Structural basis of GM1 recognition by simian virus 40

Ursula Neu*, Karin Woellner†, Guenter Gauglitz†, and Thilo Stehle*‡§

*Interfaculty Institute for Biochemistry, University of Tu¨bingen, Hoppe-Seyler-Strasse 4, D-72076 Tu¨bingen, Germany; †Institute of Physical and Theoretical Chemistry, University of Tu¨bingen, Auf der Morgenstelle 8, D-72076 Tu¨bingen, Germany; and ‡Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232

Edited by Stephen C. Harrison, Children’s Hospital Boston, Boston, MA, and approved January 23, 2008 (received for review October 30, 2007) Simian virus 40 (SV40) has been a paradigm for understanding class I molecules (8, 9), these are not endocytosed with the virus attachment and entry of nonenveloped viruses, viral DNA replica- (10). By contrast, engagement of leads to virion tion, and virus assembly, as well as for endocytosis pathways uptake via cholesterol-dependent endocytosis and transport to associated with caveolin and cholesterol. We find by array the endoplasmic reticulum (ER), an essential step on the infec- screening that SV40 recognizes its ganglioside receptor GM1 with tious route (11–14). SV40 uses ganglioside GM1, whereas BKV a quite narrow specificity, but isothermal titration calorimetry binds GD1b and GT1b, and Polyoma attaches to GD1a and shows that individual binding sites have a relatively low affinity, GT1b (Fig. 1) (6, 7). In simians, the natural hosts of SV40, GM1 with a millimolar dissociation constant. The high-resolution crystal contains a terminal ␣-5-N-glycolyl- (NeuNGc), structure of recombinantly produced SV40 capsid protein, VP1, in whereas in humans, who are unable to synthesize this , complex with the portion of GM1, reveals that the ␣-5-N-acetyl-neuraminic acid (NeuNAc) is found at the equiv- receptor is bound in a shallow solvent-exposed groove at the outer alent position (reviewed in ref. 15). Both GM1 variants can serve surface of the capsid. Through a complex network of interactions, as receptors for SV40, but the presence of NeuNGc considerably VP1 recognizes a conformation of GM1 that is the dominant one in increases binding (16). Structural studies with Polyoma (17–19) solution. Analysis of contacts provides a structural basis for the reveal that the VP1 protein binds the portion of observed specificity and suggests binding mechanisms for addi- gangliosides in shallow surface pockets on top of the pentamer, tional physiologically relevant GM1 variants. Comparison with corresponding to the outer edge of the capsid. This surface of (Polyoma) receptor complexes reveals that VP1 is formed by the loops connecting ␤-strands B and C SV40 uses a different mechanism of sialic acid binding, which has (BC-loop), D and E (DE-loop), and H and I (HI-loop). Unlike implications for receptor binding of human polyomaviruses. The the well conserved VP1 core structure, these loops exhibit SV40–GM1 complex reveals a parallel to toxin, which uses considerable sequence variability among polyomaviruses, ac- a similar cell entry pathway and binds GM1 in the same counting for their different receptor specificities. conformation. Here we present a structure–function analysis of GM1 binding to SV40. Affinity-binding data and glycan array results show a crystal structure ͉ glycan array ͉ polyomaviruses ͉ viral attachment ͉ highly specific interaction of intermediate affinity. We have protein–carbohydrate complex crystallized a complex between a SV40 VP1 pentamer and a GM1-derived oligosaccharide and determined its structure at iruses must attach to specific receptors on their host cells to 2.25-Å resolution. Our results provide a structural platform for Vinitiate entry, but receptor binding that is too tight prevents understanding the observed affinity and specificity for GM1 as viral progeny from spreading to new host cells. As a result, well as for NeuNGc-containing GM1 (NeuNGc-GM1). Further- attachment and release processes depend on precisely regulated more, they reveal an oligosaccharide-binding mechanism distinct contacts and affinities between viral proteins and their cognate from Polyoma and have implications for carbohydrate binding of ligands at the cell surface. human BKV and JCV. MEDICAL SCIENCES Simian virus 40 (SV40) and the closely related murine Poly- omavirus (Polyoma) belong to the polyomavirus family, a group Results of small nonenveloped DNA viruses. Both can transform cells in Carbohydrate Specificity of SV40 VP1. SV40 VP1 pentamers that culture and cause cancer in animals (1, 2) and are highly are unable to form capsids were produced by omitting both the homologous to the human BK and JC polyomaviruses (BKV and flexible N terminus and the C-terminal arm from the expression JCV, respectively). In the context of an impaired immune system, construct [supporting information (SI) Text]. To define the BKV and JCV infection can lead to kidney transplant loss or spectrum of that can be recognized by SV40 VP1, progressive multifocal leukoencephalopathy (3, 4). SV40 serves the protein was analyzed by glycan array screening (Fig. 1A). The as a paradigm for cholesterol-dependent endocytosis and as a array contained six and 258 synthetic physiologi- useful vector for gene transfer into eukaryotic cells. cally relevant carbohydrates. Although some binding could be The atomic structure of the complete SV40 virion has been determined by x-ray crystallography (5). The T ϭ 7d icosahedral Author contributions: U.N. and T.S. designed research; U.N. and K.W. performed research; capsid is constructed from 360 copies of the major structural G.G. contributed new reagents/analytic tools; U.N., K.W., and T.S. analyzed data; and U.N. protein VP1. Each VP1 monomer folds into a ␤-sandwich and T.S. wrote the paper. structure, termed jelly roll, that assembles with four other VP1 The authors declare no conflict of interest. monomers into a highly stable pentameric unit. A C-terminal This article is a PNAS Direct Submission. arm emerges from the base of each VP1 monomer, and part of ␤ Data deposition: The coordinates and structure-factor amplitudes have been deposited in this arm inserts into the -sheet of a VP1 molecule in a the RCSB Protein Data Bank (www.rcsb.org) with accession nos. 3BWQ (SV40 VP1) and neighboring pentamer, thus tying together the pentamers in the 3BWR (SV40 VP1–GM1 complex). capsid. §To whom correspondence should be addressed. E-mail: [email protected]. The functional receptors for SV40, Polyoma, and BKV are This article contains supporting information online at www.pnas.org/cgi/content/full/ gangliosides, complex sialic acid-containing (6, 7). 0710301105/DC1. Although SV40 can attach to major histocompatibility complex © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710301105 PNAS ͉ April 1, 2008 ͉ vol. 105 ͉ no. 13 ͉ 5219–5224 Downloaded by guest on September 28, 2021 such carbohydrates tested, SV40 VP1 exclusively recognized the oligosaccharide portion of ganglioside GM1, which was present twice on the array and which, for reasons of simplicity, will be referred to as GM1 throughout the remaining text. GM1 is a branched compound that contains two galactose (Gal) residues, a glucose (Glc) residue, an N-acetylgalactosamine (GalNAc) group, and a NeuNAc group that are linked in the following manner: Gal-(␤1,3)-GalNAc-(␤1,4)-[NeuNAc-(␣2,3)]-Gal- (␤1,4)-Glc. The array contained several GM1-related structures (Fig. 1B), but none of these interacted with SV40 VP1 in a detectable manner.

Affinity of GM1 for SV40 VP1. We used isothermal titration calo- rimetry to determine the affinity of one binding site of SV40 VP1 to GM1 (SI Fig. 5). Data from different measurements and data integration procedures yield dissociation constants between 1 and 5 mM; this scattering is due to the low observed affinity. Consistent with this result, concentrations of Ϸ5 mM were required to obtain complex by soaking VP1 crystals with GM1.

Overall Structure of the VP1–GM1 Complex. The structure of an SV40 VP1 pentamer bound to GM1 was solved at 2.25-Å resolution (Fig. 2, SI Table 1). The crystallized VP1 protein consists of amino acids 30–297, of which amino acids 43–297 are visible for all five chains in the final electron density map (SI Text). Each VP1 assumes the established ␤-sandwich fold, with two four-stranded antiparallel ␤-sheets (containing ␤-strands C,H,E,F and B,I,D,G) forming the core structure. The ‘‘unas- sembled’’ VP1 structure solved here is very similar to that of the assembled VP1 protein in the structure of the SV40 capsid (5). The rmsd between the two proteins is 1.2 Å for the C␣ atoms of residues 45–297. Three VP1 monomers bound ligand; access to the GM1- binding sites of the remaining two VP1 proteins is blocked by crystal contacts with neighboring VP1 pentamers. We solved VP1 structures with and without ligand at a similar resolution (SI Table 1). They can be superposed with an rmsd of 0.4 Å (C␣ atoms), indicating that no major conformational changes occur upon ligand binding.

Structure of GM1. The GM1 ligand used in this study contains five carbohydrate residues (Figs. 1B and 2B). Its overall shape resembles the letter ‘‘Y,’’ with the Gal-(␤1,4)-Glc moiety form- ing the stem and the NeuNAc and Gal-(␤1,3)-GalNAc moieties forming the two branches. The oligosaccharide lacks the mem- brane-anchoring , which would be attached to the terminal Glc in the GM1 ganglioside. GM1 has essentially the same conformation in all three binding sites, and all five sugar moieties are well defined by electron density (Fig. 2B). The conformation of GM1 in the complex is very similar to its Fig. 1. Specificity of SV40 VP1 for GM1. (A) Analysis of specificity by glycan conformation in solution (20), indicating that SV40 VP1 does not array screening. Glycan array data are given as mean fluorescence signal for each glycan (see Materials and Methods). Error bars correspond to the stan- induce a major structural change in its ligand. dard error of the mean of six replicates for each glycan, with the highest and lowest signals omitted from analysis to reduce bias from extreme values. The Interaction of VP1 with GM1. The GM1 ligand binds to a shallow signals on the array either come from glycoproteins (blue) or from synthesized groove formed by the BC-, DE-, and HI-loops, all of which carbohydrates (red). (B) Schematic view of gangliosides and related structures emanate from the ␤-sandwich at the outer surface of VP1. For present on the array. GM1 is highlighted with a black box, and selected reasons of clarity, the BC-loop is further subdivided here into gangliosides not included in the array but discussed in the text are shaded two consecutive loops, BC1 and BC2, that face in different gray. directions (Fig. 2B). The BC2- and DE-loops of the clockwise and counterclockwise VP1 neighbors, respectively (viewed from the outside of the virion), complete the binding site at each end detected for transferrin and ceruloplasmin, the observed signal (Fig. 3A). Both branches of GM1 make extensive contacts with could not be attributed to a specific carbohydrate structure. The VP1, and their temperature factors are in accordance with those most likely reasons for this are the heterogeneous glycosylation of surrounding amino acids. The Gal-(␤1,4)-Glc stem, which of glycoproteins and unspecific protein–protein interactions. would be attached to the ceramide and anchored into the The binding signal for synthetic is considered less membrane in a physiological setting, faces away from the protein ambiguous, because defined glycans are evaluated. Of the 258 and does not make any contacts with VP1. The stem exhibits

5220 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710301105 Neu et al. Downloaded by guest on September 28, 2021 3B). The methyl group of the N-acetyl chain partially inserts into a deep cavity that is lined with the hydrophobic side chains of Phe-270 and Leu-65 from one monomer as well as Phe-75 from the clockwise neighbor (Fig. 3B). The amide nitrogen of the N-acetyl chain is hydrogen bonded to OD1 of Asn-272 on the polar rim of the cavity. The glycerol side chain lies in a shallow groove on the surface. It points toward the aliphatic portion of the Lys-67 side chain and makes extensive polar interactions with its three hydroxyl groups (at O7, O8, and O9) and residues in the BC1- and BC2-loop. On one side, the O7 hydroxyl makes van der Waals interactions with Gln-62, whereas on the other side, the hydroxyl group of Ser-68 can hydrogen bond to the O8 or the O9 hydroxyl of NeuNAc. There is some ambiguity to this interaction, because the Ser-68 side chain assumes two alternative confor- mations. The side chain of Gln-278 points upward toward NeuNAc and forms the bottom of the binding pocket. It orga- nizes a network of hydrogen bonds that lie beneath the NeuNAc moiety, thereby helping to orient other amino acids for binding (Fig. 3B). The Gal-(␤1,3)-GalNAc arm of GM1 interacts with residues in the HI- and BC2-loops and also contacts the DE-loop of the counterclockwise neighboring monomer (Fig. 3C). The terminal Gal is held in place by hydrogen bonds to Ser-68 and Gln-84 in the BC-loop as well as van der Waals interactions with Ala-70 in the BC-loop and Asn-138 in the DE-loop of the counterclock- wise neighbor. The N-acetyl group of the GalNAc residue contacts the hydrophobic portions of the Ser-274 and Thr-276 side chains. Discussion Affinity of Binding. The structure of the complex between SV40 VP1 and GM1, presented here at 2.25-Å resolution, shows that the virus recognizes its ganglioside receptor via a complex network of interactions. Isothermal titration calorimetry reveals a relatively low affinity of one binding site on VP1 for its ligand. However, affinities in the millimolar range have been observed for several other viral attachment proteins that interact with oligosaccharide receptors, such as Polyoma VP1 or (18, 21). A lower binding affinity is thought to aid Fig. 2. Structure of the SV40 VP1–GM1 complex. (A) Overvall structure. The virus spread by facilitating release from the cell surface after five VP1 chains are shown as ribbon tracings, with one VP1 monomer high- lysis. Moreover, the affinity of intact SV40 particles for GM1 lighted in blue and the others colored gray. Bound GM1 is shown in stick would likely be much higher, because many binding sites could representation, with carbons drawn in orange, oxygens in red and nitrogens be engaged simultaneously. in blue. The GM1 molecules bind on top of the VP1 pentamer, corresponding MEDICAL SCIENCES to the capsid surface. (B) Composite annealed omit difference electron density Ligand Specificity. Glycan screening demonstrates that binding to ␴ map for GM1, calculated at 2.25-Å resolution, contoured at 2.5 and displayed GM1 occurs with narrow specificity. that con- 4 Å around GM1. One VP1 monomer is colored blue, the clockwise (cw) and counterclockwise (ccw) neighbors are colored gray. tain similar structural motifs in different contexts do not interact with SV40 VP1. Analysis of the crystal structure confirms these findings: addition of carbohydrate residues to either one of the substantially higher temperature factors, indicating higher branches would lead to steric clashes with protein residues. Both mobility. branches of GM1 are required for interaction with VP1. NMR The NeuNAc and Gal-(␤1,3)-GalNAc branches of GM1 re- studies show that GM1 assumes one dominant conformation in ␤ ␣ semble a bridge that leads over a ridge on the VP1 surface solution (20). The GalNAc-( 1,4)-[NeuNAc-( 2,3)]-Gal portion separating the binding sites for the NeuNAc moiety and the of the oligosaccharide forms a rigid entity because of close terminal Gal (Fig. 3 A and C). The ridge is formed primarily by contacts between the GalNAc and NeuNAc moieties, which are attached to neighboring carbons on the Gal. This arrangement the fully extended side chain of Lys-67, which is stabilized by a severely limits the rotational freedom of all three sugar residues. salt bridge with Asp-81 at the end of the BC2-loop (Fig. 3C). 2 By contrast, unbranched oligosaccharides are more likely to The VP1–GM1 interactions bury a total area of 404 Å from adopt different conformations. This is the case, for example, for solvent, 60% of which is contributed by NeuNAc. the linear ␣2,3-sialyllactose, whose conformation has been stud- The NeuNAc arm of GM1 is contacted by residues from the ied both in solution and complexed to different protein ligands HI- and BC1-loops from one monomer, as well as residues from (17, 22, 23). Because the conformation of GM1 in the VP1 the BC2-loop of the clockwise neighbor. The interaction features complex is very similar to its principal structure in solution, we a marked shape complementarity between all three protruding think it likely that interactions of one branch facilitate interac- functional groups (carboxyl, N-acetyl, and glycerol chains) of tions of the other branch with VP1 residues, and that this bivalent NeuNAc and sites on VP1 that accommodate each group (Fig. binding mode is a foundation for the high specificity of SV40 for 3A). The negatively charged carboxyl group points toward the GM1. HI-loop and forms hydrogen bonds to Ser-274 and Thr-276 (Fig. Glycan array screening also detected binding signals for two

Neu et al. PNAS ͉ April 1, 2008 ͉ vol. 105 ͉ no. 13 ͉ 5221 Downloaded by guest on September 28, 2021 Fig. 3. Interactions between SV40 VP1 and GM1. (A) Surface architecture of VP1 and shape complementarity with GM1. Protein chains are colored using the code in Fig. 2. (B) Contacts between NeuNAc and SV40 VP1. (C) Contacts between Gal-(␤1,3)-GalNAc and SV40 VP1. In B and C, only residues that contact the sugar are shown in stick representation. Hydrogen bonds are shown as broken lines. The cavity between one monomer of VP1 and its clockwise neighbor is shaded gray.

glycoproteins. However, we consider these to be nonspecific surface of VP1. However, the actual ligand structures differ: because of infection studies with Vibrio cholerae neuraminidase- whereas SV40 recognizes a ganglioside that carries a terminal treated cells (24). Although the NeuNAc in GM1 is protected NeuNAc residue on one branch only (GM1), Polyoma binds to from cleavage by this neuraminidase (25), the terminal sialic compounds that carry either two (GD1a) or three (GT1b) acids on glycoproteins are cleaved. Because neuraminidase NeuNAc residues distributed over both branches (Fig. 1B). treatment did not abolish infection, the glycans on glycoproteins Nevertheless, Polyoma recognizes only the NeuNAc-(␣2,3)-Gal are probably dispensable for binding. structure on the longer branch, in which the NeuNAc is linked to an unbranched Gal (7, 17–19). Interaction of SV40 with Its Receptor in Simians, NeuNGc-GM1. The In both SV40 and Polyoma, a terminal ␣2,3-linked NeuNAc glycan screen did not include more subtly modified GM1 ana- serves as a major contact point, and in both cases, VP1 residues logues, such as those containing substituted neuraminic acids. at equivalent locations are used for interactions with NeuNAc However, it was recently shown that SV40 binds to NeuNGc- (Fig. 4 A and B). Despite these similarities, the NeuNAc moiety GM1 more strongly than to NeuNAc-GM1, indicating that is bound in different orientations by the two proteins. In NeuNGc-GM1 is the natural receptor of this virus (16). Polyoma, the NeuNAc carboxylate faces away from the fivefold The N-acetyl group of NeuNAc faces toward a deep oval axis of the pentamer, forming a key salt bridge with Arg-77, and cavity, which is formed by the BC1-loop of one subunit and the the glycerol side chain points away from the virion into solution BC2-loop of its clockwise neighbor (Fig. 3B). The cavity is lined (Fig. 4 B and D). By contrast, the glycerol chain of NeuNAc in by both polar and hydrophobic residues. The NeuNGc variant GM1 faces toward VP1, whereas its carboxylate group faces carries a CH2–OH group instead of a CH3 group on the sialic acid toward the fivefold axis and does not engage in a salt bridge (Fig. amide group. The additional hydroxyl group of the NeuNGc 4 A and C). Lys-67, which is the SV40 residue equivalent to glycolyl chain does not alter the overall conformational prop- Arg-77 in Polyoma, does not directly contact NeuNAc but erties of GM1 (26), and a GM1 receptor carrying a terminal instead forms the ridge that separates the binding pockets for the NeuNGc residue could be accommodated in our structure two branches of GM1. The conformation of Arg-77 in Polyoma without requiring any alteration of either the protein or the is stabilized by Gln-59 and Tyr-72, whereas Lys-67 in SV40 is ligand. The glycolyl moiety would insert further into the cavity, held in place by a salt bridge to Asp-81. The conformation of most likely pointing toward the polar residues at its rim. There NeuNAc and Gal in the Polyoma binding site does not support are two possible energetically favorable conformations of the a branch at the axial O4 of the Gal residue, whereas this branch CH2–OH group, both of which would result in additional hy- and the rigid conformation it imposes are recognized by SV40. drogen bond formation and stabilizing van der Waals interac- Thus, residues at equivalent positions in the sequence and in tions. Because these interactions would occur in a partly hydro- space have different contacts in SV40 and Polyoma, leading to phobic environment, they would nicely explain the observed different surfaces and ligand specificities. stronger interaction between SV40 and NeuNGc-GM1 com- pared with NeuNAc-GM1 (16). Although it is, of course, pos- Implications for Receptor Binding of Human BKV and JCV. Both JCV sible that NeuNGc-GM1 and NeuNAc-GM1 have totally differ- and BKV attach to sialic acids (27, 28), and their VP1 proteins ent modes of binding, we consider this possibility unlikely. are highly homologous in sequence to SV40 VP1. Although the The binding mode for NeuNGc-GM1 postulated here differs Polyoma and SV40 VP1 surface loops differ in length and from a recently published model of a NeuNGc–GM1 complex sequence, the surface loops of BKV, JCV and SV40 VP1 all have with SV40 (16). The published model, which is based on the the same length (Fig. 4E). Furthermore, 6 and 7 of the 10 known structures of SV40 virions and Polyomavirus–receptor NeuNAc-contacting residues of SV40 are identical in BKV and complexes (5, 19), makes the assumption that SV40 and Polyoma JCV, respectively, whereas none are identical in Polyoma. This bind the sialic acid portions of their ganglioside receptors in level of conservation suggests that SV40 can serve as a model for generally similar orientations. As discussed below, this assump- BKV and JCV, and that the three proteins will have similar tion is not correct. structures for their receptor-binding sites. We therefore propose that the human polyomaviruses bind NeuNAc in a similar Comparison with Polyoma VP1–Receptor Interactions. Polyoma and position and orientation as SV40. Furthermore, studies with cells SV40 VP1 both bind to ganglioside receptors (7), and in both expressing modified sialic acids showed that the chain attached cases the ligands are recognized at similar locations on the outer to the amide nitrogen of neuraminic acids contributes to BKV

5222 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710301105 Neu et al. Downloaded by guest on September 28, 2021 Fig. 4. SV40 and Polyoma bind their ganglioside receptors in different orientations and conformations. (A and B) Comparison of specific interactions between VP1 from SV40 (A) and Polyoma (B) with their ligands. Polyoma was crystallized with an oligosaccharide that contained a fragment also present in GD1a and GT1b (shown in color). The additional ␣2,6-linked NeuNAc present in the ligand, but not on a ganglioside receptor, is shown in gray. The same view was used for both proteins, and residues at equivalent positions in both proteins have the same color. The Arg-Gly motif of Polyoma and its SV40 counterpart are colored yellow, and the residues holding them in place are shown in green. Hydrophobic residues lining the deep cavity in SV40 and their charged Polyoma equivalents are turquoise. Additional residues contacting NeuNAc in both complexes are gray. (C and D) Different binding surfaces of SV40 (C) and Polyoma VP1 (D) for their ligands. The same view from top of the pentamer was used for both complexes, and the color scheme is as in A and B. The terminal Glc was omitted from the SV40 VP1-GM1 complex for clarity. (E) Alignment of the VP1 sequences of SV40, Polyoma, BKV, and JCV. A sequence alignment of SV40, BKV, and JCV VP1 was

performed with T-Coffee (www.ebi.ac.uk/t-coffee), then the aligned sequences were arranged according to a structure-based alignment of SV40 and Polyoma MEDICAL SCIENCES (18). SV40 residues identical to both BKV and JCV are marked with an asterisk; those identical to either BKV or JCV are marked with a dot. The color scheme is as in A and B, with additional SV40 residues in contact with GM1 in gray.

attachment and suggest that it inserts into a pocket on VP1 (29). systems consist of a pentameric structure (VP1 in polyomavi- Sequence conservation suggests that JCV and BKV contain ruses, the B5 pentamer in toxins) that binds a monomeric protein cavities similar to the one in SV40. (VP2 in polyomaviruses, protein A in toxins) at its center (32, Although JCV can bind sialic acids on several , only 33); thus they both exhibit an unusual AB5 symmetry mismatch GT1b can inhibit infection (27). In addition, JCV attachment (SI Fig. 6). Furthermore, both systems are thought to undergo and entry into human glial cells proceed via engagement of a conformational change during entry that exposes the central serotonin receptors (30). The closely related BKV can use monomeric protein and leads to membrane penetration. Finally, gangliosides GD1b and GT1b as receptors (6). Both GD1b and both systems use gangliosides as functional receptors. In fact, the GT1b share the rigid GalNAc-(␤1,4)-[NeuNAc-(␣2,3)]-Gal oli- receptor for is GM1, and a comparison of the cholera toxin B protein with SV40 shows that both bind GM1 in gosaccharide core structure of GM1 (Fig. 1B), and this part of essentially the same conformation (SI Fig. 6), which is the the carbohydrate has a structure similar to GM1 (31). Both principal conformation in solution (20, 32). Despite these sim- GD1b and GT1b carry an extra NeuNAc residue attached via an ␣ ilarities, there is no structural conservation of the individual 2,8-linkage to the glycerol chain of the NeuNAc found in GM1. interactions (SI Fig. 6 B and D), and cholera toxin has a much Adding a second NeuNAc in this manner to the NeuNAc bound higher affinity for GM1 (34). However, the many structural and by SV40 would not be possible due to steric clashes. functional similarities between the two systems suggest a distant link in evolution. Comparison with Cholera Toxin. Very few infectious agents enter cells via rafts and the ER. Bacterial toxins such as cholera Conclusions toxin or enterotoxin are known to use this pathway, and they Our structure–function analysis shows how SV40 VP1 interacts share some interesting properties with polyomaviruses. Both with its receptor GM1 and provides a platform for studies to

Neu et al. PNAS ͉ April 1, 2008 ͉ vol. 105 ͉ no. 13 ͉ 5223 Downloaded by guest on September 28, 2021 probe this interaction. The complex reveals several unexpected tained 264 glycans covalently linked to a chip by variable linkers in six repli- features. First, it exhibits a marked specificity, coupled to a cates each. rather modest affinity, of VP1 for GM1. Both can readily be explained by the structural data. Second, a partially filled pocket Crystallization and Structure Determination. SV40 VP1 (10.5–11.5 mg/ml) was crystallized by hanging-drop vapor diffusion against a reservoir of 100 mM in the binding site indicates that physiologically relevant modi- Tris, pH 8.5, and 24% PEG 3350 (wt/vol). For setting up the drops, the reservoir fied gangliosides can also bind to VP1 and probably with higher solution was mixed 4:1 with 30% ethylene glycol (vol/vol), and this was mixed affinity. Third, the structure demonstrates that the closely 1:1 with the protein. Crystals were harvested into 100 mM Tris, pH 8.5; 20% related SV40 and Polyoma VP1 proteins, which both bind PEG 3350 (wt/vol); and 6% ethylene glycol (vol/vol), successively soaked for ganglioside receptors, recognize terminal sialic acid residues in 5–30 s in harvesting solution containing 12.5% and 25% (vol/vol) glycerol as a cryoprotectant, and flash-frozen in liquid nitrogen. For complex formation, a different manner. Fourth, infections with human JCV and crystals were soaked for 16 min in harvesting solution containing 5 mM GM1 BKV severely affect transplant recipients and individuals with (GM1 oligosaccharide sodium salt, Alexis). Cryoprotectant solutions were also compromised immune systems due to HIV infection. An im- supplemented with 5 mM GM1. proved understanding of BKV and JCV attachment could Diffraction data were processed with HKL (HKL Research), and the structure provide a foundation for probing these interactions by mutagen- was solved by molecular replacement with AMoRe in CCP4 (35) using the VP1 esis and directed ligand-binding studies. We show here that SV40 core of the SV40 virion structure [1SVA (5)] as a search model. Structures were refined by alternating rounds of model building in Coot (36) and restrained can serve as a plausible model for the interactions of JCV and refinement, using fivefold noncrystallographic symmetry restraints, with Ref- BKV with their carbohydrate receptors. mac5 (35). After initial refinement of the native structure, GM1 was located in FsoakedϪFnative difference Fourier maps and refined with restraints from the Materials and Methods Refmac5 monomer library. Waters were located with Coot and arp࿝waters in Protein Expression and Purification. DNA coding for amino acids 30–297 of CCP4 (35). The final model of the complex has good stereochemistry and a low SV40 VP1 was cloned into the pET15b expression vector (Novagen) in frame Rfree value of 23.4% (SI Table 1) (37). Figs. 2–4 were prepared with PyMol with an N-terminal hexahistidine tag (His-tag) and a cleavage site. (DeLano Scientific). An expanded version of the methods used can be found in SI Text. The protein was overexpressed in Escherichia coli BL21(DE3) and purified by nickel affinity chromatography and gel filtration on Superdex-200. For crys- ACKNOWLEDGMENTS. We thank members of our laboratory, especially Dr. tallization, the tag was cleaved with thrombin before the gel filtration step, Pierre Schelling, for help and discussions. We thank Dr. David F. Smith at Core leaving the nonnative amino acids Gly-Ser-His-Met at the N terminus. H of the Consortium for Functional Glycomics (National Institutes of Health Grant GM62116) for glycan array screening. We also thank Dr. Clemens Glycan Array Screening. His-tagged VP1 pentamers (0.2 or 0.5 mg/ml) were Schulze-Briese at beamline X06SA of the SLS (Villigen, Switzerland) for assis- assayed by Core H of the Consortium for Functional Glycomics on its printed tance with data collection and Eleanor Dodson and Garib Murshudov at the University of York (York, U.K.) for advice on Refmac. We also gratefully array (Ver. 2.1) in 20 mM Tris, pH 7.5; 150 mM NaCl; 2 mM CaCl2; 2 mM MgCl2; acknowledge Dr. Robert Garcea (University of Colorado, Boulder) for his gift 0.05% Tween-20; 1% BSA; 1 mM DTT. After washing, bound protein was of a vector expressing SV40 VP1. This project was supported by the Deutsche detected with AlexaFluor-conjugated anti-His-tag . The array con- Forschungsgemeinschaft (SFB-685).

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