JVI Accepts, published online ahead of print on 26 November 2014 J. Virol. doi:10.1128/JVI.02968-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

1

Title:

2

Human Noroviruses’ Fondness of Histo-Blood Group Antigens

3 4

Authors:

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Bishal K. Singh1,2, Mila M. Leuthold1,2, and Grant S. Hansman1,2,*

6 7

Affiliations:

8

1

9

Heidelberg 69120

Schaller Research Group at the University of Heidelberg and the DKFZ, Germany,

10

2

11

Heidelberg 69120

Department of Infectious Diseases, Virology, University of Heidelberg, Germany,

12 13

*Corresponding author

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CHS Foundation, University of Heidelberg, and German Cancer Research Center.

15

Norovirus Study Group.

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Im Neuenheimer Feld 242, Heidelberg 69120, Germany.

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Email: [email protected]

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Phone: +49 (0) 6221-1520

19 20

Abstract:

21

Human noroviruses are the dominant cause of outbreaks of gastroenteritis around the

22

world. Human norovirus interacts with the polymorphic human histo-blood group

23

antigens (HBGAs) and this interaction is thought to be important for infection.

24

Indeed, synthetic HBGAs or HBGA-expressing enteric bacteria were shown to

25

enhance norovirus infection in B cells. A number of studies have found a possible

1

26

relationship between HBGA type and norovirus susceptibility. The genogroup II

27

genotype 4 (GII.4) noroviruses are the dominant cluster, evolve every other year, and

28

are thought to modify their binding interactions to different HBGA types. Here, we

29

show the high-resolution X-ray crystal structures of the capsid protruding (P) domains

30

from epidemic GII.4 variants in 2004, 2006, and 2012, co-crystallized with a panel of

31

HBGA types (H type 2, Lewis Y, Lewis B, Lewis A, Lewis X, A-type, and B-type).

32

Many of the HBGA binding interactions were found to be complex, involving capsid

33

loop movements, alternative HBGA conformations, and HBGA rotations. We showed

34

that a loop (residues 391-395) was elegantly repositioned in order to allow for Lewis

35

Y binding. This loop was also slightly shifted to provide direct hydrogen and water-

36

mediated bonds with Lewis B. We considered that the flexible loop modulated Lewis

37

HBGA binding. The GII.4 noroviruses have dominated outbreaks over the past

38

decade, which may be explained by their exquisite HBGA binding mechanisms, their

39

fondness for Lewis HBGAs, and their temporal amino acid modifications.

40 41

Importance:

42

Our data provides a comprehensive picture of GII.4 P domain and HBGA binding

43

interactions. The exceptionally high resolutions of our X-ray crystal structures

44

allowed us to accurately recognize novel GII.4 P domain interactions with numerous

45

HBGA types. We showed that the GII.4 P domain HBGA interactions involved

46

complex binding mechanisms that were not previously observed in norovirus

47

structural studies. Many of the GII.4 P domain HBGA interactions we identified were

48

negative in earlier ELISA-based studies. Altogether, we showed that the GII.4

49

norovirus P domains could accommodate numerous HBGA types.

50

2

51

One Sentence Summary:

52

Norovirus GII.4 P domains have elaborate HBGA binding mechanisms.

53 54

Introduction:

55

Human norovirus is responsible for most epidemic outbreaks of gastroenteritis. There

56

are still no antivirals or vaccines approved, despite their discovery over four decades

57

ago (1). Noroviruses are genetically and antigenically diverse (2), yet a single genetic

58

cluster (genogroup II genotype 4, GII.4) has dominated over the past decade. (3). The

59

GII.4 noroviruses evolve ~5% every year and are believed to have a mechanism that

60

allows the virus to evade the immune system or alter receptor binding profiles (4-6).

61

However, immunity to norovirus is still poorly understood (7).

62 63

Human norovirus interacts with histo-blood group antigens (HBGAs), which is

64

thought to be important for viral infections (8-11). A recent report showed, for the

65

first time, that human norovirus infects B cells and that HBGAs (synthetic or HBGA-

66

expressing enteric bacteria) can enhance the infection (12). HBGAs are also found as

67

soluble antigens in saliva and are expressed on epithelial cells. Genetic

68

polymorphisms in genes that controls their synthesis is known to provide an

69

intraspecies diversity (13). To date, based on the ABH- and Lewis-HBGA types, at

70

least nine different HBGAs were found to interact with human norovirus. Individuals

71

expressing the O type are thought to have a significantly higher infection rate than

72

other blood types (11). The GII noroviruses are thought to have a preference to

73

HBGAs in a strain dependent manner (14-19).

74

3

75

Expression of the norovirus capsid protein in insect cells results in the formation of

76

virus-like particles (VLPs) that are antigenically similar to native virions. The X-ray

77

crystal structure of the prototype (GI.1) norovirus VLP identified two domains, shell

78

(S) and protruding (P) domain (20). The S domain forms a scaffold surrounding the

79

viral RNA, whereas the P domain is thought to contain the determinants for cell

80

attachment and strain diversity. The P domain can be further subdivided into P1 and

81

P2 subdomains and each subdomain likely has unique functions. In this study, we

82

determined the X-ray crystal structures of P domains from three epidemic GII.4

83

variants in 2004, 2006, and 2012 in complex with a panel of HBGAs in order to

84

elucidate HBGA binding mechanisms. Our data showed that the GII.4 norovirus

85

bound numerous HBGA types and that binding involved complex interactions,

86

including P domain loop movements and alternative HBGA conformations.

87

Importantly, many of our new findings challenge previous ELISA-based studies and

88

revealed interactions that have not been recognized so far (4-6). Altogether, we

89

showed that the GII.4 noroviruses were capable of binding diverse HBGA types,

90

which may correlate with a larger proportion of the human population susceptible to

91

GII.4 infections.

92 93

Materials and methods:

94

Sequence analysis and expression and purification of P domain

95

The P domain amino acid sequences from four GII.4 variants in 1998, 2004, 2006,

96

and 2012 [termed VA387-1998 (PDBID 2OBS), Farm-2004 (Genbank accession

97

number

98

respectively, were aligned using Clustal X. Farm-2004, Saga-2006, and NSW-2012 P

99

domains (residues 224 to 538) were expressed in E. coli and purified as previously

JQ478408),

Saga-2006

(AB447457),

and

NSW-2012

(JX459908),

4

100

described (21). Briefly, the codon optimized P domains were cloned into a modified

101

expression vector pMal-c2X and transformed into BL21 cells for protein expression.

102

Transformed cells were grown in LB medium supplemented with 100 μg/ml

103

ampicillin for 4 hours at 37°C. Expression was induced with IPTG (0.75 mM) at an

104

OD600 of 0.7 for 18 h at 22°C. Cells were harvested by centrifugation at 6000 rpm for

105

15 min and disrupted by sonication on ice. A His-tagged fusion-P domain protein was

106

purified from a Ni- column (Qiagen) and digested with HRV-3C protease (Novagen)

107

overnight at 4°C. Cleaved P domains were separated on the Ni-column and dialyzed

108

in gel filtration buffer (25 mM Tris-HCl and 300 mM NaCl) overnight at 4°C. The P

109

domains were purified by size exclusion chromatography, concentrated to 3-7 mg/ml,

110

and stored in gel filtration buffer at 4°C.

111 112

Crystallization of norovirus P domains

113

Crystals were grown in a 1:1 mixture of protein sample and mother liquor for 2-6

114

days at 18°C. Farm-2004 P domain crystals were grown in 20% PEG3350 and 0.2 M

115

magnesium formate; Saga-2006 crystals were grown in 3 M sodium acetate (pH 6.9);

116

and NSW-2012 crystals were grown in 20% PEG3350 and 0.2 M sodium formate.

117

Farm-2004, Saga-2006, and NSW-2012 formed long rod-shaped crystals, diamond-

118

shaped crystals, or both diamond- and plate-shaped crystals, respectively. For the P

119

domain and HBGA complexes, we co-crystallized 30-60 molar excess of HBGAs

120

(Dextra, UK). Prior to flash freezing, crystals were transferred to a cryoprotectant

121

containing mother liquor, 30 molar excess of HBGAs, and 30% ethylene glycol or

122

glycerol. Unfortunately, we were unable to produce complex crystals for all P

123

domains and HBGAs; and soaking experiments with HBGAs produced crystals with

124

high mosaicity and/ or the crystals dissolved.

5

125 126

Data collection, structure solution, and refinement

127

X-ray diffraction data were collected at the European Synchrotron Radiation Facility,

128

France at beamline BM30A and ID23-1 and processed with XDS (22). Structures

129

were solved using molecular replacement in PHASER (23). Saga-2006 P domain was

130

determined by molecular replacement using the previously solved GII.10 P domain as

131

a search model (21). Saga-2006 P domain was then used for determining the

132

structures of Farm-2004 and NSW-2012 P domains. Farm-2004 P domain formed

133

crystals in space group P212121, while Saga-2006 and NSW-2012 were both solved in

134

space group C2. Structures were refined in multiple rounds of manual model building

135

in COOT (24) with subsequent refinement with PHENIX (25). The HBGAs were

136

added to the models at the final stages of structural refinement in order to reduce bias

137

during refinement. Structures were validated with Molprobity (26) and Procheck

138

(27). HBGA interactions were analyzed using Accelrys Discovery Studio (Version

139

4.1), with hydrogen bonding interactions distances between 2.4-3.5Å and

140

hydrophobic interactions distances between 3.4-4.5Å. Figures and protein contact

141

potentials were generated using PyMOL (Version 1.12r3pre). Atomic coordinates and

142

structure factors are deposited in the Protein Databank.

143 144

Results:

145

Structures of unliganded GII.4 P domains

146

Three globally important epidemic GII.4 noroviruses in 2004, 2006, and 2012 [termed

147

Farm-2004, Saga-2006, and NSW-2012 (also known as Sydney 2012), respectively]

148

were selected for P domain and HBGA binding analysis using X-ray crystallography

149

(Fig. 1). Most of the amino acid variations were observed in the P2 subdomains. Data

6

150

statistics for GII.4 P domain apo structures are provided in Table 1. The P1

151

subdomains comprised of residues 224-274 and 418-530, whereas the P2 subdomains

152

were between residues 275-417 (Fig. 2). Similar to other human noroviruses, the P1

153

subdomain comprised of β-sheets and one α-helix, while the P2 subdomain contained

154

six antiparallel β-strands that formed a barrel-like structure. Overall, the GII.4 P dimer

155

structures were similar, with a maximum RMSD of 0.52 (Fig. 2D). This result

156

corresponded well with the high sequence identities (93-95%) and the amino acid

157

alignment (Fig. 1). In order to follow GII.4 P domain evolution, amino acid changes

158

from an earlier GII.4 P domain (in 1997) were projected onto Farm-2004, Saga-2006,

159

and NSW-2012 P dimer surfaces (Fig. 3). Most amino acid changes were surface

160

exposed and ~50% of these became fixed over the years. The region immediately

161

beneath the HBGA binding pocket showed little variation, whereas the surrounding

162

regions only contained a few amino changes. This result suggested that the HBGA

163

pocket was stable and likely contained important functions.

164 165

Structure of 2006 GII.4 P domain H2-trisaccharide (H2-tri) complex

166

The HBGAs chosen for this study were involved in a primary biosynthetic pathway

167

and were previously analyzed in ELISA-based studies (8, 10, 11, 28-31). Data

168

collection and refinement statistics for P domain HBGA complex structures are

169

provided in Tables 2, 3, and 4. The H2-trisaccharide (H2-tri) contains a single ABH-

170

fucose moiety. Two H2-trisaccharides (H2-tri) bound to the Saga-2006 P dimer. The

171

electron density was well defined for all three saccharide units, indicting the HBGA

172

was firmly held by the P domain (Fig. 4). The H2-tri was held in place by a network

173

of hydrophilic and hydrophobic interactions at the dimeric interface (Figs. 5A and

174

5B). The fucose was held by six direct hydrogen bonds, two from the side chain of

7

175

Asp374, two from the side chain of Arg345, one from the main chain of Thr344, and

176

one from the main chain of Gly443. A hydrophobic interaction was provided from

177

Tyr444. These five amino acids (Thr344, Arg345, Asp374, Gly443, and Tyr444) were

178

the common set of residues involved in other GII HBGA binding interactions at this

179

“regular pocket” (21). The central galactose of H2-tri was held by two hydrogen

180

bonds from the side chain of Ser442, while the terminal N-acetylglucosamine was

181

held by one hydrogen bond from the main chain of Gly392. A number of water-

182

mediated interactions were also observed in Saga-2006 H2-tri.

183 184

Comparisons with other GII.4 H2-tri complex structure were not possible, since the

185

Saga-2006 H2-tri represented the first known GII.4 H2-tri complex structure.

186

Superposition of a GII.4 2004 P dimer H1 pentasaccharide structure (3SLN) on the

187

Saga-2006 H2-tri revealed that the first three saccharides were similarly positioned as

188

H2-tri on the P dimers (Figs. 5C and 5D). However, the N-acetylglucosamine in

189

Saga-2006 H2-tri was flipped 180° compared to the H1 pentasaccharide. The two

190

remaining saccharides of H1 pentasaccharide were raised off the P domain, though

191

not held by any residues (19). This result showed that the H type orientation was

192

variable among GII.4 variants; or the longer pentasaccharide and H type influenced

193

the binding orientation.

194 195

Structure of 2006 GII.4 P domain Lewisy-tetrasaccharide (Ley-tetra) complex

196

Lewisy-tetrasaccharide (Ley-tetra) contains both an ABH and a Lewis fucose moiety.

197

Two Ley-tetra bound to the Saga-2006 P dimer. The electron density was well defined

198

for all four saccharide units (Fig. 4). The Lewis-fucose of Ley-tetra bound at the

199

regular pocket and was held by the common set of residues (Figs. 6A and 6B). The N-

8

200

acetylglucosamine was held by one hydrogen bond from the side chain of Ser442, the

201

galactose was held by one direct hydrogen bond from the hydroxyl group of Tyr444.

202

The ABH-fucose was not held with any direct hydrogen bonds. Several P domain

203

water-mediated interactions with fucose and galactose were also observed. In order

204

for Ley-tetra to bind to Saga-2006, a loop (residues 391-394) was shifted from the apo

205

position to an alternative conformation (Fig. 6C).

206 207

Other GII.4 P domain Ley-tetra complex structures were not available, however

208

comparison of the GII.10 P domain Ley-tetra structure (21) revealed different Ley-

209

tetra orientations on the P domains (Figs. 6D and 6E). The Lewis-fucose of Saga-

210

2006 Ley-tetra bound at the regular pocket, whereas the ABH-fucose of GII.10 Ley-

211

tetra bound at the regular pocket. Also, the terminal saccharides of GII.10 Ley-tetra

212

were directed towards the center of the P dimer, while the terminal saccharides of

213

Saga-2006 Ley-tetra were leaning towards the edge of the P dimer. Interestingly,

214

Saga-2006 had fewer direct hydrogen bonds with Ley-tetra than GII.10 (6 and 10,

215

respectively). Together, these findings suggested that there was a Ley-tetra placement

216

constraint among the different GII P domains.

217 218

Structure of 2004 and 2006 GII.4 P domain Lewisb-tetrasaccharide (Leb-tetra)

219

complexes

220

Lewisb-tetrasaccharide (Leb-tetra) contains both ABH and Lewis fucose moieties. One

221

Leb-tetra bound to the Farm-2004 P dimer, whereas two Leb-tetra bound to Saga-2006

222

P dimer. The electron density was well defined for all four saccharide units (Fig. 4)

223

The ABH-fucose of Farm-2004 Leb-tetra bound at the regular pocket and was held by

224

the common set of residues (Figs. 7A and 7B). The galactose of Farm-2004 Leb-tetra

9

225

was held by one hydrogen bond from the side chain of Ser442, while the N-

226

acetylglucosamine was not held with any hydrogen bonds. The Lewis-fucose of

227

Farm-2004 Leb-tetra was held by one hydrogen bond from the side chain of Asp391

228

and one hydrogen bond from the main chain of Gly392. A similar set of direct

229

hydrogen bonds was found in the Saga-2006 Leb-tetra structure (Figs. 7C and 7D).

230

Several P domain water-mediated interactions with ABH- and Lewis-fucose were also

231

observed.

232 233

In order to better understand Leb-tetra binding interactions, we superpositioned chains

234

A and B of Farm-2004 apo and Farm-2004 Leb-tetra (Fig. 7E). The loop in chain A

235

interacting with the Lewis-fucose of Farm-2004 Leb-tetra (residues 391-394) was in a

236

suitable position to allow direct hydrogen bonds with the Lewis-fucose. The

237

equivalent loop in chain A of Farm-2004 apo was in a slightly different conformation.

238

This result suggested that the loop was re-positioned to support the Lewis-fucose

239

binding. The loop at the unoccupied HBGA binding site of Farm-2004 Leb-tetra

240

(chain B) had a similar conformation to the equivalent loop (chain B) of Farm-2004

241

apo. The reason the second Leb-tetra did not bind to Farm-2004 was not determined,

242

although steric hindrance from the neighboring molecule could have played a role as

243

previously discussed (21). Nevertheless, these results highlighted the complexity and

244

importance of the flexible loop in Leb-tetra binding.

245 246

Structure of 2006 GII.4 P domain Lewisa-trisaccharide (Lea-tri) complex

247

Lewisa-trisaccharide (Lea-tri) contains a single Lewis fucose moiety. The electron

248

density was well defined for the Lewis-fucose and less defined for the two other

249

saccharides, which indicted that these saccharides were only loosely held on the P

10

250

domain (Fig. 4). Two Lea-tri bound to the Saga-2006 P dimer. The Lewis-fucose

251

bound at the regular pocket and was held by the common set of residues (Figs. 8A and

252

8B). The N-acetylglucosamine was held by one hydrogen bond from the side chain of

253

Ser442. Galactose was held by two hydrogen bonds from the hydroxyl group of

254

Tyr444. Several P domain water-mediated interactions were also observed with all

255

three saccharides.

256 257

Other GII P domain Lea-tri complex structures have yet to be determined.

258

Superposition of Farm-2004, Saga-2006, and NSW-2012 GII.4 apo structures showed

259

that the conformations of the side chains that interacted with N-acetylglucosamine

260

and galactose (i.e., Ser442 and Tyr444) were similarly orientated. This result

261

suggested that Farm-2004 and NSW-2012 were also capable of Lea-tri binding (Figs.

262

8C and 8D), although further studies are required.

263 264

Structure of 2012 GII.4 P domain Lewisx-trisaccharide (Lex-tri) complex

265

Lewisx-trisaccharide (Lex-tri) contains a single Lewis fucose moiety. Two Lex-tri

266

bound to the NSW-2012 P dimer. The electron density was well defined for all three

267

saccharide units, which indicated that the HBGA was firmly held by the P domain

268

(Fig. 4). The Lewis-fucose bound at the regular pocket and was held by the common

269

set of residues (Fig. 9). The N-acetylglucosamine was held by two hydrogen bonds

270

with the side chain of Ser442, while the galactose was held by one hydrogen bond

271

from the hydroxyl group of Tyr444. Interestingly, the terminal galactose of one Lex-

272

tri, was held in two conformations, ~1.5Å shift (data not shown; see PBD). However,

273

this shift did not result in any additional binding interactions.

274

11

275

Previous GII P domain and Lex-tri complex structures have not yet been determined.

276

Nevertheless, the side chains that interacted with N-acetylglucosamine and galactose

277

were similarly orientated in all three GII.4 P domains (see Figs. 8C and 8D), which

278

suggested Farm-2004 and Saga-2006 may also bind Lex-tri.

279 280

Structure of 2006 and 2012 GII.4 P domain A-trisaccharides (A-tri) complexes

281

Two A-trisaccharides (A-tri) bound to both Saga-2006 and NSW-2012 P dimers. The

282

electron density was well defined for all three saccharide units (Fig. 4). The

283

orientations of the A-tri in Saga-2006 and NSW-2012 P domains were similar. Fucose

284

was held by the common set of residues, while the galactose and N-

285

acetylgalactosamine were not supported with any direct hydrogen bonds (Fig. 10).

286

Compared to other GII structures, the A-tri saccharide units were similarly orientated

287

on the P dimers (21).

288 289

Structure of 2004, 2006, and 2012 GII.4 P domain B-trisaccharides (B-tri)

290

complexes

291

Two B-trisaccharides (B-tri) bound to Farm-2004 (Figs. 11A and 11B), Saga-2006

292

(Figs. 11C and 11D), and NSW-2012 P dimers (Figs. 11E and 11F). The electron

293

density was well defined for all three saccharide units (Fig. 4). The fucose was held

294

by the common set of residues in all complex structures, while the central and

295

terminal galactose were not held with any direct hydrogen bonds (Fig. 11). Similar to

296

NSW-2012 Lex-tri, the terminal galactose of B-tri bound to Saga-2006 was held in

297

two conformations, ~1.5Å shift, and this resulted in several new water-mediated

298

interactions (Figs. 11C and 11D). Interestingly, the loop described earlier (residues

299

391-394; see Figs. 8C and 8D) was found in two different positions in the Saga-2006

12

300

B-tri structure. In one conformation, the loop was orientated as in the Saga-2006 apo

301

structure, while the alternative conformation was similarly positioned as Saga-2006

302

Ley-tetra structure (see Figure 6C). The loop movement did not result in any

303

additional binding interactions, but merely indicated that the loop had a preference for

304

at least two conformations. Comparing to other GII structures, the B-tri saccharide

305

moieties were similarly positioned on the P domains (21).

306 307

Protein contact potential

308

The protein contact potential was calculated on a panel of GII.4 P dimers in order to

309

better understand the temporal variations in surface charge that might alter

310

antigenicity and HBGA binding (Fig. 12). The region ahead of the regular pocket and

311

towards the center of the P dimer (binding sites of A and B types) remained virtually

312

unchanged, mostly negatively charged. The regions that contributed to binding

313

terminal saccharide moieties of Lewis HBGAs underwent a modification, i.e., from

314

small areas of negative and positive charge (in 1998) to large areas of mostly negative

315

charge. In this view, it appeared that the more recent GII.4 HBGA binding pocket

316

became more negatively charged.

317 318

Discussion:

319

There is considerable debate on norovirus GII.4 evolution and their corresponding

320

interactions with different HBGA types (4-6, 8, 11, 28-31). In this study, we

321

determined the X-ray crystal structures of three P domains from epidemic GII.4

322

variants in 2004, 2006, and 2012 with a panel of HBGAs. The exceptionally high

323

resolutions of our structures allowed us to accurately define HBGA interactions,

324

several of which were not previously determined for GII.4 P domains (i.e., H2-tri,

13

325

Ley-tetra, Lea-tri, and Lex-tri). A common set of conserved residues (i.e., Asp374,

326

Arg345, Thr344, Tyr444, and Gly443) firmly held both the ABH- and Lewis-fucose.

327

The GII.4 variants were capable of binding numerous Lewis HBGA types and we

328

discovered that the Lewis HBGA, particularly Ley-tetra and LeB-tetra, binding

329

mechanisms involved more complex interactions than A-tri and B-tri binding

330

interactions. A flexible loop (residues 391-395) on the P dimer appeared versatile and

331

acted like a helping hand with Lewis HBGA tetrasaccharides. In one example, the

332

loop was cleverly repositioned to allow Ley-tetra binding (Fig. 6C). In another

333

example, the loop provided direct hydrogen and water-mediated bonds with Leb-tetra

334

after a slight repositioning (Fig. 7). This flexible loop likely modulates binding of

335

Lewis HBGAs, although in vivo interactions may involve additional mechanisms.

336 337

When comparing the sequences of the variant GII.4 P domains, we found that most

338

amino acid changes were surface exposed and ~50% became fixed (Fig. 3). The

339

region immediately beneath the HBGA binding pocket showed few amino acid

340

changes. On the other hand, the regions that contributed to binding terminal

341

saccharides of Lewis HBGAs underwent a more noticeable modification, i.e., from

342

small areas of negative and positive charge (in 1998) to larger areas of mostly

343

negative charge (Fig. 12). The amino acid variations likely corresponded with

344

temporal changes in antigenicity as previously described (32), but how these changes

345

related to apparent alterations in HBGA binding remains unclear.

346 347

Even though not all complex structures could be determined, we considered that these

348

three GII.4 P domains were capable of binding to all HBGA types examined, since

349

binding interactions were similar and only a few amino acid changes surrounding the

14

350

HBGA pocket were observed. We previously showed that the rarely detected GII.10

351

strain also firmly bound a number of HBGA types (H2-tri, A-tri, B-tri, and Ley-tetra),

352

but only weakly bound Leb-tetra and was unable to bind Lea-tri and Lex-tri (21).

353

Therefore, it is tempting to speculate that the GII.4 P domains were better adapted to

354

bind numerous HBGA types, whereas the rarely detected GII.10 virus was less

355

capable, which may also convey to the lower prevalence of the GII.10 strains in the

356

general population (21) and the world-wide distribution of the GII.4 viruses.

357 358

The affinity between norovirus and HBGAs is weak and in the high micromolar range

359

(33). We previously showed that the GII.10 P domain bound H2-tri with an affinity of

360

390 μM (33). Similarly, we found that Saga-2006 P domain had weak affinities to

361

HBGAs (~100 μM) using saturation transfer difference-NMR (unpublished data,

362

Mallagaray, Hansman, and Peters). In addition, a recent study found that a GII.4 P

363

domain (VA387 strain) had comparable affinities among different HBGA types in

364

vitro (34). Based on the number of direct hydrogen bonds and water-mediated

365

interactions, small changes in P domain affinities to HBGAs may exist and these

366

could be important in vivo.

367 368

The precise roles of HBGAs in a norovirus infection are still poorly understood,

369

although synthetic HBGAs or HBGA-expressing enteric bacteria were found to

370

enhance human norovirus infection in B cells (12). Interestingly, the synthetic HBGA

371

(H type disaccharide) in the infection experiment was conjugated to poly-acrylic acid

372

(PAA). Several studies have found that conjugated linkers may affect and/ or

373

influence HBGA binding interactions (35, 36). Further structural studies with

15

374

norovirus VLPs in complex with HBGAs could help explain the possible binding

375

mechanisms in vivo.

376 377

Many of our newly determined HBGA binding results challenged previous ELISA-

378

based findings (4-6). We found that Farm-2004 bound Leb-tetra and B-tri, whereas an

379

ELISA study showed that GII.4 VLPs with an identical P domain sequence (termed

380

2002) did not bind Leb-tetra and only weakly bound B-tri (6). We also found that

381

Saga-2006 bound H2-tri, A-tri and B-tri, Lea-tri, Leb-tetra, and Ley-tetra, while several

382

ELISA studies showed that GII.4 VLPs with an almost identical P domain sequence

383

(termed 2006) did not bind to Ley-tetra (5), A-tri (5), H2-tri (4), and Lea-tri (4).

384

Finally, we showed that NSW-2012 bound A-tri, B-tri, and Lex-tri, whereas a recent

385

ELISA study showed that GII.4 2012 VLPs with an identical P domain sequence

386

(termed GII.4-2012) did not to bind Lex-tri (4). Certainly, in vivo interactions may

387

also be different to the X-ray crystallography and ELISA-based studies. Nevertheless,

388

these new data provide a new focal point for improving HBGA binding assays in

389

order to increase our understanding of norovirus and HBGA interactions.

390 391

Acknowledgements:

392

The funding for this study was provided by the CHS foundation and the Helmholtz-

393

Chinese Academy of Sciences. G.S.H designed the research; M.L. and Anne-Kathrin

394

Herrmann performed initial Farm-2004 structural refinement; and G.S.H. and B.K.S.

395

finalized all structures. We acknowledge the European Synchrotron Radiation Facility

396

(ID23-1 and BM30A) for provision of synchrotron radiation facilities. We thank

397

Thomas Peters and Alvaro Mallagaray for performing the STD-NMR experiments

16

398

(unpublished). We also thank members of the Norovirus Study Group, Joel Sussman,

399

and Henri-Jacques Delecluse for critical comments of the manuscript.

400 401

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402

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403

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521

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522

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523

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524 525

Figure legends:

526

Figure 1. An amino acid alignment of norovirus GII.4 variants. The P domain

527

amino acid sequences of four GII.4 variants in 1998, 2004, 2006, and 2012 (termed

528

VA398, Farm-2004, Saga-2006, and NSW-2012) were aligned using Clustal X. The

529

capsid sequences shared between 93-95% amino acid identity. The S domain was

530

highly conserved with only seven amino acid differences (not shown), whereas the P1

531

(red) and P2 (yellow) subdomains were more variable. The common set of amino

532

acids interacting with HBGAs were shaded blue (chain A) and green (chain B).

533

Compared to the earlier discovered GII.4 P domain (VA387-1998), one amino acid

534

insertion was observed in 2004 and remained in 2006 and 2012.

535 536

Figure 2. X-ray crystal structures of unbound GII.4 P domains. (A) Farm-2004 P

537

domain apo structure contained one dimer per asymmetric unit. The P domain was

538

subdivided into P1 (chain A, pink and chain B, pale cyan) and P2 (chain A, green

539

cyan and chain B, light magenta). (B) Saga-2006 P domain apo structure contained

540

one monomer per asymmetric unit (dimer shown) and was subdivided into P1 (chain

541

A, brown and chain B, yellow orange) and P2 (chain A, deep teal and chain B, dirty

542

violet). (C) NSW-2012 P domain apo structure contained one monomer per

22

543

asymmetric unit (dimer shown) and was subdivided into P1 (chain A, lime and chain

544

B, marine) and P2 (chain A, blue white and chain B, teal). (D) Superposition of Farm-

545

2004, Saga-2006, and NSW-2012 P dimers revealed their overall structures were

546

similar.

547 548

Figure 3. Amino acid variations in GII.4 P dimers in 2004, 2006, and 2012.

549

Amino acid changes (red) were highlighted on GII.4 P dimers (side and top views).

550

The changes were numbered according to a change from 1998 to the respective year

551

(labeled once). A cumulative addition of amino acid changes was found. (A) Farm-

552

2004 contained a single amino acid insertion, Gly394, and this remained in 2012. A

553

small number of amino acid changes surrounding the HBGA pocket (black circle)

554

was observed, i.e., I389V, L375F, and Q376E. (B) Saga-2006 contained additional

555

changes, several of which became fixed, e.g., L375F and Q376E. (C) NSW-2012

556

showed the majority of changes, including several changes in the P1 subdomain.

557 558

Figure 4. Representative simulated annealing difference omit maps. The omit

559

map (blue) was contoured between 2.5-2.0 σ. The H2-tri is an α-L-fucose-(1-2)-β-D-

560

galactose-(1-4)-N-acetyl-β/α-D-glucosamine; A-tri is an α-L-fucose-(1-2)-α-D-

561

galactose-(3-1)-N-acetyl-α-D-galactosamine; B-tri is an α-L-fucose-(1-2)-β/α-D-

562

galactose-(3-1)-α-D-galactose; Ley-tetra is α-L-fucose-(1-2)-β-D-galactose-(1-4)-N-

563

acetyl-β/α-D-glucosamine-(3-1)-α-L-fucose; Leb-tetra is an α-L-fucose-(1-2)-β-D-

564

galactose-(1-3)-N-acetyl-β-D-glucosamine-(4-1)-α-L-fucose;

565

galactose-(1-3)-N-acetyl-β/α-D-glucosamine-(4-1)-α-L-fucose (the electron density

566

was well defined for fucose and partially defined for the N-acetylglucosamine and

567

galactose); and Lex-tri is a β-D-galactose-(1-4)-N-acetyl-β/α-D-glucosamine-(3-1)-α-

Lea-tri

is

a

β-D-

23

568

L-fucose. The underlined β represents the position of the reducing end hydroxyl

569

group, which was fixed in α position in the crystal structures.

570 571

Figure 5. Saga-2006 P dimer binding interactions with H2-tri. (A) Close-up

572

surface and ribbon representation of the Saga-2006 H2-tri complex structure, showing

573

the hydrogen bonds (black lines) with H2-tri (cyan sticks) and water-mediated

574

interactions (red sphere). (B) Saga-2006 P dimer and H2-tri binding interactions (α-

575

fucose, FUC; β-galactose, GAL; and α-N-acetylglucosamine, NDG). The black lines

576

represent the hydrogen bonds, the red line represents the hydrophobic interaction with

577

the hydroxyl group of Tyr444, and the sphere represents water. (C) The ABH-fucose

578

of Saga-2006 H2-tri bound at the regular pocket. The acetyl group of N-

579

acetylglucosamine was leaning towards the edge of the P dimer. (D) The ABH-fucose

580

of TCH05-2004 H1-pentasaccharide bound at the regular pocket. The acetyl group of

581

N-glucosamine was leaning towards the center of the P dimer.

582 583

Figure 6. Saga-2006 P dimer binding interactions with Ley-tetra. (A) A close-up

584

surface and ribbon representation of the Saga-2006 Ley-tetra complex structure,

585

showing the hydrogen bonds with Ley-tetra (green sticks) and water-mediated

586

interactions. (B) Saga-2006 P dimer and Ley-tetra binding interactions. (C) A loop in

587

the Saga-2006 P2 subdomain (residues 391-394) was repositioned from an apo

588

position (gray) to an alternative position (deep teal) in order for Ley-tetra binding. (D)

589

The Lewis-fucose of Saga-2004 Ley-tetra bound at the regular pocket on the P domain

590

and was leaning towards the edge of the P dimer. (E) The ABH-fucose of GII.10 Ley-

591

tetra bound at the regular pocket and was orientated towards the center of the P dimer.

592

24

593

Figure 7. Farm-2004 and Saga-2006 P dimer binding interactions with Leb-tetra.

594

(A) Close-up surface and ribbon representation of the Farm-2004 Leb-tetra complex

595

structure, showing the hydrogen bonds with Leb-tetra (marine sticks) and water-

596

mediated interactions. (B) Farm-2004 P dimer and Leb-tetra binding interactions (α-

597

fucose, FUC; β-galactose, GAL; and β-N-acetylglucosamine, NAG). The black lines

598

represent the hydrogen bonds, the red line represents the hydrophobic interaction with

599

the hydroxyl group of Tyr444, and the sphere represents water. (C) Close-up surface

600

and ribbon representation of the Saga-2006 Leb-tetra complex structure, showing the

601

hydrogen bonds with Leb-tetra and water-mediated interactions. (D) Saga-2006 P

602

dimer and Leb-tetra binding interactions. (E) Superposition of both A and B chains

603

from Farm-2004 apo (gray and black) and Farm-2004 Leb-tetra (cyan and pink)

604

structures.

605 606

Figure 8. Saga-2006 P dimer binding interactions with Lea-tri and superposition

607

of GII.4 P domains. (A) A close-up surface and ribbon representation of the Saga-

608

2006 Lea-tri complex structure, showing hydrogen bonds with Lea-tri (camel sticks)

609

and water-mediated interactions. (B) Saga-2006 P dimer and Lea-tri binding

610

interactions (α-fucose, FUC; α-N-acetylgalactosamine, NDG; and β-galactose, GAL).

611

(C) Superposition of apo and HBGA-bound Farm-2004, Saga-2006, and NSW-2012 P

612

dimer structures (HBGAs removed from the structures). The circle represents the

613

HBGA binding pocket. Farm-2004 P1 subdomains (chain A, pink and chain B, pale

614

cyan) and P2 subdomains (chain A, green cyan and chain B, light magenta); Saga-

615

2006 P1 subdomains (chain A, brown and chain B, yellow orange) and P2

616

subdomains (chain A, deep teal and chain B, dirty violet); NSW-2012 P1 subdomains

617

(chain A, lime and chain B, marine) and P2 subdomains (chain A, blue white and

25

618

chain B, teal) were colored accordingly. (D) Close-up of the P2 subdomain flexible

619

loop (residues 391-394). In the case of H2-tri, Ley-tetra, Lea-tri, and Lex-tri, the N-

620

acetylglucosamine was held with the side chain of Ser442, while the galactose was

621

held with a hydrogen bond from the hydroxyl group of Tyr444. The loop required for

622

the Lewis HBGA-tetrasaccharide interactions was found in multiple conformations on

623

both A and B chains.

624 625

Figure 9. NSW-2012 P dimer interaction with Lex-tri. (A) A close-up surface and

626

ribbon representation of the NSW-2012 Lex-tri complex structure, showing the

627

hydrogen bonds with Lex-tri (salmon sticks) and water-mediated interactions. (B)

628

NSW-2012 and Lex-tri binding interactions (α-fucose, FUC; α-N-acetylglucosamine,

629

NDG; and β-galactose, GAL). The black lines represent the hydrogen bonds, the red

630

line represents the hydrophobic interaction with the hydroxyl group of Tyr444, and

631

the sphere represents water.

632 633

Figure 10. Saga-2006 and NSW-2012 P dimer interactions with A-tri. (A) Close-

634

up surface and ribbon representation of the Saga-2006 A-tri complex structure,

635

showing the hydrogen bonds with A-tri (yellow sticks) and water-mediated

636

interactions. (B) Saga-2006 and A-tri binding interactions (α-fucose, FUC; α-

637

galactose, GLA; and α-N-acetylgalactosamine, A2G). The black lines represent the

638

hydrogen bonds, the red line represents the hydrophobic interaction with the hydroxyl

639

group of Tyr444, and the sphere represents water. (C) A close-up surface and ribbon

640

representation of the NSW-2012 A-tri complex structure, showing the hydrogen

641

bonds with A-tri and water-mediated interactions. (D) NSW-2012 and A-tri binding

642

interactions.

26

643 644

Figure 11. Farm-2004, Saga-2006, and NSW-2012 P dimer interactions with B-

645

tri. (A) Close-up surface and ribbon representation of the Farm-2004 B-tri complex

646

structure, showing hydrogen bonds with B-tri (pink sticks) and water-mediated

647

interactions. (B) Farm-2004 P dimer and B-tri binding interactions (α-fucose, FUC;

648

α-galactose, GLA). The black lines represent the hydrogen bonds, the red line

649

represents the hydrophobic interaction with the hydroxyl group of Tyr444, and the

650

sphere represents water. (C) Close-up surface and ribbon representation of the Saga-

651

2006 B-tri complex structure, showing hydrogen bonds with B-tri and water-mediated

652

interactions. The galactose was found in two different conformations (gray and pink

653

sticks) (D) Saga-2006 P dimer and B-tri binding interactions showing newly formed

654

hydrogen bonds (blue lines) with the alternative galactose position. (E) Close-up

655

surface and ribbon representation of the NSW-2012 B-tri complex structure, showing

656

hydrogen bonds with B-tri and water-mediated interactions. (F) NSW-2012 P dimer

657

and B-tri binding interactions.

658 659

Figure 12. Surface representation of protein contact potential of GII.4 P dimers.

660

The protein contact potential (where red represented negative charge, white

661

represented neutral charge, and blue represented positive charge; ~ -55 to +55 kT/e)

662

was calculated for VA387-1997 (2OBT), TCH-2004 (3SLD), Farm-2004, Saga-2006,

663

and NSW-2012 (top view and close-up of the HBGA pocket). Leb-tetra of Farm-2004

664

Leb-tetra structure (marine sticks) was modeled into VA387, TCH-05, Saga-2006, and

665

NSW-2012. B-tri (pink sticks) and A-tri (yellow sticks) were complex structures. The

666

regions surrounding the regular ABH-fucose binding pocket remained mostly

667

unchanged and negatively charged. The regions binding terminal saccharides of

27

668

Lewis HBGAs changed from small patches of negative/ positive charge to larger

669

areas of negative charge.

28

Figure 1

VA387-1998 Farm-2004 Saga-2006 NSW-2012

KPFTVPILTVEEMSNSRFPIPLEKLYTGPSSAFVVQPQNGRCTTDGVLLGTTQLSAVNICTFRGDVT .............T...........F.............................P........... .............T...........F....G........................P........... ...S..V......T...........F.............................P...........

291 291 291 291

VA387-1998 Farm-2004 Saga-2006 NSW-2012

HIAGSHDYIMNLASQNWNNYDPTEEIPAPLGTPDFVGKIQGMLTQTTREDGSTRAHKATVSTGSVHF ....T.N.T............................R..........G.....G........D... .....RN.T.....L..........................L.....KG.....G.....Y...AP. ..T..RN.T.........D......................V......T.....G.....Y...AD.

358 358 358 358

VA387-1998 Farm-2004 Saga-2006 NSW-2012

TPKLGSVQYTTDTNNDLQTGQNTKFTPVGVIQDGN-NHQNEPQQWVLPNYSGRTGHNVHLAPAVAPT ......I.FN......FE............V....GA...........S.................. ........FS...E..FE.H..............STT.R.........S....NV............ A....R..FE...DR.FEAN..............GTT.R.........S....NT............

423 424 424 424

VA387-1998 Farm-2004 Saga-2006 NSW-2012

FPGEQLLFFRSTMPGCSGYPNMNLDCLLPQEWVQHFYQEAAPAQSDVALLRFVNPDTGRVLFECKLH ................................................................... ......................D............................................ ......................D...........Y................................

490 491 491 491

VA387-1998 Farm-2004 Saga-2006 NSW-2012

KSGYVTVAHTGPHDLVIPPNGYFRFDSWVNQFYTLAPM ...........Q.......................... ...........Q.......................... ...........Q..........................

529 530 530 530

Figure 2 A B: P2

A: P2 90°

B: P1

A: P1 C

C N

N

B B: P2

A: P2 90°

B: P1

A: P1 C

C N N

C B: P2

A: P2 90°

B: P1

A: P1 C

C N

D

N

Figure 3 A S296T, D298N, I300T A’346G’ -394G, N395A

L’375F’, Q’376E’ S’296T’ H’297Q’, D’298N’ I’300T’, V’365I’, Y’367F’ -394G’, N’395A’

V365I, Y367F, T368N N407S

E’340G’

S296T, D298N, I300T L375F, Q376E A340G E346G

S355D

P504Q

90° Y250F

K329R I389V N395A P504Q Y250F

B L’375F’, Q’376E’ G’378H’ N’372E’ N393S, -394T, N395T E’340G’ R’339K’ T412N Q397R N448D P504Q

I’300T’ H’297R’, D’298N’ N’393S’, -394T’, N’395T’ V’356A’, H’357P’

V356A, H257P H297R N372E G378H T412N, G413V

Y367F, T368S S352Y N393S, -394T, N395T

T’412N’, G’413V’ Q’306L ’ S’255G 90° ’

S255G

L375F, Q376E R339K E340G A346G M333L

S255G P504Q

Y250F

Y250F

Q397R

C T’377A’, G’378N’ L’375F’, Q’376E’ N393G, -394T, N395T E’340T’ T412N Q397R N448D P504Q Y250F N’310D’ I231V T228S

N297R N’372D’, N’373R’ A’294T’ H’297R’, D’298N’ N’393G’, -394T’, N’395T’ I’300T’ V’356A’, H’357D’ T’412N’, G’413T’

S364R A294T N310D G378N V356A, H257D T412N, G413T Y367F, T368E S352Y

90°

N393G, -394T, N395T P504Q Y250F Q397R

L375F, Q376E, T377A N372E H297R E340T A346G M333L

H2-tri Saga-2006

Ley-tetra Saga-2006

Leb-tetra Farm-2004

Leb-tetra Saga-2006

Lex-tri NSW-2012

Lea-tri Saga-2006

A-tri Saga-2006

A-tri NSW-2012

B-tri Farm-2004

B-tri Saga-2006

Figure 4

B-tri NSW-2012

Figure 5

A

B

C

D N-acetylglucosamine

N-acetylglucosamine

ABH-fucose

ABH-fucose

Figure 6 A

B

C apo loop position Saga-2006-Ley-tetra loop position

D

E

Lewis-fucose

ABH-fucose

Lewis-fucose

ABH-fucose

Figure 7 B

A

C

E

D

Farm-2004 Leb-chain B (unbound) Farm-2004 Leb-chain A (bound) Farm-2004 apo-chain A

Farm-2004 apo-chain B

Lewis fucose

Figure 8

A

C

D

B

Chain A

Chain B

Loop 391-394 Chain A Tyr444

Loop 391-394 Chain B Tyr444

Ser442

Ser442

Figure 9

A

B

Figure 10

A

C

B

D

Figure 11 A

B

C

D

E

F

VA387-1997

TCH05-2004

Farm-2004

Saga-2006

NSW-2012

~ -55

55 kT/e

Figure 12

Table 1. Data collection and refinement statistics of apo GII.4 P domain structures

Data collection Space group Cell dimensions a, b, c (Å) α, β, γ (°) Resolution range (Å) Rmerge I/σI Completeness (%) Redundancy

Farm-2004 (4OOV)

Saga-2006 (4OOX)

NSW-2012 (4OOS)

P212121

C2

C2

62.95 90.12 109.21 90 90 90 46.70-1.50 (1.55-1.50)* 7.48 (54.74)* 14.18 (2.37)* 99.24 (97.64)* 4.9 (4.1)*

96.72 58.94 62.14 90 119.88 90 48.22-1.03 (1.07-1.03)* 3.40 (52.85)* 15.12 (1.93)* 96.03 (90.00)* 3.0 (2.7)*

98.48 55.07 63.46 90 120.10 90 46.25-1.60 (1.66-1.60)* 4.43 (24.53)* 20.63 (5.12)* 98.14 (94.97)* 3.5 (3.3)*

Refinement Resolution range (Å) 46.70-1.53 27.17-1.20 No. of reflections 93719 92504 Rwork/Rfree 14.08/16.41 11.96/14.32 No. of atoms 10090 4975 Protein 4755 2436 Ligand/ion 32 40 Water 778 295 2 Average B factors (Å ) Protein 13.80 13.90 Ligand/ion 22.30 22.90 Water 26.40 26.00 RMSD Bond length (Å) 0.009 0.010 Bond angle (°) 1.29 1.38 Each data set was collected from single crystals, respectively. *Values in parentheses are for highest-resolution shell.

42.60-1.64 35573 13.77/16.15 5021 2406 24 318 12.90 18.80 23.30 0.005 1.09

Table 2. Data collection and refinement statistics of Farm-2004 P domain and HBGA complex structures

Data collection Space group Cell dimensions a, b, c (Å) α, β, γ (°) Resolution range (Å) Rmerge I/σI Completeness (%) Redundancy Refinement Resolution range (Å) No. of reflections Rwork/Rfree No. of atoms Protein Ligand/ion Water Average B factors (Å2) Protein Ligand/ion Water RMSD Bond lengths (Å) Bond angles (°)

B-tri (4X05)

Leb-tetra (4OPS)

C2

P212121

175.11 89.54 106.73 90 127.55 90 19.89-1.96 (2.01-1.96)* 15.8 (113.90)* 8.89 (1.29)* 99.10 (99.30)* 5.5 (4.7)*

71.45 90.11 91.87 90 90 90 47.81-1.75 (1.79-1.75)* 11.20 (53.53)* 9.88 (2.41)* 97.30 (95.10)* 5.5 (5.7)*

19.89-1.98 90505 16.30/ 20.31 10774 9437 132 12045

47.81-1.76 57869 18.50/21.64 9541 4723 46 324

22.70 43.00 31.20

23.20 57.20 26.80

0.008 1.10

0.013 1.36

Each data set was collected from single crystals, respectively. *Values in parentheses are for highest-resolution shell.

Table 3. Data collection and refinement statistics of Saga-2006 P domain and HBGA complex structures Data collection Space group Cell dimensions a, b, c (Å) α, β, γ (°) Resolution range (Å) Rmerge I/σI Completeness (%) Redundancy

H2-tri (4WZK)

A-tri (4X07)

B-tri (4X06)

Lea-tri (4WZL)

Leb-tetra (4OPO)

Ley-tetra (4WZE)

C2

C2

C2

C2

C2

C2

114.99 58.81 98.02 90 108.11 90 46.58-1.47 (1.51-1.47)* 3.091 (48.00)* 17.31 (2.27)* 97.30 (95.10)* 2.9 (2.9)*

97.96 58.64 114.59 90 105.49 90 48.01-1.28 (1.32-1.28)* 6.353 (41.88) 7.40 (1.96)* 98.00 (92.70)* 4.5 (3.6)*

113.83 58.65 97.31 90 107.34 90 48.10-1.22 (1.25-1.22)* 3.519 (35.40)* 13.92 (2.54)* 96.70 (90.20)* 2.6 (2.5)*

96.87 58.83 124.44 90 119.8 90 49.59-1.57 (1.61-1.57)* 5.205 (48.73)* 8.11 (1.99)* 97.61 (88.60)* 3.0 (2.9)*

113.66 58.6 97.17 90 107.15 90 46.42-1.38 (1.42-1.38)* 7.537 (93.06)* 12.76 (1.71)* 99.20 (97.04)* 3.7 (3.6)

97.02 58.50 113.86 90 108.1 90 48.16-1.45 (1.49-1.45)* 2.8 (45.5)* 10.16 (1.84)* 95.17 (93.87)* 2.3 (2.2)*

32.32-1.22 176446 13.41/16.12 10037 4865 140 716

42.47-1.70 65702 17.03/19.55 9985 4812 124 569

46.42-1.40 119485 15.08/18.54 10227 4850 120 812

31.76-1.46 100015 17.74/21.17 9826 4801 100 542

15.80 27.90 25.60

17.10 37.40 28.20

12.90 30.40 25.30

22.80 41.70 32.30

0.010 1.36

0.006 1.08

0.007 1.20

0.033 1.73

Refinement Resolution range (Å) 29.66-1.49 43.18-1.46 No. of reflections 99198 106175 Rwork/Rfree 13.88/17.35 16.53/19.30 No. of atoms 9013 10143 Protein 4803 4842 Ligand/ion 80 80 Water 540 799 2 Average B factors (Å ) Protein 24.20 15.50 Ligand/ion 47.90 36.00 Water 33.10 27.40 RMSD Bond lengths (Å) 0.011 0.008 Bond angles (°) 1.29 1.20 Each data set was collected from single crystals, respectively. *Values in parentheses are for highest-resolution shell.

Table 4. Data collection and refinement statistics of NSW-2012 P domain and HBGA complex structures

Data collection Space group Cell dimensions a, b, c (Å) α, β, γ (°) Resolution range (Å) Rmerge I/σI Completeness (%) Redundancy

A-tri (4WZT)

B-tri (4OP7)

Lex-tri (4X0C)

P41212

P41212

P41212

104.74 104.74 190.85 90 90 90 48.26-1.85 (1.90-1.85)* 8.758 (98.50)* 16.73 (1.60)* 98.80 (98.30)* 6.5 (6.5)*

104.61 104.61 190.54 90 90 90 48.19-1.90 (1.97-1.90)* 11.31 (115.40)* 10.70 (1.09)* 93.80 (93.70)* 4.3 (4.2)*

104.83 104.83 191 90 90 90 45.95-1.70 (1.75-1.70)* 6.856 (128.60)* 15.62 (1.20)* 99.20 (97.60)* 3.6 (3.6)*

Refinement Resolution range (Å) 48.26-1.91 48.19-1.92 No. of reflections 81872 76345 Rwork/Rfree 15.37/17.91 18.47/20.81 No. of atoms 10006 9760 Protein 4803 4762 Ligand/ion 88 76 Water 718 492 2 Average B factors (Å ) Protein 26.30 26.90 Ligand/ion 42.80 37.90 Water 34.50 31.50 RMSD Bond lengths (Å) 0.008 0.007 Bond angles (°) 1.13 1.11 Each data set was collected from single crystals, respectively. *Values in parentheses are for highest-resolution shell.

40.14-1.72 112461 15.96/17.95 10085 4802 142 747 23.10 43.60 34.70 0.013 1.32