which the model was based. It is the unexpected helix in this linker segment that gives rise to the long central helix and in turn the unusual dumbbell shape of these molecules. An examination of the factors that induce and stabilize the central helix-especially the linker segment-is, therefore, essential to understanding the conformation and dynamics of these Ca2l-binding proteins.Based on our investigation of the x-ray structure of troponin C from chicken skeletal muscle, we suggest that the integrity of the long central helix is derived from numerous intrahelical electrostatic and/or salt-bridge interactions between the basic and acidic amino acid side chains, especially in the exposed DE-linker segment. We support this hypothesis both by reference to our x-ray structure oftroponin C and by phylogenetic considerations of the amino acid sequences of various calcium-binding proteins.It has been shown that the frequency of occ...
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