We constructed structural models of the catalytic domain and the surrounding region of human wildtype acid a-glucosidase and the enzyme with amino acid substitutions by means of homology modeling, and exam-

Phosphorylation of endogenous inhibitor proteins for type-1 Ser/Thr phosphatase (PP1) provides a mechanism for reciprocal coordination of kinase and phosphatase activities. A myosin phosphatase inhibitor protein CPI-17 is phosphorylated at Thr38 through G-protein-mediated signals, resulting in a >1000-fold increase in inhibitory potency. We show here the solution NMR structure of phospho-T38-CPI-17 with rmsd of 0.36 +/- 0.06 A for the backbone secondary structure, which reveals how phosphorylation triggers a conformational change and exposes an inhibitory surface. This active conformation is stabilized by the formation of a hydrophobic core of intercalated side chains, which is not formed in a phospho-mimetic D38 form of CPI-17. Thus, the profound increase in potency of CPI-17 arises from phosphorylation, conformational change, and hydrophobic stabilization of a rigid structure that poses the phosphorylated residue on the protein surface and restricts its hydrolysis by myosin phosphatase. Our results provide structural insights into transduction of kinase signals by PP1 inhibitor proteins.

Fabry disease comprises classic and variant phenotypes. The former needs early enzyme replacement therapy, and galactose infusion is effective for some variant cases. Attempts of early diagnosis before manifestations appear will begin in the near future. However, it is difficult to predict the phenotype, to determine the therapeutic approach, only from genetic information. Thus we attempted structural analysis from a novel viewpoint. We built structural models of mutant alpha-galactosidases resulting from 161 missense mutations (147 classic and 14 variant), and evaluated the influence of each replacement on the structure by calculating the numbers of atoms affected. Among them, 11 mutants, biochemically characterized, were further investigated by color imaging of the influenced atoms. In the variant group, the number of atoms influenced by amino-acid replacement was small, especially in the main chain. In 85% of the cases, less than three atoms in the main chain are influenced. In this group, small structural changes, located apart from the active site, result in destabilization of the mutant enzymes, but galactose can stabilize them. Structural changes caused by classic Fabry mutations are generally large or are located in functionally important regions. In 82% of the cases, three atoms or more in the main chain are affected. The classic group comprises dysfunctional and unstable types, and galactose is not expected to stabilize the mutant enzymes. This study demonstrated the correlation of structural changes, and clinical and biochemical phenotypes. Structural investigation is useful for elucidating the bases of Fabry disease and clinical treatment.