Crystal Structure of the Autocatalytic Initiator of Glycogen Biosynthesis, Glycogenin

doi:10.1016/S0022-2836(02)00305-4 available online at http://www.idealibrary.com on

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J. Mol. Biol. (2002) 319, 463–477

Crystal Structure of the Autocatalytic Initiator of Glycogen Biosynthesis, Glycogenin Brian J. Gibbons, Peter J. Roach and Thomas D. Hurley* Department of Biochemistry and Molecular Biology and Center for Diabetes Research Indiana University School of Medicine, 635 Barnhill Drive Indianapolis, IN 46202-5122 USA

Glycogen is an important storage reserve of glucose present in many organisms, from bacteria to humans. Its biosynthesis is initiated by a specialized protein, glycogenin, which has the unusual property of transferring glucose from UDP –glucose to form an oligosaccharide covalently attached to itself at Tyr194. Glycogen synthase and the branching enzyme complete the synthesis of the polysaccharide. The structure of glycogenin was solved in two different crystal forms. Tetragonal crystals contained a pentamer of dimers in the asymmetric unit arranged in an improper non-crystallographic 10-fold relationship, and orthorhombic crystals contained a monomer in the asymmetric unit that is arranged about a 2-fold crystallographic axis to form a dimer. The structure was first solved ˚ using the tetragonal crystal form and a three-wavelength Se –Met to 3.4 A multi-wavelength anomalous diffraction (MAD) experiment. Subsequently, an apo-enzyme structure and a complex between glycogenin ˚ and UDP – glucose/Mn2þ were solved by molecular replacement to 1.9 A using the orthorhombic crystal form. Glycogenin contains a conserved D £ D motif and an N-terminal b-a-b Rossmann-like fold that are common to the nucleotide-binding domains of most glycosyltransferases. Although sequence identity amongst glycosyltransferases is minimal, the overall folds are similar. In all of these enzymes, the D £ D motif is essential for coordination of the catalytic divalent cation, most commonly Mn2þ. We propose a mechanism in which the Mn2þ that associates with the UDP –glucose molecule functions as a Lewis acid to stabilize the leaving group UDP and to facilitate the transfer of the glucose moiety to an intermediate nucleophilic acceptor in the enzyme active site, most likely Asp162. Following transient transfer to Asp162, the glucose moiety is then delivered to the final acceptor, either directly to Tyr194 or to glucose residues already attached to Tyr194. The positioning of the bound UDP – glucose far from Tyr194 in the glycogenin structure raises questions as to the mechanism for the attachment of the first glucose residues. Possibly the initial glucosylation is via inter-dimeric catalysis with an intramolecular mechanism employed later in oligosaccharide synthesis. q 2002 Elsevier Science Ltd. All rights reserved

*Corresponding author

Keywords: crystal structure; glycogenin; glycosyltransferase; glycogen; cis peptide bond

Introduction Glycogen is a branched polymer of glucose that is present as a storage reserve in most organisms.1 It is synthesized during periods of nutritional sufAbbreviations used: MAD, multi-wavelength anomalous diffraction; NCS, non-crystallographic symmetry; UDP, uridine 50 -diphosphate; UDPG, uridine 50 -diphosphoglucose. E-mail address of the corresponding author: [email protected]

ficiency and, in mammals, is stored primarily in the liver and muscle. Glycogen metabolism is a major component of whole-body glucose metabolism, and defective glycogen storage is associated with several diseases, including type 2 diabetes. The biogenesis of glycogen is characteristic of biopolymer synthesis, in that it has distinct initiation and elongation phases. Initiation is mediated by glycogenin (EC 2.4.1.186) and elongation by glycogen synthase in conjunction with the branching enzyme. As the initiator of glycogen synthesis, glycogenin is in a key position to play a regulatory

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

464

Crystal Structure of Glycogenin

Figure 1. The two chemically distinct reactions catalyzed by glycogenin. (a) The initial glucosylation of the hydroxyl group of Tyr194 resulting in the formation of a glucose 1-O-tyrosyl linkage. (b) The subsequent glucosylation of the C40 -hydroxyl group of the terminal glucose on the nascent glycogen polymer resulting in the formation of a-1,4-glycosidic linkages.

role; but the available evidence is inconclusive.2 Rabbit muscle glycogenin is a 332 amino acid residue, 38 kDa protein that forms , 80 kDa dimers in solution.3 Humans have a second isoform of glycogenin (called glycogenin-2) that is found predominantly in liver,4 but non-primates seem to have a single glycogenin gene expressed in muscle and liver.5 Proven functional homologues are present in Saccharomyces cerevisiae, and genes with highly related sequences are found in worms, flies, and fish. As an enzyme, glycogenin is unusual if not unique. It is the substrate, the catalyst, and the product of the reaction. There is true catalysis, since multiple turnovers occur at the same active site. Two chemically distinct reactions are catalyzed, the initial glucosylation of Tyr194 through the formation of a glucose 1-O-tyrosyl linkage and the subsequent formation of the a-1,4-glycosidic linkages (Figure 1). In fact, there was initially debate as to whether a single enzyme was responsible for both reactions. Recombinant glycogenin produced in Escherichia coli was both already glucosylated and active,6,7 but this did not preclude the existence in E. coli of an activity capable of introducing the first glucose molecule. When the protein was produced in an E. coli strain unable to synthesize UDP – glucose, no glucose was detected in the glycogenin that was, nonetheless, capable of self-glucosylation.8 This result is the strongest evidence that both reactions are catalyzed by glycogenin.

Glycogenin can be classified as a retaining glycosyltransferase, based on the relative anomeric stereochemistry of the substrate and product in the reaction catalyzed.9 It is placed in glycosyltransferase family 810,11 which includes lipopolysaccharide glucose and galactose transferases and galactinol synthases, proteins only distantly related in overall sequence but containing certain highly conserved motifs common to glycosyltransferases.12 – 14 To date, relatively little unequivocal data has been obtained regarding the chemical mechanism of glycogenin or any other glycosyltransferase. However, it is likely that the retaining and inverting transferase mechanisms will be similar to either the retaining or inverting glycosidase mechanisms, which have been studied in more detail.9,15 – 17 The structures of several other glycosyltransferases have been solved18 – 26 and, although sequence identity amongst these glycosyltransferases is minimal, there are overall similarities in the protein fold that allow this group of enzymes to be placed into families and super-families. Glycogenin has the greatest structural similarity to LgtC, a galactosyltransferase from Neisseria meningitidis involved in lipooligosaccharide biosynthesis,23 and to a3GalT, a bovine galactosyltransferase involved in biosynthesis of the xenoantigen,19 both also belonging to the retaining class. Here, we report the determination of the structure of glycogenin in two different crystal forms.

Table 1. Data collection

Space group Unit cell ˚) a (A ˚) b (A ˚) c (A a ¼ b ¼ g (deg.) ˚) Resolution (A Total observations Unique reflections Completeness (%)a kIl/skIla Rmerge (%)b a b

Se–Met (peak)

Se–Met (inflection)

Se– Met (remote)

Native

Apo-enzyme

UDPG/Mn2þ

P43212

P43212

P43212

P43212

I222

I222

140.48 140.48 417.36 90.0 3.80 591,580 78,294 99.5 (97.0) 24.3 (5.8) 7.8 (38.4)

140.7 140.7 417.8 90.0 3.81 297,705 78,433 99.5 (97.9) 17.0 (3.3) 7.5 (42.6)

140.2 140.2 416.7 90.0 3.80 295,180 41,660 99.5 (97.5) 15.9 (3.3) 8.2 (41.6)

140.5 140.5 416.5 90.0 3.43 403,861 56,152 99.6 (99.3) 26.0 (4.2) 8.8 (48.4)

59.1 104.8 120.6 90.0 1.90 162,821 29,807 98.4 (92.9) 23.1 (2.4) 6.2 (55.9)

57.9 106.9 122.3 90.0 1.90 93,616 29,472 96.7 (88.8) 22.1 (2.7) 4.5 (37.2)

Values in parentheses for completeness, kIl/skIl, and Rmerge are for the highest-resolution shells. Rmerge ¼ ShkllIi 2 Inl/SIn where Ii is an observed intensity and In is the average of the observed equivalents.

465

Crystal Structure of Glycogenin

Table 2. Refinement statistics ˚) Resolution range (A Molecules/asymmetric unit Rwork/Rfreea Reflections Solvent atoms ˚ 2) Average B (A ˚) rmsd ideal bonds (A rmsd ideal angles (deg.) ˚) sA est. coord. error (A a

Native (P43212)

Apo (I222)

UDP–Glc/Mn2þ

25–3.4 10 0.252/0.286 53,593 0 79.0 0.011 1.45 0.54

30 –1.9 1 0.212/0.246 29,664 249 39.6 0.005 1.19 0.24

30–1.9 1 0.194/0.227 29,435 275 34.4 0.006 1.19 0.23

R ¼ (ShllFobsl 2 lFcalcll)/(SlFobsl).

Initially, the apo-enzyme structure was solved to ˚ using a crystal form that contained five 3.4 A dimers in the asymmetric unit. Subsequently, a second crystal form yielded crystals that diffracted ˚ , both in the apo-enzyme form and in a to 1.9 A complex with UDP –Glucose and Mn2þ. This structure, the first of glycogenin and of a selfglucosylating enzyme, provides new mechanistic insight into glycogen synthesis and the mechanism of retaining glycosyltransferase reactions.

Results and Discussion Structure determination The crystal structure of rabbit muscle glycogenin was determined from crystals composed of a seleno-methionine derivative of the enzyme using multi-wavelength anomalous diffraction (MAD) methods. Data collection and refinement statistics are presented in Table 1 and Table 2, respectively. The initial structure was solved from crystals that were tetragonal (P43212) and diffracted X-rays ˚ 10-fold averaged map weakly. The resulting 3.4 A was of high quality and permitted the construction of a model for glycogenin containing residues 1– 231 and 241– 259. Subsequently, orthorhombic (I222) crystals with a single subunit in the asymmetric unit were obtained. Molecular replacement and refinement with CNS27 produced high-quality maps and models for these structures. Residues 232– 240 and residues , 260 –332 are absent from the structure of glycogenin obtained from both the tetragonal and orthorhombic crystal forms. Residues 232– 240 are crystallographically disordered, while residues , 260 – 332 appear to have a more complex explanation. An examination of the contents of redissolved crystals by SDSPAGE (not shown) reveals that approximately 50% of the glycogenin subunits in the tetragonal form exist as a lower molecular mass species, while the remaining 50% of the molecules are identical in size with the full-length molecule. Redissolved orthorhombic crystals show less of the proteolyzed fragment, but it still represents between 30% and 40% of the total mass. Careful examination of the unaveraged, density-modified map of the decameric structure in the tetragonal crystal form failed

to reveal any consistent electron density extending beyond residue 259. However, several of the ten subunits contain additional electron density with the farthest extending to residue 269. The orthorhombic crystal form contains a single monomer in the asymmetric unit, so that a mixture of species in this crystal form implies statistical disorder of this portion of the structure in order to keep all molecules crystallographically identical. In fact, recently, seleno-methionine-substituted glycogenin ˚ in the orthorwas used to obtain phases to 1.9 A hombic crystal form, and these maps showed no evidence for additional electron density. The orthorhombic apo-glycogenin monomer and the complex with UDP –glucose and Mn2þ contain electron density to residues 265 and 262, respectively. The apparent size of the lower molecular mass species observed in the SDS-PAGE analysis is identical with a fragment previously identified in samples prepared in the absence of peptidase inhibitors.28 Both species have been shown to possess full catalytic activity,28 indicating that the current structure contains the entire catalytic portion of the enzyme. Overall protein fold of glycogenin A pentamer of dimers was present in the asymmetric unit of the tetragonal crystal form, which provided the data for the initial structure solution. A monomer was present in the asymmetric unit of the orthorhombic crystal form, which produced the highest-resolution data. The monomer from both crystal forms has a single domain with a mixed a þ b fold that contains one six-stranded and one three-stranded mixed b-sheet, both flanked by a-helices, arranged as an ab 4-layer sandwich. The six-stranded b-sheet forming the core of the domain is composed of a four-stranded parallel b-sheet that continues into a fifth parallel strand and sixth anti-parallel strand in the topological order 3,2,1,4,6,5 (Figure 2). These mixed sheets are surrounded by a-helices. The single domain of the glycogenin monomer has an extended, mostly open, groove running along the C-terminal ends of the parallel b-strands in the central b-sheet that forms the substrate-binding cleft (Figure 3). This cleft contains the binding site

466

Crystal Structure of Glycogenin

Figure 2. Topology diagram of the glycogenin structure. Arrows indicate b-strands; cylinders indicate helices. The beginning and ending residues for each secondary structure element are indicated. The N-terminal domain comprising the nucleotide-binding fold has strands colored magenta and helices colored violet and is outlined with a box. Regions involved in the decamer interface are circled in green and regions involved in the dimer interface are circled in red. The approximate positions of the bound Mn2þ and UDPG are shown relative to the DAD motif. The helix containing Tyr194 is indicated.

Figure 3. Stereo ribbon diagram of the glycogenin monomer with bound Mn2þ and UDP – glucose. The N-terminal Rossman-like nucleotide-binding fold is color-coordinated with the topology diagram in Figure 2. The substrate-binding cleft is an extended, mostly open groove that runs along the C-terminal ends of the parallel b-strands in the central b-sheet. This Figure was generated using SwissPdb Viewer54 and rendered using POV-Ray for Windows (downloaded from: www.povray.org).

467

Crystal Structure of Glycogenin

for the sugar-donor UDP – glucose and catalytic Mn2þ, identifying it as the putative active site of the enzyme. The UDP – glucose and Mn2þ are located in an energetically favorable association with the D £ D motif and the N-terminal b-a-b Rossmann-like fold. The overall fold is very similar to the catalytic domain of most glycosyltransferases solved to date, including SpsA,18 a3GalT,19 LgtC,23 b-4-GalT1,20 Gn T1,21 MurG,22 Mob A,24 T4 b-gt,25 GlcATI26 and GtfB.29 These enzymes contain a common fold that has been called the SGC domain (SpsA Gn T1 core domain), due to its being identified first in the structures of SpsA and Gn T1.30 It has also been referred to as the UBD (UDP binding domain).19 The current list of enzymes with a SGC/UBD fold includes both retaining and inverting glycosyltransferases, from prokaryotes and eukaryotes, and sequence comparisons predict that it is probably found in enzymes from at least ten out of the 52 families of glycosyltransferases.30 One notable difference between the SGC/UBD folds of retaining and inverting glycosyltransferases is the depth of the active-site groove. This groove is deeper in the retaining glycosyltransferases, possibly due to a need for more effective solvent exclusion in double-displacement catalysis utilizing a reactive glycosyl-enzyme intermediate. The structure of glycogenin is most similar to the other retaining glycosyltransferase structures that are currently available: LgtC, and a3GalT. The gross structural similarity of these enzymes can be observed readily through a topological comparison (Figure 4). Recognition of UDP– glucose The binding of UDP – glucose is stabilized by a number of specific interactions (Figure 5(a)). The uracil ring forms the majority of its specific interactions with residues 10 through 14, which are among the residues contained within the conserved sequence domain I (as defined by Roach & Skurat2). The uracil ring is bound between the hydrophobic surfaces of two residues, Val82 and Tyr14, while the edge of the uracil ring makes a number of specific hydrogen bonds with surrounding amino acid side-chains. Thr10 makes two separate interactions with the uracil ring, its mainchain nitrogen atom donates a hydrogen bond to the O2 carbonyl atom and its side-chain hydroxyl group accepts a hydrogen bond from the N3 atom. These two interactions would not necessarily be specific for uracil and could be utilized to bind cytidine if the side-chain hydroxyl group donated a hydrogen bond, rather than accepted a hydrogen bond, from the N3 atom. The specificity for uracil appears to be solely due to the interaction with Asn11. The side-chain amide nitrogen atom of Asn11 donates a hydrogen bond to the O4 carbonyl group. The selectivity for uridine is achieved because Asn11 could not easily rotate its carboxamide group to become a hydrogen bond acceptor

and accommodate the exocyclic amine group of cytosine. This restriction is due to the extensive interactions between the side-chain carbonyl oxygen atom of Asn11 and the N terminus of a-helix A. The side-chain of Asn11 effectively caps the free main-chain nitrogen atoms of this helix by forming hydrogen bonds with two of the three available peptide nitrogen atoms. Thus, to specifically recognize cytidine, Asn11 would have to break the two hydrogen bonds capping this helix and create only one new compensating interaction with the cytidine ring. This hydrogen bonding pattern is consistent with the known preference for UDP – glucose over CDP– glucose as a substrate for glycogenin.31,32 The uridine ribose ring is held in place by interactions between the 20 -hydroxyl group and the peptide carbonyl group of Leu8 and the peptide nitrogen atom of Ala102, and between the 30 -hydroxyl group and the peptide nitrogen atom of Asp103, part of the sequence DAD within conserved sequence domain II. The diphosphate group is held in position primarily by direct coordination to the bound Mn2þ (discussed below), but additional hydrogen bonds are accepted from Tyr14, Lys217 and the peptide nitrogen atom of Gly214. The latter two residues are found within conserved sequence domain IV. In our current structure, the glucose forms hydrogen bonds between its 40 -hydroxyl group and residues Asp124, Asn132 and Gln163, and between its 60 -hydroxyl group and Ser133. Residues 132, 133 and 163 are found within conserved sequence domain III. The configuration of the 40 -hydroxyl group is the only difference between glucose and galactose, so the interactions between this hydroxyl group and the enzyme may contribute to substrate specificity. Consistent with this, no interaction is observed between the enzyme and the 20 or 30 -hydroxyl group of glucose. There are a total of four ordered water molecules between the b-face of the glucose and the putative catalytic residues located at the N terminus of a-helix G (see below), that we believe must be displaced prior to catalysis. D 3 D motif/manganese binding Most prokaryotic and eukaryotic glycosyltransferases have a D £ D sequence motif despite the lack of other sequence similarities,11,14,33 In all of the currently known glycosyltransferase structures, this motif is located within the SGC/UBD fold and appears to function primarily in the coordination of the divalent cation essential for the binding of the nucleotide sugar-donor substrate. The D £ D motif is clearly important for catalysis, since mutation of just one of the conserved Asp residue results in complete loss of enzymatic activity for glycosyltransferases from several species,12,13,34,35 including rabbit muscle glycogenin (J. Zhou & P.J.R., unpublished results). As is the case for several other glycosyltransferases,20,36,37 Mn2þ has been shown to be required for enzyme activity.3

Figure 4. Structural comparison of the retaining glycosyltransferases. Simplified topology diagrams are shown on top with strands represented by triangles and helices represented by circles. As in previous Figures, the strands and helices are color-coded to indicate the N-terminal Rossman-like nucleotide-binding fold. Ribbons diagrams are shown underneath with corresponding colors. LgtC is shown complexed with substrate analogs UDP-2-deoxy-2-fluoro-galactose and 40 -deoxylactose as well as Mn2þ,23 glycogenin is shown complexed with UDP – glucose and Mn2þ; and a3GalT is shown complexed with UDP – galactose and Mn2þ19 These Figures were generated using SwissPdb Viewer54 and rendered using POV-Ray for Windows (downloaded from: www.povray.org).

469

Crystal Structure of Glycogenin

˚ . Conserved Figure 5. (a) The interactions between glycogenin and UDP – glucose/Mn2þ. Distances shown are in A residues from LgtC and a3GalT are indicated. (b) A stereoview of UDP – glucose bound to the D £ D motif. The electron density shown is a refined 2Fo 2 Fc simulated annealing omit map contoured at 1.0s in which the contents of the Figure were omitted from the structure factor calculations. UDP– glucose, Mn2þ, and residues Asp101, Asp103, and His211 are shown as a ball-and-stick model with carbon colored gray, nitrogen colored blue, oxygen colored red, phosphorus colored yellow, and manganese colored magenta. This Figure was generated using SwissPdb Viewer54 and rendered using POV-Ray for Windows (downloaded from: www.povray.org).

Mg2þ and Ca2þ resulted in 20-fold and tenfold lower activity, respectively.38 The single wellordered Mn2þ in our structure of glycogenin is coordinated in a somewhat irregular octahedral arrangement composed of the two phosphate oxygen atoms of UDP – glucose, OD1 of Asp101, a bidentate interaction with both carboxylate oxygen atoms of Asp103, and the NE2 nitrogen atom of His211 (Figure 5(a) and (b)). The ligand distances are characteristic of octahedral coordination, ˚ .39 Glycogenin is a ranging from 2.10 to 2.35 A member of the glycosyltransferase family 8, throughout which these three amino acid side chains, Asp101, Asp103, and His211 are well conserved,23 and among glycogenin sequences

they are found within domains III and IV.

conserved

sequence

Dimeric and decameric oligomerization The tetragonal crystal form of glycogenin has a decamer in the asymmetric unit that is actually a pentamer of dimers arranged in an improper noncrystallographic tenfold relationship (Figure 6). The glycogenin protomers in this crystal form are components of dimers that have a 2-fold noncrystallographic axis identical with the crystallographic 2-fold axis that creates the dimer in the orthorhombic crystal form. The dimer interface is composed of contacts between the loop region

470

Figure 6. Glycogenin decamer shown with each subunit indicated by color. This Figure was generated using MOLSCRIPT55 and rendered using Raster3D.56,57

Crystal Structure of Glycogenin

for crystal formation than a biologically relevant arrangement. The second crystal form, orthorhombic I222, has a monomer in the asymmetric unit. In this crystal form, the glycogenin protomers are arranged about a 2-fold crystallographic axis, forming dimers (Figure 7). The dimer interface contacts are the same as in the tetragonal crystal form. Upon ˚ 2 of solvent-accessible dimer formation, , 2400 A ˚ 2 (, 20%) of total surface area per , 12,000 A monomer surface area is buried. This is comparable to other protein– protein interactions that have a high-affinity association.40 Both analytical ultracentrifugation data and gel-filtration data indicate that the primary species present in solution has a molecular mass of , 80,000 Da (data not shown) consistent with dimers. Kinetic and mutational studies support mechanistic models for self-glucosylation involving dimeric glycogenin molecules.3,41 It is, therefore, quite likely that the dimeric form of the enzyme is important, if not essential, for enzyme function. Cis-peptide bond

from b-strand 5 to b-strand 6 (residues 124 – 131), the loop region from a-helix F to a-helix G (residues 159 – 164), the loop region from a-helix H to b-sheet 2 strand 2 (residues 175– 192), and a-helices I (residues 193, 196, and 197) and J (residues 199 – 201, 203and 204) from both subunits (Figure 2). These dimers are organized about the 5-fold non-crystallographic axis of the decamer forming a concave star-shaped pentamer of dimers each having an inner and an outer subunit with all active sites directed inward toward the concave surface of the star (Figure 6). Only the “inner” subunits form the contacts that stabilize this decamer. ˚ 2 of Decamer formation would bury , 1700 A ˚2 solvent-accessible surface area per , 24,000 A (, 7%) of total dimer surface area at each of the five interfaces between the inner subunits of the dimers, consistent with moderate affinity.40 Analytical ultracentrifuge data also suggest the presence of a small amount of a higher-order complex with a molecular mass of , 300,000 Da, which could be accounted for by glycogenin decamers (data not shown). Nonetheless, the decameric form of glycogenin may not be physiologically significant for several reasons. In the decamer, the active sites of the glycogenin monomers would be confined to the inner surface of a bowl-shaped ˚ across. structure (Figure 6) approximately 100 A This could restrict access of glycogen synthase to the newly formed short glycogen polymers, an essential step in the formation of mature glycogen. In addition, there is no obvious way for a decamer to fit into the current kinetic and structural models for the mechanism of self-glucosylation (discussed below). Despite its aesthetically pleasing appearance, the decameric form is more likely to be an artifact of the high enzyme concentrations required

An additional finding of interest is the presence of a cis peptide bond between Glu118 and Leu119. The natural occurrence of non-proline cis peptide bonds is very low (about 0.05% of all peptide bonds) and they are usually associated with functionally important structural regions.42 – 44 In glycogenin, the cis peptide bond is clearly evident only in the I222 crystal form data (Figure 8). The P43212 crystal form data is too low in resolution to confidently assign the cis conformation. However, when the model for the low-resolution data is refined with a trans peptide bond between Glu118 and Leu119, it has geometric values that, when compared to other structures containing nonproline cis peptide bonds, suggest that the bond should be built as the cis conformation.43 The bond between Glu118 and Leu119 is located near the outer surface of the protein. These two residues do not participate in any crystal contacts nor are they near any of the obvious regions of catalytic importance, such as the active site or the dimerization domain. The cis peptide bond conformation is energetically unfavorable, and is, therefore, stabilized by several interactions with the surrounding structure (Figure 8(c)). Glu118 makes three hydrogen bonds with the surrounding residues. One of its side-chain carboxyl oxygen atoms is positioned appropriately to form hydrogen bonds with the main-chain nitrogen atom of Gln139 and with the NE1 nitrogen atom of Trp89; and the main-chain carbonyl group of Glu118 accepts a hydrogen bond from the side-chain nitrogen atom of Lys180. The side-chain of Leu119 extends into a hydrophobic pocket formed by Trp89, Phe131, Phe136, Leu166, and Phe170. The multiple hydrogen bonds holding Glu118, combined with the hydrophobic pocket holding Leu119, should provide enough stability to account

Crystal Structure of Glycogenin

471

Figure 7. Glycogenin dimer ribbons diagram with a-helices colored blue and green, b-sheets colored red and yellow, and coils colored gray. UDP– glucose molecules and Tyr194 side-chains are shown as ball-and-stick models and Mn2þ is colored magenta. The distances between the C100 atom of the glucose in UDP– glucose and the Tyr194 hydroxyls are indicated with arrows. This Figure was generated using SwissPdb Viewer54 and rendered using POV-Ray for Windows (downloaded from: www.povray.org).

for the presence of the cis peptide bond. There are several factors that would make a trans conformation of this peptide bond unfavorable. If the bond were to isomerize to the trans conformation via a Glu118 carbonyl flip, the carbonyl group would first have to break its hydrogen bond with Lys180, after which it would then meet steric hindrance from the main-chain carbonyl group of Leu137. The only other way for this bond to isomerize to the trans conformation would be for Leu119 to rotate 1808 around the peptide bond. This would require the hydrophobic side-chain of Leu119 to move out of the hydrophobic pocket and into an energetically unfavorable position on the solvent-exposed surface of the protein. Either of these isomerizations would likely cause substantial conformational changes that might possibly be exploited by the enzyme during catalysis. Catalytic mechanism The currently favored catalytic mechanism for retaining glycosyltransferases is a double displacement mechanism with a glycosyl-enzyme intermediate and an oxocarbenium-ion transition state that involves two separate nucleophilic substitution reactions (Figure 9).30 For glycogenin, the first reaction would transfer the glucose moiety from the UDP –glucose via a transient covalent linkage to a side-chain acceptor appropriately positioned within the active site. The second reaction would immediately transfer the glucose to the ultimate acceptor, in this case either Tyr194 or the C40 -hydroxyl group of glucose on the nascent

glycogen polymer. Each of the two steps of this proposed mechanism would cause an inversion of the anomeric configuration of the glucose being transferred, ultimately resulting in the retention of configuration from the UDP – glucose donor to the final acceptor. The active site of glycogenin reveals clues that support the mechanism proposed for retaining glycosyltransferases. The UDP – glucose molecule is bound to the enzyme through ionic interactions with a manganese ion that could function as a Lewis acid. This would aid in drawing a pair of electrons from the C100 carbon of the glucose to stabilize the UDP leaving group, weaken the phosphate– C1 bond, and facilitate the formation of the oxocarbenium ion and the subsequent transfer of the glucose moiety to a nucleophilic acceptor. However, in our structures, the active site appears to be devoid of an obvious and appropriately positioned intermediate glucose acceptor required for the first reaction. The ideal intermediate acceptor would be a nucleophilic side-chain located adjacent to the correct (b) face of the anomeric carbon atom (C100 ) of the UDP –glucose to allow direct displacement of the UDP leaving group. If the mechanism is comparable to the retaining glycosidases, the best candidate residues are ˚ and 6 A ˚, Asp159 and Asp162, which are 6.5 A 00 respectively, from the glucose C1 position (Figures 5(a) and 8(c)). Of these two potential nucleophiles, Asp162 is more likely: it is found in all glycogenin sequences studied to date2 and it is located near another conserved residue that has been shown to be essential for glycogenin catalysis, Lys85.3 Asp162 and Lys85 are in locations structurally

472

Crystal Structure of Glycogenin

Figure 8. (a) A stereoview of the cis peptide bond between Glu118 and Leu119 is shown in a refined 2Fo 2 Fc simulated annealing omit map contoured at 1.0s in which the contents of the Figure were omitted from the structure factor calculations. (b). A stereo view showing an Fo 2 Fc difference density map contoured at 3.0s for the peptide bond between Glu118 and Leu119 refined in the trans conformation. The aligned cis conformation is shown in gold for reference. (c) The cis peptide bond and surrounding residues are shown, in stereo, as ball-and-stick models. The amino acid residues interacting with Glu118 and Leu119 are labeled. The helix between Phe170 and Asp162 is shown as a green ribbon. These Figures were generated using SwissPdb Viewer54 and rendered using POV-Ray for Windows (downloaded from: www.povray.org).

Crystal Structure of Glycogenin

473

Figure 9. Retention of configuration via double displacement. In the first reaction, a glucose residue is transferred from UDP– glucose to the intermediate acceptor, shown here as Asp162, in the active site of glycogenin, with inversion of configuration. In the second reaction, glucose is transferred from the intermediate acceptor to the reactive hydroxyl group of the ultimate acceptor, shown here as the C4 hydroxyl group of glucose on the nascent glycogen polymer. This causes a second inversion of configuration resulting in retention of configuration from the UDP –glucose donor to the final acceptor.

homologous to those of Asp188 and Arg86 in the active site of LgtC, and Asp316 and Arg202 in the active site of a3GalT, the other two retaining glycosyltransferase structures (Figure 5(a)). The residues equivalent to Lys85 and Asp162 are conserved in the other family 8 retaining glycosyltransferases.23 In order for Asp162 to be the primary acceptor, however, the active site would have to undergo conformational changes in order to bring the sidechain oxygen atom of Asp162 close enough to the C100 position of the glucose moiety for efficient catalysis. A movement of the helical bundle, in which Asp162 is located (a-helix G in Figure 2), toward the bound UDP – glucose molecule by ˚ should be sufficient. about 3 A It is possible, although purely speculative, that this type of movement could be provided by a cis – trans isomerization of the non-proline cis peptide bond located between Glu118 and Leu119 in our structures of glycogenin. As discussed above, isomerization to the trans isomer, perhaps mediated by terminal acceptor binding, could cause the Leu119 side-chain to flip out of the hydrophobic pocket. This would break its interaction with Phe170 and Leu166 in the hydrophobic pocket and would be likely to result in a structural rearrangement of one or more of the groups forming the hydrophobic pocket, possibly allowing a-helix G to slide a few a˚ngstro¨m units towards the active site. Because Phe170 and Leu166 are located on one end of a-helix G and Asp162 is located on the other, a positional shift of this helical bundle could place Asp162 close enough to the active site for participation in catalysis (Figure 8(c)). A possible conformational change upon terminal acceptor binding is suggested, but not proven, by our observation that the addition of both the alternate acceptor molecule dodecyl-maltoside and UDP –glucose/Mn2þ caused instantaneous shattering of the apo-enzyme crystals, while addition of only UDP – glucose/Mn2þ yielded the complex reported here. The involvement of cis – trans isomerizations of cis peptide bonds in enzyme-activating conformational rearrangements has been discussed.43,45,46 It remains to be seen if mutations at positions 118 or 119 of glycogenin will affect enzyme activity.

An alternative mechanism could use Gln163 as the intermediate nucleophilic glucose acceptor, even though amide groups are not considered to be good nucleophiles. The carbonyl oxygen atom ˚ from the C100 of Gln163 is slightly less than 6 A position, and is located on the b-face of the glucose (Figure 5). The involvement of a residue at this position in the structure has been suggested for a3GalT, since it has electron density attached to Glu317 that resembles galactose.19 This suggests that, at least for a3GalT, the Glu residue at this position is the intermediate sugar acceptor. Glycogenin and all other family 8 glycosyltransferases, however, have Gln at this position and, although this mechanism has been suggested for the structurally homologous Gln189 in LgtC23 and for glycogen phosphorylase,47 it is unlikely considering that mutation of this residue did not abolish activity completely. Evidence supporting involvement of Asp162 and/or Gln163 in the catalytic mechanism of glycogenin comes from Ala scanning mutagenesis in which the simultaneous substitution of Asp162, Gln163, Leu166 and Asn167 by Ala resulted in an enzyme with less than 0.5% of wildtype activity (J. Zhou and G.P.R., unpublished results). However, considering the less than conservative nature of the mutations and the possibility of local conformational changes, it serves only to suggest further mutational and kinetic studies. The second reaction requires that the glucose be transferred from the intermediate acceptor (Asp162) to the ultimate acceptor, either the reactive hydroxyl group of Y194 or the C40 -hydroxyl group of glucose on the nascent glycogen polymer. In order for this reaction to occur, the acceptor hydroxyl group would first have to be deprotonated. This would require the assistance of a general base, but there is no obvious group present in the active site to perform this function. Lacking any better candidates, it is possible that the UDP leaving group could function as a general base to deprotonate the hydroxyl (Figure 9). It is likely that the UDP remains bound to the active site until after the glucose moiety is transferred to help shield the reaction from hydrolysis. If so, it should be located near enough to the hydroxyl group of the glucose acceptor to facilitate deprotonation.

474

Although we favor the double displacement mechanism involving two separate transfer reactions, we cannot rule out the possibility that the mechanism might not involve double displacement, but rather proceed through a single frontside displacement reaction. For this to occur, the nucleophile (either the reactive hydroxyl group of Tyr194 or of the acceptor glucose) would have to approach the C100 position from the same side as the UDP leaving group. This type of reaction, known as an SNi mechanism, has little precedence. Nonetheless, it has been invoked for LgtC23 and glycogen phosphorylase9 in the absence of experimental evidence for the double displacement mechanism. Mechanism of initial versus subsequent glucosylation reactions Self-glucosylation in solution follows zero-order kinetics except at very low protein concentrations, consistent with an intramolecular reaction.3,8,41,48 The fact that a Tyr194Phe mutant can transfer glucose to a catalytically inactive mutant3 demonstrates the occurrence of intersubunit transfer, and so the model proposed for self-glucosylation was for one subunit within a dimer to transfer glucose to the other.3,41 However, our structure of glycogenin does not easily support this intersubunit model, as the location of Tyr194 relative to the sugar-donor UDP – glucose in the active sites of the dimer seems to preclude this mechanism, at least for the attachments of the initial glucose residues. The hydroxyl group on Tyr194 is at least ˚ from the C100 position of the glucose residue 15 A on the UDP –glucose molecule bound in the same ˚ from the same atom on monomer and about 12 A the opposing subunit of the dimer (Figure 7). If the catalytic mechanism does indeed involve a nucleophilic substitution reaction, then the donor UDP – glucose would need to be within bonding distance of the Tyr194 acceptor. The distances within the structure would not permit intramonomeric self-glucosylation and, unless a large positional shift can occur within the dimer to bring Tyr194 closer to the UDP –glucose bound in the opposing subunit, inter-subunit self-glucosylation would appear implausible. We suggested above that a conformational change to bring Asp162 into play during catalysis was consistent with the addition an acceptor molecule disrupting the lattice of our apo-enzyme crystals, and the extent of motion we suggested appears plausible on the basis of the position of the helical subdomain adjacent to the active site. However, we have not been able to identify a likely hinge point or conformational switch that could permit the amount of motion necessary to bring Tyr194 into direct contact with either UDP – glucose molecule in the glycogenin dimer. The kinetic data supporting an intramolecular reaction were obtained with glycogenin that was already partially glucosylated. Another way for

Crystal Structure of Glycogenin

the observed catalytic-site geometry of the glycogenin to support the initial glucosylations is if the initial reaction is mechanistically different. Specifically, the initial glucosylation(s) could be intermolecular until the glucosylation switches over to an entropically favored intramolecular reaction. Since the distance between Tyr194 of one subunit and the C100 position of the UDP – glucose ˚ of the opposing subunit of the dimer is about 12 A (Figure 7), it should be possible for intramolecular glucosylation to occur once three or more glucose residues have been transferred. The species participating in this postulated initial phase could be either dimers or monomers. There is some evidence for this mechanism, since zero-order kinetics was lost at low enzyme concentrations.8 This result could be explained if the concentration-dependence observed in the kinetic experiments indicates association of either inactive dimers into active tetramers or inactive monomers into active dimers.

Conclusion The structure of glycogenin provides the first glimpse into the enzymatic mechanisms for the initiation phase of glycogen synthesis. Our structure shows that the basic functional unit of glycogenin is a dimer and that the basic structural architecture of the enzyme is very similar to the large family of glycosyltransferases to which it belongs. We have identified important contacts between the enzyme and its substrate UDP – glucose/Mn2þ and proposed a chemical mechanism by which Asp162 may be the important intermediate nucleophile on the enzyme responsible for the retention of anomeric configuration of the glucose molecule during catalysis. Most interestingly, the structure suggests that there may be two mechanistically distinct phases in the addition of glucose residues to Tyr194. Transfer of the first one to four glucose units would be intermolecular until the subsequent attachment of glucose units can be achieved by an intramolecular reaction within a dimer.

Materials and Methods Enzyme expression and purification Wild-type glycogenin. Wild-type rabbit skeletal muscle glycogenin was expressed and purified using published procedures.6 A His-tagged version of glycogenin was expressed in Escherichia coli, pET15b/GN3 with the following modifications. Two-liter cultures of transformed BL21/DE3 cells were grown at 37 8C in TY medium containing 100 mg/ml of ampicillin. When cultures reached A600 ¼ 0.6, 100 mg/ml of IPTG was added and the cells were grown at 37 8C for three hours to induce expression. Cells were pelleted by centrifugation at 4500g, frozen, thawed, resuspended in 20 ml of lysis/ homogenization buffer, and passed twice through a French pressure cell, operated at 1000 psi (1 psi < 6.9 kPa). The lysate was centrifuged at 109,000g for 30

475

Crystal Structure of Glycogenin

minutes and the supernatant was loaded onto a 12 ml Ni –NTA– agarose (Qaigen) column equilibrated with lysis buffer, washed to give baseline UV absorbance with lysis buffer, and eluted with a 5 mM – 100 mM imidazole gradient at 1.0 ml/minute. The fractions containing glycogenin were pooled and dialyzed against 4 l of 50 mM Hepes (pH 7.5), 1 mM EDTA, 2 mM benzamidine, and 1 mM DTT for four hours. The dialyzed pool was then loaded onto a 25 ml Q-Sepharose Fast Flow column (Pharmacia), washed to baseline UV absorbance with equilibration buffer, and eluted with a 0 M – 1 M NaCl gradient in the same buffer at a flowrate of 1.5 ml/minute. Fractions containing glycogenin were pooled and stored in 50% (v/v) glycerol at 220 8C. Enzyme purification was monitored and confirmed using SDS-PAGE. Seleno-methionine substituted glycogenin. Seleno-methionine rabbit muscle glycogenin was expressed and purified as described above but with the following modifications. The same transformed cells as above were used except that they were grown in selenomethionine defined M9ZB medium with no NaCl and with seleno-methionine substituted for methionine. Expression of the enzyme was induced and the cells grown overnight at room temperature (25 8C) or for three hours at 37 8C. The Ni– NTA column was run without DTT in the column buffers. The Q-Sepharose column buffer contained 8 mM DTT and the Q-Sepharose pool was dialyzed against 15 mM Hepes (pH 7.5), 5 mM DTT before storage in 50% glycerol at 220 8C. Crystal growth All crystals of glycogenin were grown as the apoenzyme form. The crystals grew either as tetragonal˚, bipyramids in the space group P43212 (a ¼ b ¼ 140 A ˚ c ¼ 417 A) or as rectangular rods in the space group ˚ , b ¼ 105 A ˚ , c ¼ 121 A ˚ ) using the sittingI222 (a ¼ 60 A drop, vapor-diffusion method. All crystals were grown at 8 – 10 mg/ml glycogenin, 0.7 – 1.2 M ammonium sulphate, and 100 mM sodium phosphate buffer (pH 6.6– 6.9). Crystal growth occurred at both 25 8C and at 4 8C, and took from several days to several weeks. To obtain complexes with ligands, fully grown crystals were soaked in solutions containing the appropriate combinations of 5 mM MnCl2, 5 mM UDP, and 5 mM UDP –glucose in mother liquor. After a multi-step soak into cryogenic solutions of 25% glycerol in mother liquor, the crystals were flash-frozen to 2 165 8C in a liquid nitrogen stream to prevent radiation decay during data collection. Following successful crystallization and freezing, the crystals were then tested for their ability to diffract X-rays to high resolution and to determine their cell dimensions and space group using the crystallographic software associated with the Center for Structural Biology located in the Department of Biochemistry and Molecular Biology. Crystals were then stored in liquid nitrogen for subsequent data collection. Data collection ˚ Se – Met data set and a Both a three-wavelength 3.8 A ˚ native data set were collected using the tetragonal 3.4 A crystal form on the 19-ID beamline operated by the Structural Biology Center at the Advanced Photon Source located within the Argonne National Laboratory. These data were used for the MAD phasing that resulted ˚ apoin the initial structure solution. Data sets for a 2.6 A

˚ complex with UDP – glucose/Mn2þ enzyme and 1.9 A using the orthorhombic crystal form were collected on a Rigaku RU-200HB rotating anode X-ray generator equipped with Osmic confocal optics and a Rigaku ˚ apoRAXIS IIC image plate area detector. The 1.9 A enzyme data were collected on the X12B beamline at NSLS/Brookhaven National Laboratories. The data collected at beamline 19-ID were processed with HKL2000.49 All other data were processed using DENZO and SCALEPACK.49 Structure solution The structure was initially solved using the program ˚ . The Solve50,51 and the MAD data between 25 and 4.5 A ˚ on the resolution of the analysis was limited to 4.5 A basis of a comparison of the correlations between the anomalous signals at the different wavelengths. These correlations dropped below 0.3 at resolutions ˚ for all wavelengths. Solve identified a higher than 4.5 A 40 site solution (four equivalent sites per subunit) and the associated program Resolve provided a densitymodified map in which a clear non-crystallographic 5-fold axis was identified that related five separate dimers of glycogenin. This solution was then used to generate phase estimates to the limit of the MAD data ˚ ) and these phases were transferred, in the form of (3.8 A ˚ Hendrickson – Lattmann coefficients, to the native 3.4 A data set using the programs in the CCP4 suite.52 The program DM, as implemented in CCP4, was used in conjunction with the non-crystallographic symmetry (NCS) operators identified in the MAD map to perform tenfold NCS-averaging, solvent flipping and phase extension ˚ to 3.4 A ˚ using a 100-cycle procedure to obtain from 4.5 A ˚ electron density map. an interpretable 3.4 A Model building and refinement The initial model building into the experimentally ˚ map was performed using the program phased 3.4 A O53 and produced a model containing residues 1 – 231 and residues 241– 267 for a single averaged subunit of the decamer. Refinement of this model using the program CNS27 utilized strict tenfold NCS-constraints and included a single round of torsion-angle dynamics and two iterative rounds of model adjustment and positional minimization. The final decameric contents of the asymmetric unit for coordinate deposition were generated using the NCS-operators. At this point, a single round of positional minimization with tenfold NCS-restraints assigned at 500 kcal/mol (1 cal ¼ 4.184 J) for all atoms was performed and a temperature factor was refined for each residue. No evidence for non-identity between subunits was observed at this resolution. The structure of the orthorhombic crystal form was solved by molecular replacement in CNS using a single subunit from the ˚ structure as the search model. Each form of refined 3.4 A the enzyme solved in the orthorhombic form was refined to the resolution limit of the data and solvent molecules were added independently to each structure as determined from difference Fourier syntheses and local hydrogen bonding geometry using CNS. Protein Data Bank accession number The coordinates for the structure of glycogenin have been deposited in the Protein Data Bank (1LL0, 1LL2, 1LL3).

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Acknowledgments This work was supported by the National Institute of Health (RO1-DK27221) and by the Indiana University Diabetes Research and Training Center (DK20542-25). We thank Susan Carlson, Stephanie Lange, Steve Parsons, Dr Bart Pederson, Dr Wayne Wilson, and Jing Zhou for their contributions to this work. Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Energy Research, under Contract no. W-31-109-ENG-38. We specifically acknowledge the support of the staff at the SBC beamlines 19-ID: Norma E. C. Duke, PhD, Stephan L. Ginell, PhD, Younchang Kim, PhD, and Andrzej Joachimiak, PhD. We thank the beamline staff at X12B at the Brookhaven National Laboratory National Synchrotron Light Source for expert technical assistance.

13.

14. 15.

16. 17. 18.

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Edited by D. Rees (Received 17 December 2001; received in revised form 29 March 2002; accepted 1 April 2002)