The Structure and Mechanism of Phosphoprotein Phosphatases Many Phosphatases Require Two Metal Ions for Catalysis

WILLIAM P TAYLOR AND THEODORE S WIDLANSKI MINIREVIEW

Charged with meaning: the structure and mechanism of phosphoprotein phosphatases Many phosphatases require two metal ions for catalysis. New structural information on two serinekhreonine phosphatases offers insight into how the metals contribute to catalysis. A comparison with the structures of protein tyrosine phosphatases, which do not use metal ions, shows that the only similarity at the active site is that of charge.

Chemistry & Biology November 1995, 2:713-718

Many biological processes are regulated by J simple substrates; and (3) small-molecule specific - enzymes chcniical event - the cleavage or formation of phos- that hydrolyze one (or a group of structurally similar) phdte esters. In nature, these reactions are catalyzed by substrate(s), for example, phosphoinocitol nlonophos- tv,m sets of enzynws, phosphatases and kinnses. Kinases phatasc (this cnzyrne may be the clinical target of operate in the synthetic direction (phocphorylation) lithium ion therapy for depression [A]). while phosphatases catalyze the hydrolysis (cleavage) reaction. Over the last decade there has been a growing A second useful classification scheme is our according rcalizCition that phosphataws are extremely important in to the enzyme’s mechanism of action. Some phos- ccllulx and organismal functions [ 1 1. Here, we focus on phatascs use an active site nucleophile 3s the initi,J recent Ed\-antes in our understanding of phosphatases. phosphoryl group acceptor; others transfer it inuned- especially phosphoprotein phosphatases, with J vielv to atcly to water (Fig. 3). The first group cm be further clarifyiiig some of the mrchanictic and structural con- subdivided according to the phosphoryl group acceptor plcsitie and mlbiguities presented by this diverse group (cysteine. histidinc, or serine). Mculbers of the second of mzynies. The newly reported structural information group characteristically use J two-metal-ion dyad to on txvo wrine/threonine (Ser/Thr) phosphatases [2,3] is bind phosphate esters and catalyze their subsequent particularly enlightening. hydrolysis. Indeed, this metal ion motif is ubiquitous in phosphoryl group transfer biochemistry, as it is also ust‘d Categories of phosphatases by phosphodiesterases (e.g. nucleases [5]) and phosphc>- The substrates for phosphatases range from small phos- triesterases [ 01. Two-metal-ion phosphatxes are vt’r) phoryl,ltcd nletabolites such as glucose-(,-pllosphatc and heterogeneous, however, vxying in metal-ion type, second nlessengers (e.g. phosphoinositols) all the way up protein wqueuce, structure, ligands, active-site catalytic to large phosphorylated proteins. Over a hundred residues md cvw mechanism. For example, alkaline phosphatClses xe known, and it is likely that the total phosphatasc transfers the phosphoryl group to a serinc number of thcsc enzymes is well over a thousand, or residue, not to wClter, and has a third active-site metal (a > I ‘%I of the proteins encoded by the human genome. To Mg(II) ion that does not directly contact the phosphate m,tkc scnsc of this lxge family of enzymes, a clnssifica- ester) [7]. Zn(I1) ions are frequently found in the active tion schemr is essential (Fig. l).There are three major site of tmx-metal-ion phosph:ltases, but mmy other d- groups of phosphntases: (1) nonspecific - these valent metal ions x-e also common. Manmlalian purple enzymes mill catalyze the hydrolysis of almost any pho- acid phosphatase, which contains an unusual hinuclear ph,lte ester: (3) phosphoprotein specific - enzymes that Fe(II)-Fc(ll1) mrt

Fig. 1. A hierarchy of phosphatases. Initial classification is based on substrate specificity; secondary classification is based on mechanism. The low MW acid phosphatases and the low MW dual specificity phosphatases seem to be a related family of enzymes.

0 Current Biology Ltd ISSN 1074-5521 713 714 Chemistry & Biology 1995, Vol 2 No 11

Fig. 2. Typical phosphatase reaction (a) WJ ROH mechanisms. (a) Direct transfer to \t water, normally catalyzed by active- E + RO-PO,‘- +==- E.RO-PO,‘- - E*Pi yE+Pi site metal ions. (b) Hydrolysis via a phosphoenzyme intermediate. Serine, cysteine, and histidine are nucleo- philes commonly used as the initial phosphoryl group acceptor. (b) ROH

E + RO-PO, 2---- EOR@pO zmL E-PO 2_“L EmPi A E + Pi 3- 3- -

Phosphoprotein phosphatases an enzyme in the class and the name of the class) specifi- Both phosphoprotein phosphatases and protein kinases cally dephosphorylate the B subunit of phosphorylase are important in the regulation of the phosphorylation kinase and are inhibited by two low molecular weight state of proteins. Often, phosphatases simply reverse the proteins, inhibitor-l (I-l) and inhibitor-2 (I-2). Type 2 effects of kinases, for instance by inactivating a protein enzymes prefer the (X subunit of phosphorylase kinase as a that has been activated by phosphorylation. But things substrate and are insensitive to I-l and I-2. The type 2 are not always so straightforward. Phosphatases can also enzymes are subdivided according to their divalent metal regulate kinase activity, thus indirectly regulating the ion requirements. PP-2A enzymes (like PP- 1 enzymes) phosphorylation state of the substrates of the kinase. are stimulated by Mn(II), whereas PP-2B enzymes and Some phosphatases contain SH2 domains which specifi- PP-2C enzymes use Ca(I1) and Mg(II), respectively. N-1, cally recognize and bind to phosphotyrosine residues, PP-2A, and PP-2B enzymes have highly homologous and their activity is therefore modulated by protein catalytic domains, and the differences between their activ- tyrosine kinases. Such examples of crosstalk allow for the ities are mostly caused by regulatory subunits bound to exquisite fine tuning of signal transduction cascades the catalytic domain. PP-2C enzymes, however, appear to necessary for regulating cell function. be monomeric, and are structurally unrelated to the other Ser/Thr phosphatases. Phosphoprotein phosphatases are subdivided into the Ser/Thr phosphatases 191, which are probably all two- Much is known about the biological roles of some of the metal-ion phosphatases, the protein tyrosine phosphatases Ser/Thr phosphatases, particularly those of the type 1 (PTPases) [ 101, and the dual-specificity phosphoprotein family. Early studies on the regulation of glycogen syn- phosphatases [l l-131. PTPases and dual-specificity thesis and breakdown provided seminal information on phosphoprotein phosphatases are mechanistically related the effect of protein phosphorylation/dephosphorylation and use an active-site cysteine located in a phosphate on the activities of individual enzymes involved in meta- binding loop as the phosphoryl group acceptor. True bolic and catabolic processes [ 171. Dephosphorylation by DTPases are highly specific for phosphotyrosine, whereas PP-1 reverses the activity of protein kinase A and regu- the dual-specificity enzymes hydrolyze both phospho- lates the phosphorylation state (and therefore the activ- tyrosine and phosphoserine (the low-molecular-weight ity) of three of the key enzymes involved in glycogen dual-specificity phosphatases prefer aromatic phosphate metabolism: phosphorylase kinase, glycogen synthase and esters and phosphotyrosine substrates, but can still often glycogen phosphorylase. The activity of PI’-1 is itself hydrolyze phosphoserine-containing peptides and pro- tightly regulated by levels of cyclic AMP, and the degree teins [14]). For most phosphatases the nature of the of phosphorylation of the inhibitors I-l and I-2. These physiologically rel evant substrate(s) is unknown. Thus, and other inhibitors of PP-1 (such as the polyether, the fact that a phosphatase is classified as having Ser/Thr okadaic acid, and the cyclic heptapeptide toxins known phosphatase activity (Fig. 1) does not necessarily imply as microcystins) have been useful for the study and that this is its sole activity itz viva. Indeed, calcineurin, a manipulation of signal transduction cascades. Studies prototypical Ser/Thr phosphatase, can hydrolyze phos- on inhibitors of calcineurin-A (PP-2B), such as the photyrosine-containing peptides as well as aromatic cyclosporins and FK506, have added greatly to our phosphates such as y-nitrophenyl phosphate [15]. Even understanding of T-cell activation and the design of new prostatic acid phosphatase, a so-called nonspecific phos- immunosuppressants [ 1X]. phatase, has phosphoprotein substrates (in particular, those containing phosphotyrosine) with K, values in the Structure and mechanism low nanomolar range ]I 61; perhaps a phosphoprotein is a Despite a wealth of information about the biological major target in vim. processes involving Ser/Thr phosphatases, chemical and structural information about these enzymes is sparse and Functions of Ser/Thr phosphoprotein phosphatases little is known about how they catalyze phosphate ester The Ser/Thr phosphatases fall into four classes, which hydrolysis. Most of the mechanistic information on these account for virtually all cellular Ser/Thr phosphatase proteins stems from mutagenesis studies of bacteriophage activity [9].Type 1 enzymes (PP-I enzymes; PP-1 is both X phosphatase, a phosphatase with substantial homology Phosphoprotein phosphatases Taylor and Wdlanski

Fig. 3. Actiw-site structure of two phosf,hol)rot’iri l)hosl)h,lt,l~c,~. (a) Kiblwn cliagram of PI’-1 sho\z/ing lhcl ~-u-~-u-~ nic>ta- binding motit twcl), active-site mct,ds Iviolc>t sl)hmx~~l, ,In(l rnclt,ll ligands iviolct c ire IPSI 121. (b) B,III ,wl stick rc,l)rc,sent,~tior~ ot Yq151 phosphate hinrling loop showing the c ~~tcint,-histitliri(, pair as wll J< two ,irginines that il~nk the ,Ictive sit?. K,itkl)onc~ ,iniitles direct severdl hydrogen bonding arf-dys into the ,ictiw 5itc 11or cl;rrltb, onlv amide protons are clepictd 12 il. 716 Chemistry & Biology 1995, Vol 2 No 11

Fig. 4. Two-metal ion catalysis of phosphate ester hydrolysis. The active site ~A-Enzyme A-Enzyme A-Enzyme metals probably act to bind substrate, H’ H/ 3- R\o(H stabilize the transition state(s) of the reac- --._ tion, facilitate leaving group departure, R\o----.o M, R\o.--.Q M, -0 .e-:o Ml and activate the phosphoryl group accep- I %,,I 8’ -0<.$ tor (shown here as H,O, but possibly P--d 0’ \ an enzyme nucleophile or metal-bound I ‘< hydroxide). A variety of phosphatases ,0-----bM2 ,0“‘--QM2 have metal ions at the catalytic active site. H H I - ’ An active site base catalyzes the deprotonation of the attacking water molecule, \ H\ H\ and an active-site acid protonates the B-Enzyme Y-Enzyme $!--Enzyme leaving group.

Intermediate or transition state

analogy with other phosphatases and based on model Although many additional mechanistic questions about building) a metal-bound water molecule or hydroxyl the Ser/Thr phosphatases remain unanswered, these new group is a reasonable candidate. Alternatively, the con- structural data provide the necessary background for served His125 (coupled with Asp95) could act as the starting to understand the mechanisms of most of the nucleophile or perhaps as a general acid to promote the known Ser/Thr phosphatases, as well as a number of departure of the leaving serine or threonine residue. other enzymes that share the two-metal-ion phosphatase signature motif. Despite the noted similarities in structure and in active site composition, PP-1 and calcineurin-A have distinctly dif- Protein tyrosine phosphatases ferent biological roles and regulatory mechanisms. These This subgroup is characterized by a conserved catalytic differences apparently arise from variations in the surface domain of -250 amino acids, containing the active site structure of the catalytic domain and from regulatory signature sequence Pro-Xaa-Ile/Val-Ile/Val-His-Cys-Ser- regions near the carboxy-termini of these enzymes. The Ala-Gly-Xaa-Gly-Arg-Ser/Thr-Gly. PTPases can be surface topology of PP-1 is characterized by three distinct divided into two classes: (1) transmembrane, receptor-like grooves that radiate from the central, shallow active site: a PTPases, and (2) soluble intracellular PTPases.The trans- ‘hydrophobic’ groove, an ‘acidic’ groove, and a ‘carboxy- membrane enzymes (with two exceptions) contain two terminal’ groove (Fig. 5a). These grooves have a role in intracellular catalytic domains. The amino-terminal inhibitor binding and presumably in substrate binding as region (domain I) is thought to be constitutively active, well. Microcystin inhibits PP-1 by embedding itself in the while the carboxy-terminal region (domain II) has little hydrophobic groove and overlapping the active site. or no enzymatic activity, at least in vitvo.The amino-acid Inhibitor-l may act in a similar manner, though modeling sequence outside of the catalytic domains varies consider- indicates that its major interactions are with the acidic ably in both transmembrane and intracellular PTPases. groove. The third (carboxy-terminal) groove may be This structural diversity may allow for control of PTPase involved in the inhibition of PP-1 caused by the phospho- activity through mechanisms such as ligand binding, cell rylation ofThr320, located near the carboxyl terminus of adhesion and protein compartmentalization [ 101 and may the protein. account for substrate specificity.

The topology and architecture of the catalytic domain of There is much evidence indicating that phosphate ester calcineurin is similar to that of PP-1, with the striking hydrolysis catalyzed by PTPases proceeds through a exception of the regulatory carboxyl-terminus. Calci- phosphocysteine intermediate. X-ray structures of PTPI B neurin-A has a five-turn amphipathic helix known as 1221 and Yop51 [23] depict an active-$ite phosphate the calcineurin-B binding helix (BBH) linked to the phos- binding loop, with a cysteine positioned to attack the phatase domainThe hydrophobic face of this helix fits into phosphate ester. This residue is apparently maintained as a complementary groove in calcineurin-B, leaving the the thiolate by a flanking histidine residue (reminiscent of polar face mostly exposed. The immunophilin-immuno- the Cyr-His catalytic dyad of cysteine proteases) as well as suppressant complex FKBP12-FK506 binds at the base of an extensive hydrogen-bonding network radiating from the BBH, contacting both calcineurin-A and calcineurin-B the active site (Fig. 3b). Treatment of PTPases with ‘?I’ in the process.This brings FKEG’12 into close proximity to labeled substrate results in the incorporation of radio- the hydrophobic groove of calcineurin-A, apparently activity into the enzyme [24,25], whereas mutants in inhibiting enzymatic activity by hindering the approach of which the active site cysteine is replaced with serine have macromolecular substrates.The active site of calcineurin-A, no catalytic activity and do not incorporate label, although however, remains accessible to solvent and perhaps also to they do retain the ability to bind substrate 1251. ForYop51 small molecules. As with PP-1, the mode of substrate the breakdown of the phosphocysteine intermediate is binding to the catalytic domain has yet to be determined. apparently rate-limiting for both small aromatic phosphate Phosphoprotein phosphatases Taylor and Widlanski 717

have two arginine residues located near the surface of the active site, whereas PTPases have three. Positively charged groups in the active site (such as metal ions or arginine residues) seem to be important in the catalytic mechanisms of many phosphatases. The surfaces of both PTPases and PP-1 are largely negative or neutral, with the exception of the active site and surrounding areas, which contain a region of densely packed positive charge (Fig. S).This is not unexpected, since both proteins need to bind the negatively charged phosphate group and stabilize the negatively charged phosphorane intermediate that forms during nucleophilic attack. The charge presumably also facilitates the deprotonation of the nucleophile (cysteine for PTPases, possibly water for N-1 enzymes) and charge development on the leaving group. All the steps necessary to catalyze the hydrolysis of a phosphate ester should be promoted by the high degree of positive charge in the active sites of these two types of enzymes. Thus the general approach to phosphoester hydrolysis used by these two families of phosphatases is similar, despite the large differences in the structural and chemical details of catalysis.

Summary The new structural data obtained for PPI and calcineurirr-A resolve many of the outstanding questions about the catalytic nature of the Ser/Thr phosphatases. These enzymes may now be firmly classed as two-metal ion phosphatases.These data also provide a model for understanding a variety of phosphatases that share the two- metal-ion binding signature motif found in the Ser/Thr phosphatases. These enzymes are clearly important in the control of a variety of cellular functions and in a number of cases are important targets for therapeutic intervention. The structural data obtained for PPI and calcineurirr-A should certainly facilitate efforts to understand, and design Fig. 5. Electrostatic surface potentials of (a) PP.1 (reprinted with irrhibitors of, these proteins and related phosphatases. permission from 1211 and (b) Yop51 1231. Despite the vast differences in sequence, structure, and catalytic mechanism, these enzymes have surprisingly similar surface charges, suggesting References that they have evolved a similar strategy for active site charge I. Posada, J. & Cooper, ].A. (1992). Molecular signal integration. distribution ~ a feature no doubt dictated by the catalytic Interplay between serine, threonine. and tyrosine phosphorylation. requirements of phosphate ester hydrolysis. MO/. Bid. Cd3, 583-592. 2 Goldberg, J., Huang, H., Kwon, Y., Greengard, P., Nairn, A.C. CI Kuriyan. 1. (19951. Three-dimensional structure of the catalytic esters and for phosphopeptide substrates [2h]. Site-directed subunit of protein serine/threonine phosphatase-1. Nature 376, 745-753. mutagenesis studies have also identified a conserved aspar- .I Griffith, J. P., et a/., & Navia, M.A. (1995). X-Ray structure of cal- tate residue that apparently acts as an active-site acid 1271. cineurin inhibited by the immunophilin-immunosuppressant This residue is located on a mobile loop that covers the FKBPl2-FK506 complex. Cell 82, 507-522. 4. Pollack, S.J., et al., & Broughton, H. B. (19941. Mechanism of inosi- active site when substrate is bound. to1 monophosphatase, the putative target of lithium therapy. Proc. Nat/. Acad. SC;. USA 91, 5766-5770. Ser/Thr phosphatases compared to PTPases ‘i Beese, L.S. & Steitz, T.A. (1991). Structural basis for the 3’.5’ exonu- clease activity of Fscherichia co/i DNA polymerase I: a two-metal-ion Tyrosine kinases and Ser/Thr kinases share a great deal of mechanism. EMBOI. 10, 25-33. structural and mechanistic homology.The same cannot be 6. Benning, M.M., Kuo, J.M., Raushel, F.M. 6; Holden, H.M. (1994). said for PTPascs and Ser/Thr phosphatases. Although the Three-dimensional structure of phosphotriesterase: an enzyme capable of detoxrfying organophosphate nerve agents. biochemistry two classes of phosphatases carry out very similar reac- 33, 15001~15007. tions, PTPnses are not metalloenzymes and seem to have 7. Krm, E.E. & Wyckoff, H.W. (1991 I. Reaction mechanism of alkaline evolved a completely different strategy for catalyzing phosphatase based on crystal structures. /. Mol. Bid. 218, 449-464. 8. Strater, N., Klabunde, T., Tucker, P., Witrel, H. & Krebs, B. (1995). phosphate-ester hydrolysis from that of Ser/Thr phos- Crystal structure of a purple acid phosphatase contarnrng a dinuclear phatascs. The major structural similarity between these Fetlll)-Zn(ll) active site. Science268, 1489-1492. '9. Cohen, P. (1989). The structure and regulation of protein phosphatases. enzymes seems to be the presence of multiple arginine Annu. Rev. Biochem. 58, 453-508. r-&dues positioned at the active site. Ser/Thr phosphatases 1 Il. Walton, K.M. & Dixon, I.E. (1993). Protein tyrosine phosphatases. 718 Chemistry & Biology 1995, Vol 2 No 11

Annu. Kev. Biochem. 62, IOI-ILO. of phosphoesterases. Protein Sci. 3, 356-358. Il. Cuan, K., Broyles, S.S. & Dixon, J.E. IlctYl). ATyr/Scr protein phos- 21. Lian, Y., Haddy, A. C; Rusnak, F. 11995). Evidence that calcineurin phatase encoded by vacc inia viw. Nature 350, 359-362. accommodates an active yite hinuclear metal center. /. Am. Chem. 12. Logan, T.M., Zhou, M.-M., Netteshrim, D.C., Meadows, R.P., Van sot. 117,10147~10148. Et&n, R.L. & Fesik, S.W. 119941. Solution structure of a low molecw 22. Barford, D., Flrnt, A.J. & Tonks, N.K. (1994~. Crystal structure of Ix weight protein tyrosine phosphatase. Bioc hemistrv 33, human protein tyrosine phosphatase I B. Science 263, 1 397-I 404. I 1087-l 1096. 23. Stuckey, J.A., Schubert, H.L., Fauman. E.B., Zhang, Z.-Y., Dixon, J.E. Ii. Zhang, M., Van Etten, R.L. X Stauffachcr, C.V. 119941. Crystal strut- XI Saper, M.A. iI9941. Crystal structure of Ycrsinia lprotein tyrosinr turc of bovine heart phosphotyrosyl phosphatase at 2.2A resolution. phosphatase at 2.5 A and the complex with tungstatr. Nature 370, Biochem/sir)~ 33, 11097~1 1 105. 571-i75. 14. Zhang, Z.-Y., et ,I/., b; Cuan, K. f 19951. Purification and Lharactcrlr+ 24. Cho, H., Krishnaraj, R., Kitas, E., Bannwarth, W., Walsh, C.T. R tion of the low molecular weight protein tyrosine phosphatase, Stpl, Anderson, K.S. ( 1992). Isolation and structural elucidation of a novel from the fission yeast Schirotarrh,~rom)/crt pornhe. Hiochemistr) phosphocysteine intermediate rn the LAR protein tyrosinc phos- 34, IOihO-I 0508. phatase rnrymatic pathway. /. Am. Chem. SW. 114, 7196-7298. 1 i. Chan, C.P., Callis, B., Blumenthal, D.K., t’allcn, C.J., Wang. J.H. K 2.5. Guan, K. & Dixon, J.E. (1991). Evidence for protein-tyrosine-phos- Krebs, E.G. 119861. Characterization of the phosphotyrosyl protein phatase catalysis proceeding via a cysteine-phosphate Intermediate. phosphatase activity ofcalmodulin-tlrpendent protein phosphatase. /. Bid. Chem. 266, 17026-l 7010. /. Bid. C/wnm. 261, 9890-9895. 26. Zhang, Z.-Y., M‘llachowski, W.P., Van Ettcn, R.I.. & Dixon, J.E. 16. Lrn, M.-F. (L Clinton, C.M 119861. Human prostatic acid phos- 11994). Nature of the rate-determining steps oi the reaction cat- phatase has phosphotyrosyl protein phosphatase activity. Riothcm /. alyzed hy the Yersini,l protein-tvroslne phosphataw. /. Rio/. Cl~cm. 235,z!SlL$57. 269, 8140-8145. 17. Fischer, E.H. 5; Krebs, E.G. 119891. Commentary on ‘The phosphory- 27. Zhang, Z.-Y., Wang, Y. & Dixon, J.E. (I 9941. Dlasrcling the r atalytic lase b to c~converting cnryme of r&hit skeletal must le’. Sioc-h/r??. mechanism of protein-tyrosine lphosphatascs. frac. Nat/. Acad. Sci. Bio/~/~ys. Art