Supporting online material #1093373

Materials and Methods

Cloning

Based on H3 numbering (S1), cDNA corresponding to residues 11-329 (HA1) and 1-176 (HA2),

of the ectodomain of 18HA0 (A/South Carolina/1/18) (S2) was cloned into the baculovirus

transfer vector, pAcGP67A (BD Biosciences, Bedford, MA) to allow for efficient secretion of

recombinant protein. Initial constructs, however, produced only unstable monomers that

precipitated at each stage during purification (figure S2). A subsequent database search revealed

research which utilized a trimerizing sequence (‘foldon’) from the bacteriophage T4 fibritin for

stabilizing collagen triple helices (S3). A new construct containing this C-terminal ‘foldon’

sequence was made with a thrombin site introduced between the C-terminus of 18HA0 and the

‘foldon’. A His-Tag was also incorporated at the extreme C-terminus of the construct to enable

purification. Transfection and virus amplification were carried out according to the baculovirus

expression system manual (Pharmingen). Expression of secreted protein was assessed by

detection of the His-tag by Western blot using an anti-His6 antibody (Qiagen, Valencia, CA).

HA0 protein secreted into the cell culture media contains additional plasmid-encoded residues at

both the N- (ADPGYLLE) and C-terminus (RSLVPRGSPGSGYIPEAPRDGQAYVRKDGEW

VLLSTFLGHHHHHH, where the sequence in italics is the thrombin site, underlined is the

foldon sequence and bold is the His-Tag). The construct facilitated expression of unprocessed

HA0 homotrimers (figure S2) consistent with a previous report that Sf9 cells do not express

proteases that effectively cleave hemagglutinin to its infectious HA1/HA2 form (S4).

Expression and Purification

Suspension cultures of insect Sf9 cells were cultured in EX-CELL 420 serum-free media (JRH

Biosciences, Lenexa, KS). For a typical preparation, 12 liters of Sf9 cells at 2 x 106 cells/ml were

infected at a multiplicity of infection (MOI) of 3. After 3 days, cells were removed by

centrifugation (1500g; 20 mins) and soluble 18HA0 recovered from the cell supernatant by metal

affinity chromatography using Ni-NTA resin (Qiagen). Fractions containing 18HA0 were pooled

and dialyzed against 10 mM Tris-HCl, 50 mM NaCl; pH 8.0, then subjected to ion exchange

chromatography on a Mono-Q HR10/10 column (Pharmacia). Trimeric protein eluting at 160

mM NaCl was subsequently subjected to thrombin digest (3 units/mg protein; overnight at room

temperature) and purified further by gel filtration chromatography using a Superdex-200 16/60

column (Pharmacia). Protein eluting as a trimer was concentrated to 10-15 mg/ml in 10 mM

HEPES, 25 mM NaCl; pH 7.5. The yield of pure protein is approximately 1.5 mg/liter of infected

culture. At this stage, 18HA0 still contains the additional plasmid-encoded residues at both the N-

(ADPGYLLE) and C-terminus (RSLVPR)

Crystal structure determination

18HA0 crystals were grown by the sitting drop vapor diffusion method with a reservoir solution

(0.6 ml) of 1.68 M sodium dihydrogen phosphate, 0.32 M di-potassium hydrogen phosphate, 0.1

M phosphate-citrate, pH 5.5. Crystallization drops were set up at 22 °C, with 0.6 µl of protein

mixed with an equal volume of reservoir solution. The resulting crystals were ‘flash-cooled’ at a

temperature of 100 K in a reservoir solution containing 20% glycerol. A dataset was collected to

3.0Å resolution at the Stanford Synchotron Radiation Laboratory (SSRL) beamline 9-2 at 100 K.

The data were indexed in spacegroup C2 with unit cell dimensions a = 190.5Å, b =109.9Å, c

=136.2Å and =108.6. Statistics for data collection are presented in Table S1. Data were

processed with the DENZO-SCALEPACK suite (S5) using all observations. One hemagglutinin

trimer occupies the asymmetric unit with an estimated solvent content of 66% based on a

Matthews’ coefficient (Vm) of 3.57 Å3/Da. Rotation functions were performed with AMoRe (S6)

and the highest rotation and translation solutions were found using a polyalanine-truncated

trimeric model of the H9 swine hemagglutinin (pdb:1JSH) (S7). Rigid body refinement of the

trimer led to an overall correlation coefficient of 46.6% and an Rcryst of 53.2%. The model was

then "mutated" to the correct sequence, rebuilt using O (S8), and refined with the Crystallography

& NMR System software (CNS version 1.1) (S9) and REFMAC (S10). Tight non-

crystallographic symmetry restraints were applied throughout model building (all non-hydrogen

atoms; weight=300). Electron density maps for model building included 2Fobs–Fcalc, Fobs–Fcalc and

composite annealed omit 2Fobs–Fcalc maps. Progress during refinement was judged by monitoring

the Rfree test set, consisting of 5% of all reflections, for cross-validation (S11) At later stages of

refinement, N-linked carbohydrates were built at nine of the fifteen potential glycosylation sites

in the HA0 trimer. The final model also contains one of the eight vector-encoded residues (Glu)

at the N-terminus, and one phosphate ion derived from the crystallization buffer. Due to poor or

no density, the HA2 C-terminal Val176 and the thrombin cleavage site residues were also omitted

from the final model. The final 18HA0 structure has an Rcryst of 27.0 % and Rfree of 29.6 % (see

Table S1). The quality of the model was checked with the program PROCHECK (S12). For the

Ramachandran plot, 98.5% of the residues were in the most favored (81.9%) and additionally

allowed regions (16.6%). Although the HA0 is a single monomeric chain, the final pdb is

numbered according to H3 numbering, based on structural overlay with the H3 structure (pdb:

2hmg). Therefore, each HA0 monomer is numbered according to H3 HA1 and HA2 subunits for

ease of comparison. Residues deemed to be insertions are numbered XA where X is the

preceding residue number in the chain (e.g. Asn 19A in HA1)

Glycosylation

The protein sequence of 18HA0 predicts six possible glycosylation sites per monomer,

but one is in the cytoplasmic tail, only four residues from the membrane insertion sequence and

unlikely to be glycosylated. Electron density is observed for nine of the possible fifteen

glycosylation sites in the trimeric construct; only a single N-acetyl glucosamine was interpretable

at six of these sites (AsnA20, AsnA34, AsnB154, AsnC34, AsnC288, AsnE34), but at glycosylation site Asn95

additional N-acetyl glucosamine and mannose residues could be interpreted (one mannose for

chains A and E and two for chain C) (Figure 1) (S13).

Figure Legends

Figure S1 Structural alignment of the amino acid sequence of 18HA0 compared to sequences

of other H1 strains and as well as other pandemic and epidemic strains from different subtypes in

the 20th century. Numbering and alignment are based on the human H3 sequence (Hu_H3 1968;

A/Aichi/2/68) (S14). Sequences of human H1 (Hu_H1 1918; A/South Carolina/1/18) (S15),

swine H1 (Sw_H1 1930; A/Swine/Iowa/15/30) (S16), human H1 (Hu_H1 1934; A/Puerto

Rico/8/34) (S17), and H2 (Hu_H2 1957; A/Singapore/1/57) (S18) are shown. Sequences for two

other subtypes that crossed the species barrier, but failed to reach pandemic levels: H5 (Hu_H5

1997; A/Hong Kong/156/97) and H9 (Hu_H9 1999; A/Hong Kong/1073/99) are also shown

(S19, S20). Only residues different from the H3 subtype are shown. Residues marked < and > are

the start/stop sites for the expressed construct. Residues shaded grey indicate potential

glycosylation sites (N-X-S/T), yellow indicates the transmembrane domain and boxed is the

cleavage site arginine in each sequence. Histidine residues marked # indicate the His patch

surrounding HA2 Trp21, while those labeled * (including HA1 Lys50) indicate the proposed pH-

dependent charged patch near the E region. Antigenic sites, identified previously by Brownlee

and Fodor (S21) are marked with different highlight colors: Ca1 (red), Ca2 (purple), Cb (green),

Sa (light blue), Sb (gold). Residues labeled • indicate those implicated in receptor binding

specificity. See main text for full discussion.

Figure S2 Gel filtration analysis of 18HA0 constructs. The original construct expressed only

the HA0 gene encoded with a His-Tag for purification (18HA0), but this produced only

monomeric material, eluting at ~70kDa (dashed line) by size exclusion chromatography on a

Superdex-200 10/30 column (Pharmacia). Thus, a new clone was engineered so that the resulting

18HA0 was fused to a C-terminal linker containing a thrombin cleavage site, a trimerizing

sequence from T4 bacteriophage (S3) and a hexa His-tag to aid purification (18HA0zip). This re-

designed construct expressed trimeric material that eluted at ~200kDa (solid line), ran as a 70kDa

protein on an SDS-PAGE gel (see gel picture inset) and yielded diffractable crystals. Figure

represents superimposed elution profiles of two separate experiments overlaid with calibration

standards (dotted line).

Figure S3 Comparison of 18HA0 to other human, avian and swine HA’s. A) Comparison of

their HA1 domains, looking down the long axis of the trimer from the membrane-distal end. B)

Schematic showing the rotation of the 18HA0 (H1, red), H5 (orange) and H9 (blue) subtypes in

relation to the H3 subtype (green). Each trimer was overlaid on their HA2 domains with the

central axis of the trimer on the z-axis, and the rotation required to superimpose the HA1

domains were determined as Tait-Bryan angles (S22). Figure S3A was generated using VMD

(S23) and rendered using Tachyon (S24).

Figure S4 18HA0 patch of surface exposed histidines. The ‘His patch’ is located near the

vestigial esterase (E) domain of the HA monomer. HisA47, HisA275, HisA285, HisA298 and LysA50 are all

within close proximity and create a highly-positively charged patch, which would increase the

charge of the HA as it enters the endosome pathway, where the pH drops well below 6.0.

Figure S5 Surface representations of the monomer of the 18HA0 structure in comparison to

the human H3 (pdb:2hmg), avian H5 (pdb:1jsm), swine H9 (pdb:1jsd) and influenza C HEF

(pdb:1flc). Surface-exposed histidines are colored blue, and indicate that the 18HA0 structure is

more avian-like with respect to the positions of the surface-exposed histidines. Figure was

generated using VMD (S23) and rendered using Tachyon (S24).

Figure S6 Antigenic sites of 18HA. Residues in the HA1 chain, previously described by

Brownlee and Fodor (S21) are grouped into 5 regions: Ca1 (169 to 173, 296 to 208, 238 to 240),

Ca2 (140 to 145, 224 to 226), Cb (78 to 83), Sa (128 to 129, 156 to 160, 162 to 167), and Sb (187

to 198). The antigenic site colors are coordinated with their corresponding sequences in FigS1.

Residues which differ between 18HA and the swine HA (A/swine/Iowa/15/30), its closest

homologue based on sequence identity, are labeled. The Asn95 glycosylation site in 18HA

adjacent to Ca1 and Ca2 is labeled.

Fig

ure

S1 1 < # # * * 96

Hu_H3 1968 QDLPGNDNST ATLCLGHHA. VPNGTLVKTI TDDQIE.VTN ATELVQSSST GKICNN.PHR ILDGIDCTLI DALLGDPHCD VFQNE.TWDL FVERSKAFSN

Hu_H1 1918 .......... D-I-I-YH-N NSTD-VDTV. .LEKNVT--H SVN-LED-HN --L-KLKGIA P-QLGK-NIA GW---N-E-- LLLTASS-SY I--T-NSENG

Sw_H1 1930 .......... D---I-YH-N NSTD-VDTV. .LEKNVT--H SVN-LED-HN --L-RLGGIA P-QLGK-NIA G..--N-E-- LLLTVSS-SY I--T-NSDNG

Hu_H1 1934 .......... D-I-I-YH-N NSTD-VDTV. .LEKNVT--H SVN-LED-HN --L-RLKGIA P-QLGK-NIA GW---N-E-- PLLPVRS-SY I--TPNSENG

Hu_H2 1957 .......... DQI-I-YH-N NSTEKVDTI. .LERNVT--H -KDILEKTHN --L-KLNGIP P-ELG--SIA GW---N-E-- RLLSVPE-SY IM-KENPRDG

Hu_H5 1997 .......... DQI-I-YH-N NSTEQVDTI. .MEKNVT--H -QDILERTHN --L-DLNGVK P-ILR--SVA GW---N-M-- E-I-VPE-SY I--KASPAND

Hu_H9 1999 .......... DKI-I--QST NSTE-VDTL. .TETNVP--H -K--LHTEHN --L-ATSLGH P-ILDT--IE GLVY-N-S-- LLLGGRE-SY I----S-VNG

97 191

• • • • •

Hu_H3 1968 .CYPYDVPDY ASLRSLVASS GTLEFITEGF ...TWTGVTQ N.GGSNACKR GPGSGFFSRL NWLTKSGSTY PVLNVTMPNN DNFDKLYIWG IHHPSTNQEQ

Hu_H1 1918 T---G-FI-- EE--EQLS-V SSF-KFEIFP KTSS-PNHET TK-VTA--SY AGA-S-YRN- L----K--S- -K-SKSYV-- KGKEV-VL-- V---P-GTD-

Sw_H1 1930 T---G-FI-- EE--EQLS-V SSF-KFEIFP KTSS-PNHET TR-VTA--PY AGA-S-YRN- L--V-KENS- -K-SKSYV-- KGKEV-VL-- V---P-STD-

Hu_H1 1934 I---G-FI-- EE--EQLS-V SSF-RFEIFP KESS-PNHNT TK-VTA--SH AGK-S-YRN- L---EKEGS- -K-KNSYV-K KGKEV-VL-- -----NSKD-

Hu_H2 1957 L---GSFN-- EE-KH-LS-V KHF-KVKILP K.DR--QH-T TG-.-R--AV SGNPS-FRNM V---EK--N- --AKGSYN-T SGEQM-I--- V---NDEK--

Hu_H5 1997 L---GNFN-- EE-KH-LSRI NHF-K-QIIP K.SS-SNHDA SS-V-S--PY LGR-S-FRNV V--I-KN-A- -TIKRSYN-T NQE-L-VL-- V---NDAA--

Hu_H9 1999 T---GN-ENL EE--T-FS-A SSYQR-QIFP .DT--N.--Y TGT.-R--S. ...GS-YRSM R---QKSGF- --QDAQYT-- RGKSI-FV-- ----P-YT--

192 290

• •• • * *

Hu_H3 1968 TSLYVQASGR VTVSTRRSQQ TIIPNIGSRP WVRGLSSRIS IYWTIVKPGD VLVINSNGNL IAPRGYFKMR TGK.SSIMRS DAPIDTCISE CITPNGSIPN

Hu_H1 1918 Q---QN-DAY -S-GSSKYNR RFT-E-AA-- K--DQAG-MN Y---LLE--- TITFEAT--- ---WYA-ALN R-SG-G-IT- ---VHD-NTK -Q--H-A-NS

Sw_H1 1930 Q---QN-DAY -S-GSSKYDR RFT-E-AA-- K---QAG-MN Y---LLE--- TITFEAT--- V---YA-ALN R-SE-G-IT- ---VHD-DTK -Q--H-A-NS

Hu_H1 1934 QNI-QNENAY -S-V-SNYNR RFT-E-AE-- K--DQAG-MN Y---LL---- TIIFEA---- ----YA-ALS R-FG-G-IT- N-SMHE-NTK -Q--L-A-NS

Hu_H2 1957 RT--QNVGTY -S-G-STLNK RST-D-AT-- K-N--G--ME FS--LLDMW- TINFE-T--- ---EYG--IS KRGS-G--KT EGTLEN-ETK -Q--L-A-NT

Hu_H5 1997 -K--QNPTTY IS-G-STLN- RLV-E-AT-- K-N-Q-G-ME FF---L--N- AINFE----F ---EYAY-IV KKGD-T--K- ELEYGN-NTK -Q--M-A-NS

Hu_H9 1999 TN--IRNDTT TS-T-EDLNR -FK-V--P-- L-N--QG--D Y--SVL---Q T-RVR----- ---WYGHVLS G-SHGR-LKT -LKGGN-VVQ -Q-EK-GLNS

291 * 328 1 57

Hu_H3 1968 DKPFQNVNKI TYGACPKYVK QNTLKLATGM RNVPEKQT.. ..RGLFGAIA GFIENGWEGM IDGWYGFRHQ NSEGTGQAAD LKSTQAAIDQ INGKLNRVIE

Hu_H1 1918 SL----IHPV -I-E-----R STK-RM---L --I-SI-S.. ..-------- ----G--T-- ------YH-- -EQ-S-Y--- Q----N---G -TN-V-S---

Sw_H1 1930 SL----IHPV -I-E------ STK-RMV--L --I-SI-S.. ..-------- ----G--T-L ------YH-- -GQ-S-Y--- Q----N---G -TN-V-S---

Hu_H1 1934 SL----IHPV -I-E-----R SAK-RMV--L --I-SI-S.. ..-------- ----G--T-- ------YH-- -EQ-S-Y--- Q----N--NG -TN-V-S---

Hu_H2 1957 TL--H--HPL -I-E------ SEK-V----L ----QIES.. ..-------- ----G--Q-- ------YH-S -DQ-S-Y--- KE---K-F-G -TN-V-S---

Hu_H5 1997 SM--H-IHPL -I-E------ S-R-V----L --T-QRERRR KK-------- ----G--Q-- V-----YH-S -EQ-SCYS-- KE---K---G VTN-V-SIIN

Hu_H9 1999 TL--H-IS-Y AF-T-----R V-S----V-L ----ARSS.. ..-------- ----G--P-L VA-----Q-S -DQ-V-M--- RD---K---K -TS-V-NIVD

58 # 157

Hu_H3 1968 KTNEKFHQIE KEFSEVEGRI QDLEKYVEDT KIDLWSYNAE LLVALENQHT IDLTDSEMNK LFEKTRRQLR ENAEEMGNGC FKIYHKCDNA CIESIRNGTY

Hu_H1 1918 ---TQ-TAVG ---NNL-R-- EN-N-K-D-G FL-I-T---- ---L---ER- L-FH--NVRN -Y--VKS--K N--K-I---- -EF-----D- -M--V-----

Sw_H1 1930 ---TQ-TVVG ---NNL-R-- KN-N-K-D-G FL-V-T---- M--L---ER- L-FH--NVKN -Y--A-S--- N--K-I---- -EF-----D- -M--V-----

Hu_H1 1934 ---IQ-TAVG ---NKL-K-M EN-N-K-D-G FL-I-T---- ---L---ER- L-FH--NVKN -Y--VKS--K N--K-I---- -EF------E -M--V-----

Hu_H2 1957 ---TQ-EAVG ----NL-R-L EN-N-KM--G FL-V-T---- ---LM--ER- L-FH--NVKN -YD-V-M--- D-VK-L---- -EF-----DE -MN-VK----

Hu_H5 1997 ---TQ-EAVG R--NNL-R-- EN-N-KM--G FL-V-T---- ---LM--ER- L-FH--NVKN -YD-V-L--- D--K-L---- -EF------E -M--VK----

Hu_H9 1999 ---KQYEI-D H------T-L NMINNKID-Q IQ-V-A---- ---L----K- L-EH-ANV-N -YN-VK-A-G S--M-D-K-- -EL-----DQ -M-T------

158 > 221

Hu_H3 1968 DHDVYRDEAL NNRFQIKGVE LKSGYKDWIL WI.SFAISCF LLCVVLLGFI MWACQRGNIR CNICI

Hu_H1 1918 -YPK-SE-SK L--EE-D--K -E-MGVYQ-- A-Y-TVA-SL V-L-S-GAIS F-M-SN-SLQ -R---

Sw_H1 1930 -YPK-SE-SK L--EE-D--K -E-MMVYQ-- A-Y-TVA-SL V-L-S-GAIS F-M-SN-SLQ -R---

Hu_H1 1934 -YPK-SE-SK L--EKVD--K -E-MGIYQ-- A-Y-TVA-SL V-L-S-GAIS F-M-SN-SLQ -R---

Hu_H2 1957 -YPK-EE-SK L--NE----K -S-MGVYQ-- A-YATVAGSL S-AIMMA-IS F-M-SN-SLQ -R---

Hu_H5 1997 -YPQ-SE--R L--EE-S--K -E-MGTYQ-- S-Y-TVA-SL A-AIMVA-LS L-M-SN-SLQ -R---

Hu_H9 1999 NRRK--E-SR LE-QK-E--K -E-EGTYK-- T-Y-TVA-SL V-AMGFAA-L F--MSN-SC- -----

Figure S2

Figure S3

Figure S4

Figure S5

Figure S6

Table S1. Data collection and refinement statistics for 18HA0

Data Collection 18HA0Resolution (Å) 49.4 – 3.0No. observations 125,868Unique reflections 51,346 (3,368)a

Completeness (%) 96.4 (95.1)a

<I/σI> 7.6 (1.4)a

Rsym

b 14.6 (52.0)a

RefinementNo. reflections (Working set) 48,772No. reflections (Test set) 2,573Rcryst (%)c 27.0Rfree (%)d 29.6Average B value (Å2) 40.4

Number of atoms in trimerProtein 11,871Carbohydrate 212PO4

2- 5

Ramachandran statistics (%)e

Most favored 81.9Additional allowed 16.6Generously allowed 0.8Disallowed 0.7

R.m.s.d. from ideal geometryBond length (Å) 0.019Bond angles (º) 1.85

a Numbers in parentheses refer to the highest resolution shell.b Rsym= (•h•IIi(h)-<I(h)>I•h•IIi(h))x100, where <I(h)> is the average intensity of ‘i’ symmetry-related observations with reflections with Bragg index ‘h’.c Rcryst = (•hklFo – Fc/•hklFo) x100d Rfree was calculated as for Rcryst, but on a test set comprising 5% of the data excluded beforerefinement.e Calculated using PROCHECK (S12).

Table S2 Comparison of r.m.s.d’s (Å) for different HA domains. A) For analyzing

differences in the overall structure, r.m.s.d values were calculated between monomers or domains

of different HA’s, after the Cα atoms of the HA2 domains of H3 (pdb:1HA0), H5 (pdb:1jsm) and

H9 (pdb:1jsd) were superimposed by sequence and structural alignment onto the equivalent

domain of 18HA0. The following residues were used for superimposition and calculation of the

r.m.s.d. values for the HA2 domain: 18HA0: A11 to A51, A276 to A324, B21 to B160; H3: A11

to A51, A276 to A324, B21 to B160; H5: A1 to A41, A273 to A321, B21 to B160 and H9: A1 to

A41, A267 to A315, B1 to B160. After superimposition, the following residues were used to

compare the HA1 domains: 18HA0: A52 to A275; H3: A52 to A275; H5: A42 to A272 and H9:

A42 to A266. The HA1 domain was further divided into the Receptor (R) and Esterase (E)

regions. The following residues were used to compare to R domains: 18HA0: A117 to A265; H3:

A117 to A265; H5: A110 to A262 and H9: A110 to A256. The following residues were

compared for the E domains: 18HA0: A52 to A116, A266 to A275 ; H3: A52 to A116, A266 to

A275; H5: A42 to A109, A263 to A272 and H9: A42 to A109, A257 to A266. For the column

labeled monomer, both HA1 and HA2 were used in the calculations.

Subtype pdbentry

Monomer HA1Domain

HA1‘R region’

HA1‘E region’

hu-H3 2hmg 4.1 6.7 7.4 2.6av-H5 1jsm 2.3 2.6 2.5 2.8sw-H9 1jsd 2.9 3.8 4.1 3.2

Table S3 R.m.s.d. (Å) for individual domains. Each domain was superimposed separately to

determine how the individual 18HA0 domains compared to equivalent domains in the other

structures. Residues were selected as for Table S2.

Subtype pdbentry

HA2Domain

HA1Domain

HA1‘R region’

HA1‘E region’

hu-H3 2hmg 2.2 2.2 1.6 2.6av-H5 1jsm 1.9 1.7 1.1 2.2sw-H9 1jsd 1.7 1.8 1.6 2.0

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