J. Mol. Biol. (1992) 225, 1027-1034

Purification and Crystallization of Glycogen Phosphorylase from Saccharomyces cerevisiae Virginia L. RathT, Peter K. Hwang and Robert J. Fletterick Department

of Biochemistry

and Biophysics

University of California San Francisco, CA 94143-0448, (Received 9 October

1991; accepted

U.S.A.

6 February

1992)

Glycogen phosphorylase from Saccharomyces cerevisiae is activated by the covalent phosphorylation of a single threonine residue in the N terminus of the protein. We have hypothesized that the structural features that effect activation must be distinct from those characterized in rabbit muscle phosphorylase because the two enzymes have unrelated phosphorylation sites located in dissimilar protein contexts. To understand this potentially novel mechanism of activation by phosphorylation, we require information at atomic resolution of the phosphorylated and unphosphorylated forms of the enzyme. To this end, we have purified, characterized and crystallized glycogen phosphorylase from X. cerevisiae. The enzyme was isolated from a phosphorylase-deficient strain harboring a multicopy plasmid containing the phosphorylase gene under the control of its own promoter. One liter of cultured cells yields 12 mg of crystallizable material. The purified protein was not phosphorylated and had an activity of 47 units/mg in the presence of saturating amounts of substrate. Yeast phosphorylase was crystallized in four different crystal forms, only one of which is suitable for diffraction studies at high resolution. The latter belongs to space group P4,2,2 with unit cell constants of a = 161.1 a and c = 175.5 A. Based on the density of the crystals, the solvent content is 497%, indicating that the asymmetric unit contains the functional dimer of yeast phosphorylase. Keywords: overexpression;

crystal; X-ray diffraction;

1. Introduction Protein phosphorylation is the most common post-translational modification of proteins and a dynamic mediator in the regulation of fundamental biological processes. The modification was first described in the enzyme rabbit muscle glycogen phosphorylase (EC 2.4.1.1), whose activity is governed by its phosphorylation state (Fischer et al., 1959). Found ubiquitously in nature, glycogen phosphorylase catalyzes the degradation of glycogen to yield glucose l-phosphate (Glc-1-Pf), which subsequently enters glycolysis to fulfill the energetic requirements of the organism. The mammalian phosphorylases are allosterically controlled both by phosphorylation of a single t Current address: Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, P.O. Box 6666, New Haven, CT 06511, U.S.A. $ Abbreviations used: Glc-l-P, glucose l-phosphate; DTT, dithiothreitol; PEG, polyethylene glycol. 0022%2836/92/121027-08

$03.00/0

1027

phosphorylation;

allosteric

residue and by multiple interacting effector sites. In contrast, the enzymes isolated from Escherichia coli and potato are neither phosphorylated nor allosterically controlled. Other than the mammalian enzymes, only yeast phosphorylase and one of the isozymes from the slime mold, Dictyostelium discoideum (Rogers et al., 1992), are known to be activated by covalent phosphorylation. The yeast and rabbit muscle enzymes are 45% identical at the amino acid level, yet both the phosphorylation sites and the protein environment surrounding the phosphorylation sites (including the residues that bind the phospho-amino acid) are different (see Fig. 2). Residues in the N terminus up to position 80 of rabbit muscle glycogen phosphorylase undergo important conformational changes when the enzyme is phosphorylated (Sprang et al., 1988). The corresponding N terminus in yeast phosphorylase is not conserved relative to the muscle enzyme. Only 23 o/o of the residues up to amino acid 80 in the muscle enzyme sequence are identical in yeast phosphorylase; the identity drops to 12% if only the first 60 amino acid residues are considered. 0

1992

Academic Press Limited

V. L. Rath ee al.

1028

The yeast enzyme is also longer at the N terminus; an additional 38 residues precede the position of the starting methionine of the muscle enzyme. The disparity in length and lack of similarity in the N terminus indicates that activation by phosphorylation in yeast phosphorylase is likely to proceed by a mechanism different from that of rabbit muscle phosphorylase and suggests that reversible phosphorylation evolved independently in the yeast and vertebrates. Our goal is to understand the det.ails of this structurally distinct, but functionally equivalent mechanism at the atomic level.

2. Materials and Methods (a) PuriJcation

of yeast glycogen phosphorylase

All steps of the purification

were performed at 4 “C or on cells of strain PH5-3, genotype MATcr urad leu2 gphl(A::LEUZ) harboring the plasmid Yep24 :: GPHl, were grown under selection in ice. Xaccharomyces

cerewisiae

media lacking uracil to early stationary phase (Hwang et al., 1989) and harvested by centrifugation. The pellets were resuspended in an equal volume of homogenization buffer (150 mM-Tris (pH 7.9); 10 rnM-magnesium chloride, 2 mrvr-calcium chloride, 1 mM-EDTA, 10% (v/v) glycerol, 1 mm-dithiothreitol (DTT) and a cocktail of protease inhibitors consisting of 94 ,uiv-phenylmethylsulfonyl fluoride, 0.5 pg leupeptin/l, 0.7 pg pepstatin/l, 901 yc (w/v) benzamidine-HCI and 2 pg aprotiniml). The cells were disrupted by adding an equal volume of glass beads (0.5 mm diameter; Biospec Products) and homogenizing 4 times for 2 min each in an 80 ml Beadbeater chamber (Biospec Products) with chilling on ice in between. The homogenized mixture was t,hen centrifuged at 3000g for 10 min to remove the glass beads and cell debris. The pH of the supernatant was adjusted to 5.7 and the mixture centrifuged at 5000 g for 40 min. The resulting supernatant was decanted through glass wool to remove lipid material. Streptomycin sulfate was added to a final concentration of 0.7 o/0 and the solution stirred for 30 min. The precipitate was removed by centrifugation for 25 min at 5OOOg. The protein concentration of the supernatant was adjusted to 5 to 15 mg/ml and phosphorylase precipitated by slowly adding cold ethanol to a final concentration of 33 y0 (v/v) and stirring for 15 min. The precipitate was collected by centrifugation at 2600g for 10 min and resuspended to a final concentration of 15 mg/ml in BisTris buffer (50 mivr-Bis-Tris (pH 6.0); 1 mM-DTT plus protease inhibitors). This material was loaded onto a 25 ml DlL4E Sephacel column that had been pre-equilibrated with Bis-Tris buffer. The column was washed with 300 ml of Bis-Tris buffer/100 mM-NaCl and eluted with 300 ml of a 100 to 400 rnM linear gradient of NaCl. Fractions containing phosphorylase activity were analyzed by 10 yc (w/v) SDS/ PAGE (Laemmli, 1970) and stained with Coomassie blue or silver (Merril et al.? 1981). Central peak fractions were pooled and exchanged into phosphate buffer (100 mMpotassium phosphate (pH 60), 1 mM-DTT and protease inhibitors) using a 50 ml Amicon stirred ultrafiltration cell with a 100,000 molecular mass cutoff membrane (YMlOO; Amicon). The protein concentration was adjusted to 5 to 15 mg/ml and the material loaded on a 15 ml DEAE Sephacel column. The column was washed with 100 ml of

100 rnx-potassium phosphatfe buffer and eiuted with 300 ml of a linear gradient of 100 to 750 mM-potassium phosphat’e (pH 60). Fractions cont,aining activity were analyzed by SDSi PAGE, pooled and exchanged into a buffer of 10 mM-RiSTris (pH 6.0) containing I mu-DTT, 100 mM-XaCl; 902 % (w/v) sodium azide, using a 10 ml Amicon stirred ultrafiltration cell and a YMlOO membra,ne. The protein was finally concentrated to 50 to 60 mg/ml by vacuum dialysis employing a 25>000 molecular mass cutoff colloidon membrane (Xchleicher and Schuell) after which the protein was frozen in liquid nit,rogen and stored at -10°C. The protein concentration of the pure enzyme was determined by absorbance at 280 nm and corrected using an extinction coefficient of 149 for a 1 oh solution of yeast phosphorylase (Babul & Stellwagen, 1969). (b) Activity

assays

Enzymatic activity was determined using the method of Carney et al. (1978) essentially as described (Hwang et al.; 1989), except bhat the substrate buffer contained 50 ma-sodium succinate (pH 6.0): 57.2 rnAM-repurified oyster glycogen (Dickey-Dunkirk & Killilea, 1985; molar equivalent of glucose, Ashwell, 1957) and either 15 or 75 rnM-Glc-I-P. The assay determines the amount of phosphate released during the formation of glycogen from Glc-l-P, taking advantage of the fact that the equilibrium constant favors glycogen synthesis. One unit, of phosphorylase activity represents the release of 1 nmol of phosphate from Glc-1-P/min at 30°C. In phosphate buffers, enzymatic activity was determined by measuring [U-“4C]Glc-1-P incorporation into glycogen (Lederer &. Stahlmans, 1976). One unit of phosphorylase activity represents the addition of 1 nmol of labeled glucose to glycogemmin at, 30°C. The reaction was initiated by adding protein to a substrate mix consisting of 4 &i/ml of 250 mCijmmo1 [U-‘%]Glc-l-P, 4cJ, (w/v) glycogen and 40 rnM-Glc-1-P. At the end of the react,ion period, 75 ~1 of each reaction mix was spotted onto a filter disc. The discs were washed 3 times for 30 min each in a shaking solution of 667; (v/v) etha.nol. Aft,er drying. the filter discs were placed in IO ml of scintillation fluid and counted.

(c) Western analysis Rabbit polyclonal antibodies were prepared against intact yeast glycogen phosphorylase (Berkeley Antibody Company, Richmond, CA) and purified from serum before use (Harlow & Lane, 1988). Western analysis of immunoreactive proteins was performed according to standard methods (Harlow & Lane, 1988). (d) In vitro

phosphorylution

The purified yeast enzyme was phosphorylated ~TLait7.o using yeast phosphoryla,se kinase generously provided by B. E. Grunenberg and A. C. Schwartz (Botanisches Tnstitut der Universitat Bonn, Bonn, Germany) and analyzed for amino acid composition. In addition, kemptide (Sigma), a peptide (LRRASLG) containing the consensus sequence for cyclic AMP-dependent protein kinase, and rabbit muscle phosphorylase b (from S. B. Madsen and S. Schechosky, Department of Biochemistry. University of Alberta, Edmonton, Canada) were phosphorylated in vitro using porcine cyclic AMP-dependent protein kinase (gift of S. Taylor. Universit,y of California at San Diego, CA) and phosphorylase kinase, respectivelp.

1029

Yeast Phosphorylase in order to provide control samples for amino acid analysis. Unphosphorylated control samples were prepared in the same way except that ATP was excluded from the reaction. All reactions were performed in a buffer consisting of 10 miv-3-(N-morpholino)propanesulfonic acid (pH 7.0), 100 pg bovine serum 10 mM-magnesium chloride, albumin/ml. The reaction was started by adding substrate and [y-32P]ATP and incubating at 30°C. The extent of incorporation of radioactive label was assayed by spotting 25 ~1 of the sample onto filter paper. The filters were washed 3 times for 5 min in 10 ml/filter of a solution of 75 mw-phosphoric acid with gentle shaking. The filters were then rinsed in acetone, air dried, placed into 10 ml of scintillation fluid and counted. (e) Phosphorylated

amino

acid

analysis

Protein samples were precipitated with 10 vol. of ethanol overnight at - 20 “C. The samples were then dried in 6 mm x 50 mm glass tubes and hydrolyzed at 110°C for 1.5 h under vacuum in 6 M-HCl and phenol using the vapor method (Tarr, 1986). Following hydrolysis, the were derivatized with o-phthalaldehyde samples (Fluroaldehyde, Pierce) and separated on a Beckman C-18 column as described (Stahl et al., 1990). Amino acids were quantified by comparison to 25 and 125 pmol samples of amino acid standards (Pierce).

95% pure.

(f) Crystals Crystals were grown at room temperature, 4”C, or at 17°C by vapor diffusion against 600 ~1 of reservoir solution using hanging drops (Hampel et al., 1968; McPherson, 1982). Crystal of yeast glycogen phosphorylase were characterized by precession photography using CEA Reflex 25 X-ray film (CEA AB, Sweden) and an EnrafNonius precession camera. Monochromatic copper K, X-rays were generated from a 200 p focal spot source on a Rigaku RU-200 rotating target tube operating at 50 kV, 60 mA.

3. Results (a) Purijkation

reported. Due to low yields, earlier published purifications were designed for kilogram quantities of yeast cells (Fosset et al., 1971; Becker et al., 1983). In order to circumvent purification difficulties and to obtain sufficient quantities of material for structural studies, the yeast phosphorylase gene from the baker’s yeast, X. cerevisiae, was cloned and overexpressed (Hwang & Fletterick, 1986; Hwang et al., 1989). Using an isogenic strain of yeast as the expression host, we have purified sufficient quantities of cloned yeast phosphorylase for biochemical and crystallographic analyses. Table 1 shows the results from a typical preparation. For each step, we show the protein content and activity of the fraction containing most of the phosphorylase. In the presence of saturating amounts of the substrates (75 m&r-Glc-I-P and 1% glycogen), the activity of the purified protein was 4.7( + 1.4) units/mg. No activity could be detected in the absence of glycogen or when lower amounts of substrate (15 mM-Glc-I-P) were used. The corresponding SDS/PAGE and Western analysis of these fractions is shown in Figure 1. We routinely obtained 12 mg of crystallizable material from one liter of cultured cells. Based on silver-stained gels of the purified fractions, the protein is greater than

of yeast glycogen phosphorylase

The purification and biochemical characterization of glycogen phosphorylase from yeast has been

(b) Correction of the N-terminal yeast phosphorylase

sequence of

The cDNA-derived nucleotide sequence showed that there were two methionine residues, 11 residues apart, in the 5’ coding region (EMBL Nucleotide Sequence Data Library, accession number X04604; Hwang & Fletterick, 1986). N-terminal sequence analysis of yeast phosphorylase from two different preparations gave the sequence PPA(STST)XN demonstrating that the upstream methionine was the correct one (Fig. 2). Of the two possible start sites, the upstream methionine occupies the preferred nucleotide sequence context for initiation (Clements et al., 1989). The first three residues and

Table 1 Purifkation

of glycogen phosphorylase per liter of culture overexpressing strain

Volume (ml)

Fraction Crude homogenate

Post pH drop supernatant Post streptomycin supernatant

precipitation

Resuspended pellet from ethanol precipitation DEAE 1 peak DEAE 2 peak (corrected absorbance) The Table shows the results from were determined by the absorbance the DEAE 2 peak fraction where coefficient of 14.9 for a 1 O/osolution

Total protein (mg)

from

Specific activity (fimol/min mg-‘)

an

Total activity (units)

43 34 34

5600 2300 2750

0.2 0.4 0.3

1120 920 825

150

1340

0.4

536

28 23

38 126

1.2 4.1

456 592

a typical preparation. Protein concentrations for crude fractions at 280 nm, assuming an absorbance of 1 unit/mg ml-‘, except for the protein concentration was determined using an extinction of yeast phosphorylase.

V. L. Rath et a,l.

1030

(a)

(b)

Figure 1. SDS/PAGE and Western analysis of the purification of yeast phosphorylase. The Figure shows a 10% denaturing gel (a) of fractions from 1 of the preparations and the corresponding Western analysis (b) using antibodies prepared against native yeast phosphorylase. The band corresponding to yeast phosphorylase (calculated relative molecular mass of 103,770) migrates just above that of the rabbit muscle phosphorylase marker (relative molecular mass of 97,400). The prestained molecular mass standards used for the Western analysis migrate anomalously farther on the gel than the unstained standards. Lanes 2 and 3 show samples prepared from lo* cells in log and stationary phase using a rapid trichloroacetie acid precipitation method (Wright et aZ., 1989). Lane 4 contains 50 pg of protein from the crude homogenate fraction; lanes 5 and 6 contain 50,~g of protein from the supernatant and pellet, respectively, after centrifugation of the material titrated to pH 5%; lanes 7 and 8 contain 50 pg of protein from the supernatant, and pellet, respectively, after streptomycin sulfate precipitation; lanes 9 and 10 contain 50 @g of protein from the supernatant and pellet, respectively, after the ethanol precipitation step; and lanes II and 12 contain 25 pg (determined from the extinction coefficient for yeast phosphorylase), respectively, of the peak fractions from the 1st and 2nd DEAE columns.

the last residue were clearly identifiable from the chromatogram; the parentheses enclose residues that were represented in the chromatogram at some level; and X indicates that we could not identify the amino acid released during that cycle of Edman degradation. Our difficulty in sequencing through the stretch of serine and threonine residues may be the result of losses due to the acid lability of these two residues. (c) The purified

protein

is not phosphorylated

The phosphorylation site was previously identified as a threonine by Edman degradation of a 32P-labeled peptide (Lerch & Fischer, 1975). From this peptide sequence, the phosphorylated threonine corresponds to threonine 30 in the corrected amino acid sequence (Fig. 2). To determine the phosphorylation state of the enzyme, we looked for the presence of threonine-phosphate after a limited amino acid hydrolysis of the protein, Hydrolyzed amino acids were quantified by comparison to 25 and 125 pmol samples of serineand threonine-

phosphate standards. We estimated that we could detect as little as 25 pmol of serine- or threoninephosphate, or the amount expected if 10 to 15% of the enzyme monomers in the assays were phosphorylated. We phosphorylated rabbit muscle phosphorylase and kemptide in vitro using rabbit muscle phosphorylase kinase and porcine cyclic AMP-dependent protein kinase, respectively, to produce control samples containing phosphate covalently linked to serine residues. Based on incorporation of [Y-~~P]ATP, 60% of the commercial grade kemptide could be phosphorylated using the porcine cychc AMP-dependent protein kinase and 30% of the rabbit muscle phosphorylase could be phosphorylated using commercial phosphorylase kinase. The activity of rabbit muscle phosphorylase was increased from 1.8 units/mg before to 17.1 units/mg after phosphorylation. After correcting for losses due to the degradation of serine-phosphate during the hydrolysis period and the efficiency of the kinase reaction, hydrolysis of phosphorylated muscle phosphorylase or kemptide released the expected

Yeast Phosphorylase

1031

*

10

20

YCOII Rabbi1

MPPASTS'ITN DMITREPTSP HQIPRLTRRL TCFLPQEIKS IDmIPI;LsR ALWNKHQVKK ,.......,. _..,.,.,,. . . . . . . . . . . . M SRPLSDQEK KQISVRCLAG

YCOSl Robbi!

50 60 70 80 FIDHvm-Z RSLYNCDDMVAYEAASMSIR DNLVIDWNKT QQKFTTRDPK HWEK H CRIR VENVTELKKN NR LHF V KDRNVATPRDY F LAHTV

Year1 Robbif

120 110 90 100 RVYYLSLEFL MGRALDNALI NMKIEDPEDP AASKCKPREMIKGALDECGF KLEDVLDPEP T Q mv LAL NAC E n....... . . . . ..QL L DM ELEEI E I Y

yeast

150 130 140 DMLGNGGLC RLAACFVDSMATECIPAWGY LLAY L

1

Robbil

30

FNKAEDF~R

160

170

GLRYEYCIFA

I

F

180

QKIIDCYQVE

N

TPDYWLNSGN

CC W M EA D RY

RobBit

190 200 210 220 230 240 PWEIERNEVQIPVTFYCYVD RPECGKTTLSASQWIGCERVLAVAYDFPVP CFKTSNVNNL MP T ICAPFTL H R E HTSQ AK... VDTQV YRNNV TM

YCOSC Robbit

290 250 270 280 300 260 RLWQARPTTEFDLNKFNNGDYKNSVAQQQRAESITAVLYP NDNFAQGKELRLKQQYFWCA EW S KAPND N KD V C IQA LDRNL N SR FE

YlO¶i RoDbit

310 320 330 340 350 360 ASLHDILRRF K&SK...... ..RPmEFPD QVAIQLNDTH PTLAIVRLQR VLVDLEUDW R s FGCRDP.VRTNFDA K S P St TQ 1

Y.?OSi

370 400 410 420 360 390 HEAWDIVTKT FAYTNHTVMPEALEKWPRRLFCHLLPRHLE IIYDINWFFL EDVAKKFPKD R VH LET DK EVTV C LP E QR NR AA G Q

Year1 RoDbit

YeaIt RobBi

Yeast RoBBit

t

430 MLLSRISII RRMLV

470 i40 460 450 EENSPERQIR MAFLAINGSH KVNGWELHS ELIKTTIFKD FIKFYCPSKF CAVK 1.N HCA A AR1 IL K YELE. H

480 500 510 530 490 520 VNVTNGITPR RWLKQANPSLAKLISETLND PTEEYLLDMA KLTQLEK\VE DKEFLKKBNQ VLC c EI ARIC. IS LD Q RK LS D EA IRDVAK QK

Robbit

540 550 580 570 560 590 YKLNNKIRLV DLI~NDGV DIINREYLDD TLFDhiQVKRI HEYKRQQLNVFGIIYRYLM V QE LKFA AYLER YKVH IN......PN S V L C LHV TL NRI

Yeast

600 KNMLKNCiASIEEVARKYPRK

Yecnt

Rabbit

ycost RoDCit

Yeast Robbit

‘taorr

Rabbit

yeast Robbit

K.. .

.

650 WFVADYNVS

I LEN R

PNKFWPR

660 KAEIIIPASD L KV A

610 VSIFCCKSAP TWdI A

630 640 620 CYYMAKLIIR LINCVADIVN NDESIEHLLK H M TAIC V H PWGDR R

670 080 690 700 LSEHISTAGT EASGTSNMKFVMNCCLIIGT VXGANVEITR G MLAT M MAE Q

740 750 760 710 130 720 EICRDNVFLF GNLSENVEEL R...YNHQYH PQDLPSSLDSVLSYIESCUF SPENPNEFKP KQ DL D MflV D DR DQRG A EY YDRI .E RQ IIEQLS F A EFI 790 810 800 780 770 LVDSLKYHGDWLVSDDFES YLATHELVDQ EFHNQRSEM. KKSVLSLANV GFFSSDRCIE TA I N&&M. H RFK FA Y E VKCP R SA LYK P . T RMVIRNS TS K

820 830 EYSDlTwNVE PVT.......

QARE

G

. . . .

SRQFUPAPDEKIP

Figure 2. Amino acid sequence comparison of yeast and rabbit muscle phosphorylases. Numbering is according to the rabbit muscle phosphorylase sequence. Only amino acid differences are shown. The 2 residues that, undergo reversible phosphorylation, threonine 30 in yeast, phosphorylase and serine 14 in rabbit muscle phosphorylase, are indicated (* ), The initiating methionine was identified on the basis of the N-terminal sequence analysis of purified yeast phosphorylase. A 2nd methionine, previously thought to be the starting methionine, is located 11 residues downstream.

1032

V. L. Rath et al.

amount of serine phosphate. The background levels of phospho-amino acids in a protein not subject to phosphorylation control were determined using bovine serum albumin. The amount of serinephosphate released frdm unphosphorylated control samples of rabbit musle phosphorylase or kemptide was within the level detected for serum albumin. Five samples of yeast phosphorylase from two different enzyme preparations were analyzed. In each case, the level of threonine-phosphate was within the backgound amount detected for bovine serum albumin, as was the amount of serinephosphate present. Therefore, within the limits of our assay, the enzyme is not phosphorylated. The protein can be phosphorylated in vitro using yeast phosphorylase kinase. Phosphorylation increased the activity of the enzyme from 4.7 to 85 to 90 units/mg as expected (Fosset et al., 1971; our unpublished results). (d) Crystals

precession photographs, we est,imated that we could measure data to 3.1 .& resolution from these crystals. Form 2 triclinie crystals were grown at room temperature in pot,assium phosphate buffer using polyethylene glycol as the precipitant with the addition of either ethanol or acetone. The hanging drop contained equal volumes of protein (12.5 mg/ml in 100 mM-potassium phosphate (pH 65), I mMEDTA, 1 rnM-DTT, 0.02% sodium azide) and reservoir solution (100 mM-potassium phosphate (pH 6*5), 1 m&I-EDTA, 16% PEG 4000, 1 m&f-DTT, @02 y0 sodium azide and 4% either ethanol or acetone). These crystals were less tha,n 0.1 mm on an edge. Although we could detect diffraction to 3.6 A resolution using synchrotron radiation, for practical purposes the resolution limit, was 4.0 8. Form 3 orthorhombic crystals could be grown ab either 4 “C or 17 “C using form 1 tetragonal crystals to streak seed hanging drops (Stura & Wilson, 1990). Equal volumes of reservoir solution (50 mMTris (pH 8-O), 50 mix-GIc-6-P, 9% PEG 4000, 1 mncDTT, 0.02% sodium azide) and protein (26.8 mg/ml in a buffer of 10 m;M-Bis-Tris (pH CO), 100 mnfNaCl, 1 mM-DTT) were mixed in a drop and seeded immediately. Easily reproduced, the crystals grew to dimensions of 0.125 mm x 0.250 mm x 0.5 mm in about four days. The crystals were radiation sensitive, exhibiting the rapid loss of diffraction from Bragg spacings between 2.6 and 4.0 8, as shown by an incomplete dataset measured on film at the National Light Source Synchrotron at Brookhaven National Laboratory. Our attempts to circumvent this difficulty by freezing the crystals caused the lattice to disorder completely. Form 4 tetragonal crystals proved to be the most useful for high resolution data collection. The fir& two of these crystals grew spontaneously at 17°C and were used to determine the cell constants a,nd space group. Assuming a solvent content of SOo,b, the asymmetric unit contained only two monomers of phosphorylase, significantly less than the three previous forms. Although we were unable to obtain crystals under the original conditions, some of the

of yeast glycogen phosphorylase

Yeast phosphorylase b has been crystallized in the four different crystal forms described in Table 2. Of these, only form 4 is suitable for diffraction studies at high resolution. The other crystal habits contain four or more monomers in the asymmetric unit or else result in crystals that are too small or too for measurement of high radiation sensitive resolution data. Form 1 tetragonal crystals were grown at 4°C. Equal volumes of protein solution (8 mg/ml in 10 mM-Bis-Tris (pH 6.0), 100 rniM-NaCl, 1 mM-DTT) and reservoir solution (50 mm-Tris (pH %O), 50 m;MGlc-6-P, 9% (v/v) polyethylene glycol (PEG) 4000, 1 mM-DTT, 0.02% sodium azide) were mixed to form the hanging drop. Unscreened precession photographs revealed a 4-fold screw axis and, most likely, an additional 2-fold axis. Possible space groups were therefore P4, or P4,22 (or their enantiomers). We could not be certain about the symmetry of the crystal due to the low resolution (8 8) of the few precession photographs we were able to obtain. Despite the poor resolution of the

Table 2 Details

Crystal form 1. Tetragonal ’ 2. Triclinic 3. Orthorhombicd 4. Tetragonale~’

of crystal forms of yeast phosphorylase

Cell constants (A) 245 130.2 G(=79.3 202 161.1

245 180.5 /?=689 210 161,l

198 223.9 y=90+3 240 175.5

b Resolution (4 b

Space group P4, or P4,22 Pl

p212121 P4,2,2

16 or 8 4

31 4.0

8 2

40 27

a 2 indicates the number of phosphorylase monomers in the asymmetric unit of the crystal and, except where noted, is based on an estimated solvent content of 50%. b Resolution indicates the practical, rather than the maximum, diffraction limit of the crystals. ’ Iiow resolution precession photographs indicated that the space group was either P4, or P4122 (or their enantiomers). d Crystals were grown using form 1 tetragonal crystals as seeds. e Crystals were grown by macroseeding. ’ From density measurements, the solvent content of the crystals is 497%.

1033

Yeast Phosphorylase

drops microseeded (N. B. Madsen & S. Schechosky, personal communication; Stura & Wilson, 1990) with fragments from the crystals produced showers of small crystals which were then used to macroseed fresh drops (Thaller et al., 1981; Stura & Wilson, 1990). Seed crystals, typically 6025 mm x 0.025 mm x 0.05 mm in size, were washed in reservoir buffer (50 m;M-Tris (pH &O), 50 mM-Glc-6-P, 10% PEG 4000, 380 mM-ammonium sulfate, 1 mMDTT, O.O2o/osodium azide) before use. Two microliters of protein solution (56 mg/ml in 10 mM-BisTris (pH 60), 100 mM-NaCl, 1 mM-DTT) were added to 1 ~1 of reservoir solution containing one washed seed crystal. Single crystals could be grown to dimensions of 0.125 mm x 0.125 mm x 0.65 mm in three days. It was necessary to confirm the space group of the crystals grown from seeds, since we had previously observed that form 1 tetragonal seeds produced orthorhombic crystals. X-ray precession photographs of the hk0 and h01 reciprocal lattice demonstrated that the space group of the crystals grown from seeds was identical to the original. The density of the crystal, determined using a Ficoll gradient (Westbrook, 1985), corresponded to a solvent content of 49.7 o/O, confirming that the asymmetric unit of the crystal consisted of the functional dimer of yeast phosphorylase. The crystals diffracted well to 27 A Bragg spacings. Data to 24 A resolution could be collected at a synchrotron facility. 4. Discussion Unlike muscle phosphorylase, which is subject to both extracellular and intracellular regulation through the reciprocal action of kinases and phosphatases in the first case and by the binding of allosteric ligands in the second, the activity of the yeast enzyme is governed solely by the phosphorylation state of the protein. The activity of the unphosphorylated form of the enzyme is low and is increased through the action of phosphorylase kinase. None of the ligands known to activate the unphosphorylated form of rabbit muscle phosphorylase, such as sulfate or AMP, has any effect on the unphosphorylated yeast enzyme when tested at similar concentrations (our unpublished results). Inhibitors of muscle phosphorylase, such as glucose, caffeine and Glc-B-P, have no effect on the yeast enzyme when tested at similar levels (our unpublished results). We and others (Fosset et al., 1971; Tanabe et aZ., 1987), have observed that Glc-6-P inhibits the activity of the unphosphorylated form of the enzyme at high concentrations (>5 mM, our unpublished results). In contrast to the results presented here, previous studies reported higher specific activities (25 to 40 units/mg) for preparations of phosphorylase from wild-type strains of S. cerevisiae (Fosset et al., 1971; Tanabe et al., 1987). This higher specific activity is most likely the result of contamination of the unphosphorylated form by small amounts of the phosphorylated enzyme. Becker et al. (1983) showed

that phosphorylase preparations with a specific activity of 5 to 20 units/mg contained between 91 to 0.3 mole of covalently linked phosphate per mole of enzyme subunit, indicating that a homogeneous sample of unphosphorylated phosphorylase should have an activity of approximately five units/mg, in agreement with our results. The phosphorylation site of the yeast enzyme is unrelated to that of the muscle protein and is located in a unique protein context. Although the sequence around the phosphorylated threonine 30 in matches the consensus yeast phosphorylase sequence of substrates of cyclic AMP dependent protein kinase (Cohen, 1985), yeast phosphorylase kinase is not cyclic AMP dependent (Becker et al., 1983) and yeast phosphorylase is inefficiently phosphorylated by cyclic AMP dependent protein kinases from mammals and yeast (our unpublished results). Rabbit muscle phosphorylase kinase does not accept the yeast enzyme as a substrate and vice verSa (our unpublished results). The lack of recognition between yeast phosphorylase and muscle phosphorylase kinase, in combination with the previously discussed differences in amino acid sequence, indicates that yeast phosphorylase may be activated by a switch that is structurally distinct from the one observed in the muscle enzyme. We have measured a native data set from form 4 tetragonal crystals to 2.7 A resolution. The molecular replacement method (Rossmann, 1972), including Patterson correlation refinement, as implemented in the program X-PLOR (Brunger, 1990a,b), has been used to position the yeast phosphorylase model within the unit cell. We look forward to understanding the details of activa,tion in yeast phosphorylase and to advancing our knowledge of the evolution of allosteric features in proteins as the structure is elucidated. We thank Chris Bystroff for help in data collection and analysis of the form 3 crystals and for characterizing the form 2 crystals, Christine Luong for excellent technical support, Frank Masiarz for N-terminal amino acid sequencing, Mary McGrath for advice and consolation concerning crystallization experiments, Bryan Sutton for density measurement of the form 4 crystal and Neil Stahl for the amino acid analysis of yeast phosphorylase. We are indebted to Robert Sweet for his expertise and time on beamline X12-C at the Brookhaven National Light Source Synchrotron and to Paul Phizackerly and Mike Soltis for access and help on beamline 7-l of the Stanford Synchrotron Radiation Laboratory. This research was supported by grants from the NIH (DK26081), NSF (DMB84-20489) and the Biotechnology, Resources and Education Program from the University of California.

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by R. Huber