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Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity.

Buch MH, Liaci AM, O'Hara SD, Garcea RL, Neu U, Stehle T - PLoS Pathog. (2015)

Bottom Line: By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding.Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a.Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.

ABSTRACT
Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

No MeSH data available.


Related in: MedlinePlus

Binding of GT1a to PTA.A The PTA binding pocket and the GT1a conformation upon binding are shown from an angle parallel to the fivefold axis. A scheme of the glycan is shown in the inset, and the sugar rings are filled according to the coloring scheme from Fig 1. B Simulated annealing Fobs-Fcalc omit map (resolution 1.71 Å, calculated at 3.5 σ, carved 2.3 Å around the glycan). C Possible binding interactions of GT1a and PTA. E91 and V296 are highlighted in salmon. Hydrogen bonds are shown in black, the hydrophobic contact mediated by V296 in gold, and the van-der-Waals contacts of E91 are shown in cyan. Waters that mediate key hydrogen bonds are shown as red spheres. Unique interactions mediated by the novel GT1a-like binding motif are shown in red. D Zoomed view of the binding to the two terminal Neu5Ac moieties. The rest of the glycan is omitted for clarity. Residues except Y72 and R77 as well as waters involved in contacts with these two glycan moieties are pale grey and salmon, respectively.
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ppat.1005104.g004: Binding of GT1a to PTA.A The PTA binding pocket and the GT1a conformation upon binding are shown from an angle parallel to the fivefold axis. A scheme of the glycan is shown in the inset, and the sugar rings are filled according to the coloring scheme from Fig 1. B Simulated annealing Fobs-Fcalc omit map (resolution 1.71 Å, calculated at 3.5 σ, carved 2.3 Å around the glycan). C Possible binding interactions of GT1a and PTA. E91 and V296 are highlighted in salmon. Hydrogen bonds are shown in black, the hydrophobic contact mediated by V296 in gold, and the van-der-Waals contacts of E91 are shown in cyan. Waters that mediate key hydrogen bonds are shown as red spheres. Unique interactions mediated by the novel GT1a-like binding motif are shown in red. D Zoomed view of the binding to the two terminal Neu5Ac moieties. The rest of the glycan is omitted for clarity. Residues except Y72 and R77 as well as waters involved in contacts with these two glycan moieties are pale grey and salmon, respectively.

Mentions: In order to define the mode of recognition of GT1a, particularly to the naturally occurring PTA strain of MuPyV, we have soaked VP1 crystals with the glycan portion of GT1a and solved the structure of the complex (Table 2). While the receptor interaction pocket of RA VP1 has been described [11–13], no structural information for the pathogenicity-defining amino acids at positions 91 and 296 in the pockets of PTA and LID has been available. PTA and LID both carry a glutamate at position 91, and this side chain is being held in a characteristic position with the carboxyl group facing away from the glycan receptor due to a salt bridge formed with K186 (Fig 3), as previously predicted [12]. The GT1a glycan is a branched structure with a long disialylated arm, which has the sequence Neu5Acb-[α-2,8]-Neu5Aca-[α-2,3]-Gala-[β-1,3]-GalNAc, and a second short arm, which consists of a single Neu5Acd [α-2,3]-linked to Galb (for carbohydrate structures, nomenclature, and moiety indexing see Fig 1). The disialylated arm of GT1a is clearly visible in the crystal structure of PTA VP1; it is well defined by electron density and makes extensive contacts with the protein (Fig 4B–4D). Overall, the GT1a glycan adopts a twisted horseshoe-like shape, with Neu5Aca and Neu5Acb wrapping around the side chains of Y72 and R77 of VP1. Its longer, disialylated arm contains a Neu5Aca-[α-2,3]-Gala sequence that is also present in GD1a and simpler compounds such as 3’-sialyllactose (3SL), and the interactions of this motif with VP1 are essentially identical to those seen in previous structures [11–13]. However, our structure visualizes an additional network of contacts made by the terminal [α-2,8]-linked Neu5Acb (Fig 4C and 4D). Its carboxyl group engages Y72 and forms water-mediated hydrogen bonds with Q71, Y72, as well as D85 of the neighboring monomer (D85*). In addition, the N-acetyl nitrogen of Neu5Acb forms a hydrogen bond with the backbone carbonyl of T67, and O8 and O9 in the glycerol chain of the sugar are hydrogen-bonded with the R77 side chain. The carboxyl groups of Neu5Aca and Neu5Acb are about 4 Å apart, and the positively charged side chain of R77 counteracts their negative charges (Fig 4C and 4D). Neu5Aca and Neu5Acb contribute binding interfaces of approximately 160 Å2 and 190 Å2, respectively (calculated using the PISA server [31]). The remaining Gala-GalNAc-Galb stem of GT1a forms fewer contacts with the protein, which include a hydrogen bond between G78 and the Gala O4 hydroxyl group (Fig 4) as well as several van der Waals interactions. Notably, the Cβ and Cγ atoms of E91 are within van-der-Waals range of O6 and C6 of Gala, and the E91 carboxylate group is close to C6 of GalNAc. The total contact surface for this portion of the glycan is 142 Å2.


Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity.

Buch MH, Liaci AM, O'Hara SD, Garcea RL, Neu U, Stehle T - PLoS Pathog. (2015)

Binding of GT1a to PTA.A The PTA binding pocket and the GT1a conformation upon binding are shown from an angle parallel to the fivefold axis. A scheme of the glycan is shown in the inset, and the sugar rings are filled according to the coloring scheme from Fig 1. B Simulated annealing Fobs-Fcalc omit map (resolution 1.71 Å, calculated at 3.5 σ, carved 2.3 Å around the glycan). C Possible binding interactions of GT1a and PTA. E91 and V296 are highlighted in salmon. Hydrogen bonds are shown in black, the hydrophobic contact mediated by V296 in gold, and the van-der-Waals contacts of E91 are shown in cyan. Waters that mediate key hydrogen bonds are shown as red spheres. Unique interactions mediated by the novel GT1a-like binding motif are shown in red. D Zoomed view of the binding to the two terminal Neu5Ac moieties. The rest of the glycan is omitted for clarity. Residues except Y72 and R77 as well as waters involved in contacts with these two glycan moieties are pale grey and salmon, respectively.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608799&req=5

ppat.1005104.g004: Binding of GT1a to PTA.A The PTA binding pocket and the GT1a conformation upon binding are shown from an angle parallel to the fivefold axis. A scheme of the glycan is shown in the inset, and the sugar rings are filled according to the coloring scheme from Fig 1. B Simulated annealing Fobs-Fcalc omit map (resolution 1.71 Å, calculated at 3.5 σ, carved 2.3 Å around the glycan). C Possible binding interactions of GT1a and PTA. E91 and V296 are highlighted in salmon. Hydrogen bonds are shown in black, the hydrophobic contact mediated by V296 in gold, and the van-der-Waals contacts of E91 are shown in cyan. Waters that mediate key hydrogen bonds are shown as red spheres. Unique interactions mediated by the novel GT1a-like binding motif are shown in red. D Zoomed view of the binding to the two terminal Neu5Ac moieties. The rest of the glycan is omitted for clarity. Residues except Y72 and R77 as well as waters involved in contacts with these two glycan moieties are pale grey and salmon, respectively.
Mentions: In order to define the mode of recognition of GT1a, particularly to the naturally occurring PTA strain of MuPyV, we have soaked VP1 crystals with the glycan portion of GT1a and solved the structure of the complex (Table 2). While the receptor interaction pocket of RA VP1 has been described [11–13], no structural information for the pathogenicity-defining amino acids at positions 91 and 296 in the pockets of PTA and LID has been available. PTA and LID both carry a glutamate at position 91, and this side chain is being held in a characteristic position with the carboxyl group facing away from the glycan receptor due to a salt bridge formed with K186 (Fig 3), as previously predicted [12]. The GT1a glycan is a branched structure with a long disialylated arm, which has the sequence Neu5Acb-[α-2,8]-Neu5Aca-[α-2,3]-Gala-[β-1,3]-GalNAc, and a second short arm, which consists of a single Neu5Acd [α-2,3]-linked to Galb (for carbohydrate structures, nomenclature, and moiety indexing see Fig 1). The disialylated arm of GT1a is clearly visible in the crystal structure of PTA VP1; it is well defined by electron density and makes extensive contacts with the protein (Fig 4B–4D). Overall, the GT1a glycan adopts a twisted horseshoe-like shape, with Neu5Aca and Neu5Acb wrapping around the side chains of Y72 and R77 of VP1. Its longer, disialylated arm contains a Neu5Aca-[α-2,3]-Gala sequence that is also present in GD1a and simpler compounds such as 3’-sialyllactose (3SL), and the interactions of this motif with VP1 are essentially identical to those seen in previous structures [11–13]. However, our structure visualizes an additional network of contacts made by the terminal [α-2,8]-linked Neu5Acb (Fig 4C and 4D). Its carboxyl group engages Y72 and forms water-mediated hydrogen bonds with Q71, Y72, as well as D85 of the neighboring monomer (D85*). In addition, the N-acetyl nitrogen of Neu5Acb forms a hydrogen bond with the backbone carbonyl of T67, and O8 and O9 in the glycerol chain of the sugar are hydrogen-bonded with the R77 side chain. The carboxyl groups of Neu5Aca and Neu5Acb are about 4 Å apart, and the positively charged side chain of R77 counteracts their negative charges (Fig 4C and 4D). Neu5Aca and Neu5Acb contribute binding interfaces of approximately 160 Å2 and 190 Å2, respectively (calculated using the PISA server [31]). The remaining Gala-GalNAc-Galb stem of GT1a forms fewer contacts with the protein, which include a hydrogen bond between G78 and the Gala O4 hydroxyl group (Fig 4) as well as several van der Waals interactions. Notably, the Cβ and Cγ atoms of E91 are within van-der-Waals range of O6 and C6 of Gala, and the E91 carboxylate group is close to C6 of GalNAc. The total contact surface for this portion of the glycan is 142 Å2.

Bottom Line: By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding.Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a.Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.

ABSTRACT
Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

No MeSH data available.


Related in: MedlinePlus