Protein phosphatase-1 (PP1) plays a key role in dephosphorylation in numerous biological processes such as glycogen metabolism, cell cycle regulation, smooth muscle contraction, and protein synthesis. Microorganisms produce a variety of inhibitors of PP1, which include the microcystin class of inhibitors and okadaic acid, the latter being the major cause of diarrhetic shellfish poisoning and a powerful tumor promoter. We have determined the crystal structure of the molecular complex of okadaic acid bound to PP1 to a resolution of 1.9 Å. This structure reveals that the acid binds in a hydrophobic groove adjacent to the active site of the protein and interacts with basic residues within the active site. Okadaic acid exhibits a cyclic structure, which is maintained via an intramolecular hydrogen bond. This is reminiscent of other macrocyclic protein phosphatase inhibitors. The inhibitor-bound enzyme shows very little conformational change when compared with two other PP1 structures, except in the inhibitor-sensitive 12-13 loop region. The selectivity of okadaic acid for protein phosphatases-1 and -2A but not PP-2B (calcineurin) may be reassessed in light of this study.The phosphorylation and dephosphorylation of proteins is vital to the regulation of many cellular pathways and processes. Two classes of enzymes in the cell that catalyze cellular dephosphorylation activity are tyrosine phosphatases and serine/threonine phosphatases (1). Classification of serine/threonine phosphatases can be subdivided into four categories: protein phosphatase-1 (PP1), 1 -2A (PP2A), -2B(PP2B) and -2C (PP2C) (2). The first three of these categories comprise what is known as the PPP family of protein phosphatases since they contain extensive sequence similarity in their catalytic domains and little or no sequence homology to PP2C or to tyrosine phosphatases. There are several natural toxin inhibitors of the PPP family of enzymes. These include microcystins, calyculins, tautomycin and okadaic acid (OA) ( Fig. 1) (1).OA is a tumor-promoting C 38 polyether fatty acid produced by marine dinoflagellates (1, 3-6). OA contains acidic and hydrophobic moieties and is cyclic (via an intramolecular hydrogen bond) (6). This toxin can accumulate in filter-feeding organisms and is the principle cause of diarrhetic shellfish poisoning worldwide (4).There have been many biochemical and modeling studies on the inhibition of the PPP family of phosphatases by the natural toxins, but the lone crystal structure is of microcystin-LR (MCLR) bound to PP1 (␣ isoform) (8). Here we describe the crystal structure of OA bound to the recombinant catalytic subunit of PP1 (␥ isoform). EXPERIMENTAL PROCEDURESCrystallization-The catalytic subunit of protein phosphatase-1 ␥ isoform was purified as described previously (9, 10). OA was purified from Prorocentrum lima (9, 10). Crystals were obtained by the hanging drop vapor diffusion method at room temperature. The enzyme and inhibitor were mixed in a 1:2 molar ratio with the concentration of protein being ϳ0.4 mM. The P...
Heptapeptide microcystin and pentapeptide motuporin (nodularin-V) are equipotent inhibitors of type-1 and type-2A protein phosphatase catalytic subunits (PP-1c and PP-2Ac). Herein we describe elucidation of the molecular mechanisms involved in the interaction of these structurally similar hepatotoxins with PP-1c/PP-2Ac and identification of an important functional difference between their mode of interaction with these enzymes. Microcystin-LR, microcystin-LA, and microcystin-LL were found to interact with PP-2Ac and PP-1c by a two-step mechanism involving rapid binding and inactivation of the protein phosphatase (PPase) catalytic subunit, followed by a slower covalent interaction (within hours). Covalent adducts comprising PPase-toxin complexes were separated from free PPase by C-18 reverse-phase liquid chromatography, thus allowing the time course of covalent adduct formation to be quantitated. In contrast to microcystins, motuporin (nodularin-V) and nodularin-R were unable to form covalent complexes with either PP-1c or PP-2Ac even after 96 h incubation. Specific reduction of microcystin-LA to dihydromicrocystin-LA abolished the ability of the toxin to form a covalent adduct with PP-2Ac. Specific methyl esterification of the single Glu residue in microcystin-LR rendered this toxin inactive as a PPase inhibitor and abolished subsequent formation of a covalent adduct. Our data indicate that inactivation of PP-2Ac/PP-1c by microcystins precedes covalent modification of the PPases via a Michael addition reaction between a nucleophilic phosphatase residue and Mdha in the heptapeptide toxin. In contrast, following rapid inactivation of PP-2Ac/PP-1c by motuporin, the equivalent N-methyldehydrobutyrine residue in this toxin is unreactive and does not form a covalent bond with the PPases. These results are consistent with structural data for (i) the NMR solution structures of microcystin-LR and motuporin, which indicate a striking difference in the relative positions of their corresponding dehydroamino acids in the toxin peptide backbone, and (ii) X-ray crystallographic data on an inactive complex between PP-1c and microcystin-LR, which show a covalent bond between Cys-273 and the bound toxin.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.