Crystal Structure of the Tumor-promoter Okadaic Acid Bound to Protein Phosphatase-1

Abstract

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...

Developmental Biology of Neoplastic Growth

“…Egloff et al (1995) investigated the nature and function of the two metal ions (Mn 2+ and Fe 2+ or Fe 3+ ) and reported that metal ions promote hydrolysis reactions by activating water molecules and stabilizing nucleophilic hydroxide and oxide ions. Figure 14 shows the molecular surface of the catalytic subunit of PP1-microcystin-LR complex (Goldberg et al 1995), and similar crystal structures of the molecular complex of catalytic subunit of PP1-okadaic acid (Maynes et al 2001), PP1-calyculin A (Kita et al 2002), and PP1-motuporin (Maynes et al 2006) have also been reported.…”

“…As Uemura and Hirata (1989) suggested (Fig. 13), a hydrogen bond formed by a cyclic structure between the C-1 acid and the C-24 (Uemura and Hirata 1989) hydroxyl group was confirmed in both free okadaic acid and the PP1-okadaic acid complex (Maynes et al 2001). The acid motif in okadaic acid accepts a hydrogen bond from the hydroxyl group of Tyr 272 in PP1, and the esterification or removal of the acidic moiety in okadaic acid results in elimination of its inhibitory activity.…”

“…They found that microcystin-LR binds to the catalytic subunit of PP1 through the three regions of the surface, and the long hydrophobic Adda side chain was packed into the hydrophobic groove. Although covalent linkage between a methyldehydroalanine residue in microcystin-LR and the Sγ of Cys 273 is present, it is not the primary cause of inhibition of PP1 by microcystin-LR (Maynes et al 2001). The carboxyl group of d-erythro-β-methylaspartic acid was also found to interact with Tyr 134 (Maynes et al 2001(Maynes et al , 2006.…”

“…Although covalent linkage between a methyldehydroalanine residue in microcystin-LR and the Sγ of Cys 273 is present, it is not the primary cause of inhibition of PP1 by microcystin-LR (Maynes et al 2001). The carboxyl group of d-erythro-β-methylaspartic acid was also found to interact with Tyr 134 (Maynes et al 2001(Maynes et al , 2006. Interaction between microcystin-LR and the C-terminal groove occurs at the loop connecting the β12-β13, where the leucine side chain in microcystin-LR is packed close to the side chain of Tyr 272 (Goldberg et al 1995).…”

Okadaic acid and microcystin insensitive PPP-family phosphatases may represent novel biotechnology targets

“…1A). 9,15,16 The general lack of inhibition exhibited by the PPP-family members PP2B and PP7 in the presence of these compounds, coupled with differential inhibition of the remaining PPP-family enzymes by select toxins, has made these small molecules invaluable tools in protein phosphatase research by helping implicate distinct protein phosphatases in various cellular processes. Recently, we reported two additional PPPfamily protein phosphatases from A. thaliana (AtSLP1 and -2 phosphatases) that demonstrated complete insensitivity to inhibition by both microcystin and okadaic acid, with AtSLP1 exhibiting slight enzymatic activation.…”