Unbound structure of the GII.10 P domain.
The GII.10 P domain MBP fusion protein was expressed at a level of ~10 mg/liter in E. coli
. The cleaved GII.10 P domain formed rectangular plates that diffracted to better than 1.5-Å resolution (). A molecular replacement solution with the previously determined GII.4 P domain (4
) was obtained in space group P21
, with one P domain dimer in the asymmetric unit ( and B). Refinement of the GII.10 structure led to an Rwork
value of 0.151 (Rfree
= 0.167) and well-defined density for most of the P domain dimer (). Following the nomenclature established by Prasad and colleagues (30
), the GII.10 P1 subdomain was located between residues 222 to 277 and residues 427 to 549, whereas the P2 subdomain was between residues 278 and 426. The GII.10 P1 subdomain was formed primarily by a single α-helix, which was flanked by seven antiparallel β-strands (). The GII.10 P2 subdomain contained 12 antiparallel β-strands, 6 from each subunit, which formed 2 antiparallel β-sheets (). Overall, the secondary structure of the GII.10 P domains was highly reminiscent of previously published GI and GII structures (4
). On one of the asymmetric unit monomers, residues 344 to 351 (chain B) were disordered; these disordered residues were not modeled into the GII.10-apo structure.
Data collection and refinement statistics for structures of the GII.10 norovirus P domain alone and with various HBGAs
Unbound structure of the GII.12 P domain.
The GII.12 P domain MBP fusion protein was expressed at a level of ~2 mg/liter in E. coli. The cleaved GII.12 P domain formed rectangular parallelepipeds that diffracted to 1.75-Å resolution (). The GII.12 P domain structure was determined by molecular replacement with the GII.10 P domain; structure solution indicated that the space group was C2221, with one P domain monomer in the asymmetric unit (, with its monomeric P1 and P2 subdomain partners shown in green and cyan, respectively). Refinement of the GII.12 structure led to an Rwork value of 0.185 (Rfree = 0.203) and well-defined density for most of the P domain monomer (). The GII.12 P1 subdomain was located between residues 222 to 277 and residues 414 to 536, whereas the P2 subdomain was between residues 278 and 413.
Data collection and refinement statistics for structures of the GII.12 norovirus P domain alone and with triglycan HBGA type B
Comparisons of unbound structures of the GII.10, GII.12, GI.1, and GII.4 P domains.
Despite the great genetic diversity of noroviruses, the GII.4 strains have been responsible for the majority of outbreaks around the world over the past 10 or so years (25
). To examine whether the rare versus outbreak status had bearing on the overall structures, we compared rare and outbreak GII strains. The P domains from rare GII.10 and GII.12 were highly similar in structure, with a root mean square deviation (RMSD) for Cα atoms of 0.64 Å. However, in addition to their shared rare status, they were also more closely genetically related to each other than to the GII.4 outbreak strain. Pairwise analysis of RMSD differences in the P domain structures () found that the three GII P domain structures, two rare and one outbreak, were more similar to each other than to the GI structure. Overall structural differences thus appeared to reflect genetic distance (see Fig. S1A in the supplemental material) rather than rare or outbreak status.
Structures of HBGA H type 2-trisaccharide and -disaccharide bound to the GII.10 P domain.
HBGAs are a group of short oligosaccharides that are expressed in a polymorphic manner on cell surfaces or found as free antigens and have been shown through a number of studies, including the aforementioned crystallographic ones, to interact with norovirus () (11
). HBGAs are generated from a number of different precursor disaccharides, with additional saccharides added by enzymes, which are variably present in the human population (see Fig. S2 in the supplemental material) (22
). One distinction is made by the presence of α1,2fucosyltransferase, which adds a terminal αfucose1-2 unit; HBGAs with this saccharide are termed secretors, while those missing the terminal αfucose1-2 are termed nonsecretors.
Fig. 2. Surface comparisons of the GII.10 (PDB ID, 3ONU), GII.12, GII.4 (PDB ID, 2OBR), and GII.1 (PDB ID, 2ZL5) P domain dimer structures. The GII HBGA binding sites (black circles in panels A to C) involve a dimeric capsid interface that is formed primarily (more ...)
Because the GII.10 P domain protein was expressed to larger amounts and crystals diffracted to higher resolution than those of GII.12, we chose to examine first the GII.10 P domain by X-ray crystallography in complex with a panel of HBGAs (see Fig. S2 in the supplemental material) representing an assortment of secretor and nonsecretor HBGAs. The secretor HBGAs used were H type 2-disaccharide, H type 2-trisaccharide, A-trisaccharide, B-trisaccharide, Ley-tetrasaccharide, and Leb-tetrasaccharide, whereas the nonsecretor HBGAs used were Lea-trisaccharide and Lex-trisaccharide.
The HBGA H type 2-trisaccharide is α-l-fucose-(1-2)-β-d-galactose-(1-4)-2-N-acetyl-β-d-glucosamine, which is the first secretor in one of the major biosynthetic HBGA pathways (see Fig. S2 in the supplemental material). Cocrystallization of the GII.10 P domain with H type 2 resulted in P21 crystals that diffracted to 1.40 Å, with cell constants virtually isomorphous with those of the unbound crystals (). Structure solution and refinement with the unbound P domain resulted in a single clearly defined patch of electron density that spanned two P domain monomers ( and ). Placement of the trisaccharide was assisted by a well-defined fucose density, which led to an unambiguous orientation of this HBGA. Refinement led to an Rwork value of 0.169 (Rfree = 0.188) and well-defined density for all of the saccharide units (). No unassigned electron density was observed in the corresponding position of the HBGA on the P domain dimer, around the molecular 2-fold. Inspection of the lattice indicated a lattice contact at this position, which would occlude the presence of a second HBGA molecule (see Fig. S3A in the supplemental material).
Fig. 3. GII.10 P domain and H type 2 (trisaccharide and disaccharide) interactions. The H type 2-tri- and -disaccharide binding site is at the same location on the P domain and utilizes identical residues to bind the terminal αfucose1-2 saccharide. (A) (more ...)
The fucose showed the most well-defined density and was fixed by a network of P2 subdomain hydrogen bonds, two contributed by the side chain of Asp385, two by the side chain of Arg356, and one by the main chain of Asn355 (; see also Fig. S1B in the supplemental material). A sixth hydrogen bond was contributed from the backbone of Gly451 from across the P domain dimer interface, with the aromatic ring of Tyr452 packing over the fucose methyl. Both Gly451 and Tyr452 are located on a loop that extends from the P1 subdomain to form part of the P domain dimer interface (). Meanwhile, the galactose was fixed by one hydrogen bond, and the N-acetyl-glucosamine by three, contributed by a mix of backbone and side chain interactions, including Lys449 on the aforementioned P1-interface loop (; see also Fig. S1B).
To better understand H type 2 recognition, we also determined the structure of an H type 2-disaccharide [α-l-fucose-(1-2)-β-d-galactose] in complex with the GII.10 P domain (). The fucose appeared well ordered, but the galactose ring was substantially less well defined (). Apparently the single observed hydrogen bond to the galactose ring in the trisaccharide structure was not sufficient to fix the galactose in the disaccharide structure when not also sandwiched by an N-acetylglucosamine, as in the H type 2-trisaccharide ().
Overall, the unbound and H type 2-bound structures of the GII.10 P domain were virtually indistinguishable, except that in the bound structures, saccharides replace a number of surface waters. Within the bound H type 2 HBGAs, the primary interactions were observed to be through the terminal αfucose1-2 moiety, which was tightly held by both hydrophobic and hydrophilic interactions at the P domain dimer interface and involved the P1-interface loop from one monomer and the P2 subdomain from another monomer ( and ).
Structure of HBGA Ley-tetrasaccharide bound to the GII.10 P domain.
The Ley-tetrasaccharide HBGA is α-l-fucose-(1-2)-β-d-galactose-(1-4)-2-N-acetyl-β-d-glucosamine-(3-1)-α-l-fucose, which is the product of α1-3fucosyltransferase on H type 2-trisaccharide HBGA (see Fig. S2 in the supplemental material). Cocrystallization of the GII.10 P domain with Ley resulted in P21 crystals that diffracted to 1.48 Å, with cell constants virtually isomorphous with those of the unbound and H type 2-bound crystals (). Similar to the H type 2 structure described above, the Ley complex structure solution and refinement resulted in a single patch of electron density, which overlapped with the position of the αfucose1-2 in the H type 2 complex structure ( and ). The Ley-tetrasaccharide was tested in the following two orientations: either with αfucose1-2 or with αfucose1-3 placed in the P domain interface. Only the αfucose1-2 placement refined well. Refinement led to an Rwork value of 0.185 (Rfree = 0.204) and well-defined density for all of the saccharide units ().
Fig. 4. GII.10 P domain and Ley and Leb (tetrasaccharide) interactions. The complete Ley-tetrasaccharide easily fits into electron density and shows extensive hydrogen bonding interactions, whereas only αfucose1-2 of Leb can be fit into the observed electron (more ...)
As described for the H type 2 complex structures, the αfucose1-2 of Ley was fixed by a network of six hydrogen bonds, i.e., two by Asp385, two by Arg356, one by Asn355, and one by Gly451, and a Tyr452-hydrophobic interaction, (; see also Fig. S1B in the supplemental material). The galactose of Ley was fixed by one water-mediated hydrogen bond, the N-acetylglucosamine by two backbone hydrogen bonds, and the terminal αfucose1-3 by a hydrogen bond to the side chain of Trp381. Interestingly, the positions of the saccharides, other than αfucose1-2, in Ley were quite different from those in H type 2 (). In Ley, the galactose kinks up away from the protein, the N-acetylglucosamine swivels closer to the protein, and the terminal αfucose1-3 ends up being positioned close to the location of the third saccharide (N-acetylglucosamine) from H type 2.
Fig. 5. Stereo views of H type 2/Ley and type A/B superposition. For H type 2 and Ley HBGAs, only fucose is positioned similarly, whereas for type A and B HBGAs, all saccharides are held in practically identical positions. (A) Stereo view of the H type 2 (cyan) (more ...) HBGA Leb-tetrasaccharide bound to the GII.10 P domain as a single ordered fucose.
The Leb-tetrasaccharide HBGA is α-l-fucose-(1-2)-β-d-galactose-(1-3)-2-N-acetyl-β-d-glucosamine-(4-1)-α-l-fucose, which is the product of α1-4fucosyltransferase on H type 1-trisaccharide HBGA (see Fig. S2 in the supplemental material). Cocrystallization of the GII.10 P domain with Ley resulted in C-centered orthorhombic crystals that diffracted to 1.85 Å, and structure solution with the unbound GII.10 P domain structure revealed the crystals to be in space group C2221, with three monomers of the P domain in the asymmetric unit (see Fig. S3B in the supplemental material). These three monomers formed the previously observed dimer, with the monomer arranged around a crystallographic 2-fold, so that it also formed the standard dimer.
Refinement to an Rwork value of 0.164 (Rfree = 0.189) revealed that the molecular dimer and the crystallographic dimer were virtually identical to each other (RMSD = 0.20 Å) and to the unbound dimer (RMSDs of 0.19 and 0.21 Å for the molecular and crystallographic dimer, respectively). Each of the three independent monomers contained a single somewhat poorly ordered αfucose1-2 (average B value of 49 Å2), held in place by the standard six hydrogen bonds that spanned between two P domain monomers (). Notably, other than this single fucose, no additional saccharides were observed ( and D).
Comparison of the structures of the H type 2-di- and -trisaccharide HBGAs indicated that without a third saccharide, the intervening galactose became partially disordered (compare Gal in and C). Moreover, examination of the differences between the Leb and Ley chemistries indicated that the differences of these two could be envisioned as a swapping of the chemistries around the critical third saccharide ring, such that the two hydrogen bonds which are made at the first and second positions of that ring in the well-ordered Ley-bound HGBA would be disrupted (compare GlcNAc in and D). Thus, while we could not rule out completely different potential orientations for the bound Ley HBGA, analysis of the other bound HBGAs indicated that only the αfucose1-2 of Leb could bind in a manner similar to that of Ley, consistent with the singly ordered fucose that was observed.
Structures of HBGA type A- and B-trisaccharides bound to the GII.10 P domain.
The type A-trisaccharide HBGA is α-l-fucose-(1-2)-β-d-galactose-(3-1)-2-N-acetyl-α-d-galactosamine,whereas the type B-trisaccharide HBGA is the same as type A, except for a terminal α-d-galactose instead of an N-acetylgalactosamine [α-l-fucose-(1-2)-β-d-galactose-(3-1)-α-d-galactose]. Both of these HBGAs have the H type 2-disaccharide as a precursor (see Fig. S2 in the supplemental material). Cocrystallization of the GII.10 P domain with types A and B also resulted in P21 crystals that diffracted to 1.48 and 1.28 Å, respectively (). Similar to the structures described above, type A and B complex structure solutions and refinements resulted in a single patch of electron density, which overlapped with the position of the αfucose1-2 in the H type 2 complex structures (). Placement of the αfucose1-2 of types A and B at the P domain interface allowed for the other two saccharides to be easily built into the remaining density. Refinement led to Rwork values of 0.178 and 0.167 (Rfree = 0.198 and 0.181) for type A and B bound structures, respectively, and well-defined density for all of the saccharide units ().
Fig. 6. GII.10 P domain and type A and B (trisaccharide) interactions. The GII.10 P domain interacts with type A and B HBGAs in virtually identical ways. (A) Close-up surface and ribbon representation of the GII.10 P domain showing the bound A-trisaccharide (yellow) (more ...)
In addition to the six hydrogen bonds described above, αfucose1-2 was fixed by another water-mediated hydrogen bond to Lys449 ( and D). In total, five hydrogen bonds were contributed by one monomer of the P2 subdomain (Asn355, Arg356, and Asp385), and two were contributed by the P1-interface loop on the other monomer (Lys449 and Gly451), which also contributed the Tyr452-hydrophobic interaction (see Fig. S1B in the supplemental material). For type A, the galactose was fixed by one backbone-mediated hydrogen bond to Gly451, and the N-acetylgalactosamine by two water-mediated hydrogen bonds to Glu382. For type B, interactions were virtually identical, with the α-d-galactose also fixed by two water-mediated hydrogen bonds to Glu382.
In contrast to H type 2 and Ley, types A and B bound in remarkably similar manners, with all atoms of fucose and galactose superimposing after alignment of P domain, with an RMSD of less than 0.01 Å ().
Nonsecretor HBGAs Lea- and Lex-trisaccharides were not observed to bind to the GII.10 P domain.
The HBGAs Lea-trisaccharide and Lex-trisaccharide are the product of the α1,3/4fucosyltransferase, which adds a terminal αfucose1-3/4 unit to the standard galactose-N-acetylglucosamine precursor. These HBGAs are termed nonsecretors because they lack a αfucose1-2 unit. Cocrystallization of these with the GII.10 P domain resulted in monoclinic crystals that diffracted to 1.40 and 1.43 Å for Lea and Lex, respectively, and molecular replacement and refinement revealed the standard P21 structure (), though in both cases, the patch of electron density was quite weak and no saccharide could be fitted (structures deposited without HBGA).
Structure of HBGA type B-trisaccharide bound to the GII.12 P domain.
Having determined structures of the GII.10 P domain with a panel of HBGAs, we next turned to the GII.12 P domain. Cocrystallization of the GII.12 P domain with the type B-trisaccharide HBGA resulted in C2221 crystals that diffracted to 1.60 Å (). Structure solution and refinement with the unbound GII.12 P domain resulted in a small patch of electron density, located at the P domain interface (). Refinement led to an Rwork value of 0.219 (Rfree = 0.237). The fucose appeared very well ordered, while the two other saccharides were less well defined (). The fucose was held in place by the standard six hydrogen bonds that spanned between two P domain monomers (). However, in the case of GII.12, a main-chain hydrogen bond from cysteine (Cys345) replaced the GII.10 main-chain hydrogen bond from asparagine (Asn355).
Fig. 7. GII.12 P domain and B-trisaccharide interaction. The GII.12 P domain binds αfucose1-2 of type B HBGA with hydrogen bonds similar to those of GII.10, except that the carbonyl of Cys345 replaces that of Asn355. (A) Close-up surface and ribbon representation (more ...) Conservation of the HBGA binding motif in GII noroviruses.
The structure of the outbreak GII.4 (VA387) strain of norovirus previously determined with HBGA type A- and B-trisaccharides closely resembles the GII.10 and GII.12 norovirus structures with HBGAs described here. Taken together, they reveal a coherent picture of HBGA recognition, dominated by αfucose1-2 binding, as observed by Tan et al. (41
Of the 13 potential hydrogen bonds made by a terminal fucose, 6 are made by all 3 GII P domains in all 9 different HBGA P domain structures. These six, which are located in almost exactly the same places in all HBGA-bound structures, consist of five from a P2 subdomain and one from the P1-interface loop on another P domain monomer ( to C). These extensive contacts are quite specific for αfucose1-2, with αfucose1-3 unable to fit. The GII.10 and GII.4 interactions are further strengthened by a hydrophobic contact with the side chains of Tyr452 and Tyr443 on the P1-interface loop, respectively. Saccharides other than αfucose1-2 are attached in diverse ways, held in place by a rotating cast of surface residues.
To identify regions of high/low structural conservation, the six structures of GII.10 bound to different HBGA were further analyzed. Per-residue nonhydrogen atoms RMSDs were computed for each pair of structures, and the average RMSD among all structure pairs for each residue was obtained. The RMSD values for the GII.10 binding site residues were then compared to the RMSD values of nonbinding site residues, with a range of solvent accessibility cutoffs. In all cases, residues interacting with the different HBGAs were more conserved structurally as opposed to nonbinding site residues, though the average RMSD values were generally low for both sets of residues (see Fig. S4 in the supplemental material).
Sequence conservation of GII noroviruses and comparison with GI noroviruses.
The conserved GII recognition of HBGAs requires conservation of interacting residues. To understand the effect on sequence conservation engendered by this conserved recognition, we aligned a panel of GII norovirus sequences onto the atomic-level structures of GII.10 norovirus and analyzed conservation of surface residues relative to HBGA recognition. The residues on the surface of the P domain corresponding to the outer surface of the capsid were substantially less conserved than the inward facing surface residues (). On the outer facing surface, two major regions of high conservation were observed. These overlapped with the two dimer-equivalent regions that interact with αfucose1-2 of the HBGA (, middle, and B). Notably, the residues forming the surface of the P domain that interacts with the peripheral saccharides were generally less conserved than the αfucose1-2-interacting residues (see Fig. S5 in the supplemental material). Thus, the structure-function relationships involved in HBGA recognition appear to be reflected in the conservation of the GII norovirus surface residues.
Fig. 8. Surface representations of GII amino acid conservation and putative site of vulnerability for GII noroviruses. Antigenic diversity of noroviruses is seen primarily on the outermost surface of the capsid, although patches of conservation on the top surface (more ...)
To test whether this conservation was indeed a reflection of HBGA recognition, we aligned a panel of GI norovirus sequences (10
) onto the previously determined structures (6
) of GI.1 norovirus in complex with the HBGA type A and type H saccharides. The residues forming the surface of the GI P domain corresponding to the outer surface of the capsid were also substantially less conserved than the inward facing surface residues (see Fig. S6 in the supplemental material). On the outer facing surface, two regions of high conservation were observed. These overlapped with the dimer-equivalent regions on each monomer that interact with the HBGAs (Fig. S6). Notably, the surface patch formed by conserved residues in the GI noroviruses was in a different location than the patch in the GII noroviruses. In both cases, the sites of sequence conservation related to the regions involved in HBGA recognition, which is in agreement with previous observations (4
). Thus, the structure-function relationships involved in HBGA recognition appear to be reflected in surface-residue conservation for both GI and GII noroviruses.
The region of high conservation on the GII.10 outer facing surface included an additional residue, His358, which was not part of the identified HBGA binding sites (see Fig. S7 in the supplemental material). In our structures and in the GII.4 structures determined previously (4
), this residue was observed to make a potential hydrogen bond with the side chain of Asp385. The conservation of both Asp385 and His358 suggests that these two residues form a hydrogen bonding network that may be essential for HBGA binding of GII viruses. Due to its solvent exposure and adjacency to the fucose-binding site residues, it may be possible for His358 to also participate in direct binding interactions with some HBGAs. Likewise for GII.12, His348 (GII.12 numbering) was observed to form a similar hydrogen bond with the side chain of Asp375 (data not shown).