1

Supplemental data for

SIALIC ACID MUTAROTATION IS CATALYSED BY THE

ESCHERICHIA COLI β-PROPELLER PROTEIN YJHT

Emmanuele Severi1

, Axel Müller1,2,#

, Jennifer R. Potts1,3

, Andrew Leech4

, David

Williamson3

, Keith S Wilson2

& Gavin H Thomas1

*

1

Department of Biology (Area 10), 2

York Structural Biology Laboratory, Department of

Chemistry, 3

Department of Chemistry and 4

Technology Facility, Department of Biology

(Area 15), University of York, York YO10 5YW, UK.

#

Current address: Caltech, 157 Broad Center, MC 114-96, Pasadena, CA 91125.

*Corresponding author. Tel. +44 1904 328678. Email ght2@york.ac.uk

2

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Protein analysis by mass spectrometry

An API Qstar using an ionspray source was used for electrospray mass spectrometry (ES-

MS). For the analysis of YjhT under denaturing conditions, purified YjhT dialysed in

ddH2O was diluted to a final concentration of 1 µM into 1:1 acetonitrile:water containing

0.1% formic acid. The sample was infused at 5 µl min-1

and data collected over 5 minutes

within a m z-1

range of 550-2000. For the analysis of YjhT under native conditions, the

protein was diluted to 5 µM into dH2O containing 3% methanol. In this case the sample

was infused at 9 µl min-1

; multiply charged protein states were observed in the m z-1

ranges of 800-1700 and 1700-3200. The raw m z-1

were deconvoluted to mass spectra

using the Bayesian Protein Reconstruction routine in the BioAnalyst software provided

the manufacturer.

Filter binding assay

[14

C]-Neu5Ac binding to YjhT was investigated by a filter-binding assay as previously

described (1). Briefly, purified YjhT was added to 50 µl of 50 mM Tris-HCl pH 8 at

various concentrations (0, 1, 2, 5, and 10 µM). After incubation on ice for 10 min, 2.5

µM [14

C]-Neu5Ac (final concentration) was added to all samples, which were then

equilibrated on ice for other 10 min. The protein was precipitated by addition of 1 ml of a

saturated solution of (NH4)2SO4 followed by a 20 min incubation on ice. The samples

were then filtered through 0.22 µm pore-size nitrocellulose filters (Millipore), washed

with 4 ml of the ammonium sulphate solution, and the radioactivity within them was

measured as for the sialic acid uptake assay. 2.5 µM of purified Haemophilus influenzae

Neu5Ac-binding protein SiaP (2;3) was used as a positive control for [14

C]-Neu5Ac

binding. The efficiency of this method, calculated as the ratio of the actual amount of

precipitated bound SiaP over the theoretical one, was ~30%.

3

Analytical ultracentrifugation (AUC)

Sedimentation equilibrium experiments were conducted on a Beckman Optima XL/I

analytical ultracentrifuge, using Beckman cells with 12 mm path length six channel

charcoal-filled Epon centerpieces and quartz windows, in an AN-50Ti rotor.

Approximately 120 µl reference buffer (150 mM NaCl, 20 mM Tris-HCl pH 7.5) and a

slightly smaller volume (118 µl) of sample were loaded into the cells. Absorbance scans

were taken at 3000 rpm to check loading concentrations and that the cell contents were

uniformly distributed. The speed was increased to the required rate and absorbance scans

taken at approximately 3 hourly intervals until sedimentation equilibrium was achieved

(judged by absence of change in overlays or subtractions of successive scans). Scans

were conducted in step mode with 10 replicates per data point. Runs were conducted at

20 o

C. A range of protein concentrations from 2 mg/ml to 0.01 mg/ml was loaded into the

cells; scans were taken at 300, 280 or 230 nm as appropriate. This YjhT preparation had

been additional purified using size exclusion chromatography and eluted in reference

buffer. Data were analysed using the Beckman Origin software package supplied with the

centrifuge. Partial specific volume and buffer density were calculated using the program

SEDNTERP (4).

Sialidase activity test

NMR samples containing 1 mM sialyllactose were prepared as described in Experimental

Procedures. The samples were reacted at 37 ºC with either 0.6 U of Clostridium

perfringens sialidase for one hour or with 0.5 µM YjhT (final concentration) overnight

before the acquisition of 1

H-NMR spectra. Reference spectra were acquired for unreacted

sialyllactose and sialyllactose incubated at 37 ºC overnight with no added protein.

Growth experiments

For growth experiments starter cultures were grown as for the [14

C]-Neu5Ac uptake assay

except that M9 containing Neu5Ac 1 mg/ml was used. Overnight grown cells were

4

harvested, washed four times in M9 salts and inoculated to an initial OD650 of 0.025 in

M9 supplemented with Neu5Ac at 0.25, 0.5, or 1 mg/ml. The OD650 of the growing

cultures were measured hourly. To assay growth on colominic acid (poly-α(2-8)-

Neu5Ac, Sigma) cells were prepared as above and inoculated to an initial OD650 of 0.05

in M9 supplemented with colominic acid 0.5 mg/ml; after taking a sample to measure the

initial OD650 Clostridium perfringens sialidase was added to the cultures at a final

concentration of 0.2 U/ml, after which growth was followed hourly.

Sequence analysis and bioinformatics

Structural alignments were created using ESPript (5).

5

Supplemental figure legends

Supplemental Fig. 1. ES-MS analysis of purified YjhT. ES-MS spectrum of 5 µM YjhT

showing a small amount of dimer [molecular masses for monomeric and dimeric YjhT

after processing of the leader peptide are 38686 and 77372 atomic mass units (amu),

respectively]. Inset. Coomassie-stained SDS-polyacrylamide gel of purified YjhT (1 µg)

as used for crystallisation trials (L: protein ladder).

Supplemental Fig. 2: Analysis of the Neu5Ac-YjhT interaction by filter-binding assay.

SiaP is a high-affinity Neu5Ac-binding protein from H. influenzae (2).

Supplemental Fig. 3. 1

H-NMR investigation of the potential sialidase activity of YjhT.

A) 1

H-NMR spectrum of a 1 mM mixture of α(2-3)- and α(2-6)-sialyllactose focussing

on the H3 region. 1,2: H3eq peaks of α(2-3)- and α(2-6)-sialyllactose, respectively; 3,4:

H3ax peaks of α(2-3)- and α(2-6)-sialyllactose, respectively. B) 1

H-NMR spectrum of

the same mixture after 1 hour incubation at 37 ºC with 0.6 U Clostridium perfringens

sialidase. The hydrolysis of both sialyllactose isoforms is complete as shown by the

disappearance of the H3 characteristic peaks (compare with A)). During the reaction both

the α- and β-anomers of Neu5Ac are produced. 5,8: respectively, H3eq and H3ax peaks

of α-Neu5Ac; 6,7: respectively, H3eq and H3ax peaks of β-Neu5Ac. C) 1

H-NMR

spectrum of the sialyllactose mixture after incubation at 37 ºC with 0.5 µM YjhT o/n.

This spectrum is identical to that in A) as well as to that of the same sialyllactose mixture

after o/n incubation with no added protein (not shown).

Supplemental Fig. 4. Growth phenotypes of a ∆yjhT mutant in minimal medium

supplemented with colominic (poly-α(2-8)-sialic) acid or monomeric Neu5Ac. These

results are the average of three biological replicas ± SD, and are shown for the WT

(continuous line), the ∆yjhT (dash-dotted line) and ∆yjhS (dashed line) mutants.

A) Growth of BW25113 and isogenic mutant strains in M9 minimal medium containing

0.5 mg/ml colominic acid and Clostridium perfringens sialidase (0.2 U/ml). B) Growth of

6

the same strains in M9 minimal medium containing 0.25 mg/ml Neu5Ac. This

concentration of Neu5Ac stimulates growth at a rate comparable to that of sialidase-

treated colominic acid.

Supplemental Fig. 5. Analysis of the Kelch motifs in YjhT. A) Structure based

alignment of the 6 blades of YjhT aligned by the four -strands in each blade (a,b,c and

d). A consensus is drawn below indicating the consered twin glycine and conserved

tryptophan residue in stand d. B). View of the YjhT protein from above (the long

dimerisation helices have been removed for clarity) illustrating the positions of the twin

glycines (yellow space filling) and the conserved tryptophan (pink space filling)

Supplemental Fig. 6. AUC analysis of purified YjhT. A) A typical sedimentation

equilibrium scan, from a sample of 0.47 mg/ml at 16500 rpm, scanned at 280 nm. The

solid line is a fit to a single species model indicating a MW of 73.9 kDa, within 5% of the

expected MW for a dimer. The small discrepancy is within the error expected from the

calculated estimate of partial specific volume. Inset: residuals distribution. B) A plot of

molecular weight as a function of concentration, derived from several scans, shows no

indication of dissociation at low concentration or of formation of higher oligomers at

high concentrations.

Supplemental Fig. 7. Structural alignment of YjhT with close orthologues. The

alignment starts with the sequence QTYPD for HI0148 which is that determined by N-

terminal sequencing of soluble H. influenzae protein (6). The figure was drawn using

ESPript (5).

Supplemental Fig. 8. Surface structure of YjhT illustrating the cleft that contains the surface

exposed Glu209 (red surface) and Arg215 (blue surface) residues. The secondary structure of the

protein is visible beneath the surface with Glu209 and Arg215 shown in stick representations.

7

Supplemental Table 1 Oligonucleotides used in this study

Name Sequence (5'

EcyjhT-NdeI ACAGTCCATATGAATAAAACAATAACG

EcyjhT-XhoI CCCTCGAGGTTTTGTACTGTGACTTTA

NanMK11Afor CCATTTGCTAGCGGTACCGGAGCAATTGATAAC

NanMK11Arev ACCGCTAGCAAATGGCACAGGAGTTTCCGG

NanME209Afor AATGGCGCGGCCAAACCCGGGTTGCGAACG

NanME209Arev TTTGGCCGCGCCATTAATAAGCCAGGTTTTATC

NanMR215Afor GGATTGGCCACGGATGCCGTATTTGAACTTG

NanMR215Arev ATCCGTGGCCAATCCTGGTTTGGCTTCGCC

NanMH278Afor TATGCGGCCGAAGGCCTGAAAAAATCATATAGC

NanMH278Arev GCCTTCGGCCGCATAGTTCTTACCGTTCTG

NanMK283Afor CTGAAAGCTTCATATAGCACTGATATTCATC

NanMK283Arev ATATGAAGCTTTCAGGCCTTCATGCGCATAG

NanMY309Afor CGGGCCGCGGGAGTAAGCTTGCCCTGGAAT

NanMY309Arev TACTCCCGCGGCCCGACCTTGCGATAATTC

NanME325Afor GGCGGTGCAACGGCCGGCGGCAAAGCGGTG

NanME325Arev GGCCGTTGCACCGCCAATAATCAATAGACT

Restriction sites used for cloning or screening of recombinant constructs are underlined.

Letters in bold identify mis-sense mutations, while italicised letters indicate silent

mutations introduced to engineer novel sites in mutant yjhT (nanM) alleles.

8

Supplemental Fig. 1

38685

77371

38685

77371

9

Supplemental Fig. 2

Control YjhT SiaP

pm

ol [1

4

C]-N

eu

5A

c

0

15

30

45

10

Supplemental Fig. 3

ppm

13

5

2

4

6

7

8

C)

B)

A)

ppm

13

5

2

4

6

7

8

C)

B)

A)

11

Supplemental Fig. 4

12

Supplemental Fig. 5

a b c d

I 14 TGAIDNDTVYIGLG.....∆.........GTAWYKLDTQAKDKKWTALAA 47

II 60 TSAFIDGNLYVEGG.....∆.........FNDVHKYNP..KTNSWVKLMS 100

III 113 VTFVHNGKAYVTGG.....∆.........NKFLLSFDP..STQQWSYAGE 182

IV 195 AVVNKGDKTWLING.... ∆.........TDAVFELDFTGNNLKWNKLAP 233

V 247 FAGISNDSLIFAGG.... ∆.........STDIHLWH....NGKWDKSGE 302

VI 311 VSLPWNNSLLIIGG.... ∆.........DSVLITVK....DNKVTVQN 349

.....pp.hhhhGG..............................W.....

A)

B)

a b c d

I 14 TGAIDNDTVYIGLG.....∆.........GTAWYKLDTQAKDKKWTALAA 47

II 60 TSAFIDGNLYVEGG.....∆.........FNDVHKYNP..KTNSWVKLMS 100

III 113 VTFVHNGKAYVTGG.....∆.........NKFLLSFDP..STQQWSYAGE 182

IV 195 AVVNKGDKTWLING.... ∆.........TDAVFELDFTGNNLKWNKLAP 233

V 247 FAGISNDSLIFAGG.... ∆.........STDIHLWH....NGKWDKSGE 302

VI 311 VSLPWNNSLLIIGG.... ∆.........DSVLITVK....DNKVTVQN 349

.....pp.hhhhGG..............................W.....

A)

B)

13

Supplemental Fig. 6

A)

B)

Radius (cm)

5.85 5.90 5.95 6.00 6.05

Ab

so

rb

an

ce

(A

280)

0.0

0.2

0.4

0.6

0.8

1.0

[YjhT] (mg/ml)

0 1 2 3 4 5

Ap

pa

re

nt M

W (K

Da

)

71

72

73

74

75

76

77

A)

B)

Radius (cm)

5.85 5.90 5.95 6.00 6.05

Ab

so

rb

an

ce

(A

280)

0.0

0.2

0.4

0.6

0.8

1.0

[YjhT] (mg/ml)

0 1 2 3 4 5

Ap

pa

re

nt M

W (K

Da

)

71

72

73

74

75

76

77

14

Supplemental Fig. 7

15

Supplemental Fig. 8

16

Supplemental Reference List

1. Walmsley, A. R., Shaw, J. G., and Kelly, D. J. (1992) J.Biol.Chem. 267, 8064-8072

2. Severi, E., Randle, G., Kivlin, P., Whitfield, K., Young, R., Moxon, R., Kelly, D.,

Hood, D., and Thomas, G. H. (2005) Mol.Microbiol. 58, 1173-1185

3. Muller, A., Severi, E., Mulligan, C., Watts, A. G., Kelly, D. J., Wilson, K. S.,

Wilkinson, A. J., and Thomas, G. H. (2006) J.Biol.Chem. 281, 22212-22222

4. Laue, T. M., Shah, B. D., Ridgeway, T. M., and Pelletier, S. L. (1992) in Analytical

Ultracentrifugation in Biochemistry and Polmer Science, Eds S.E.Harding,

A.J.Rowe and J.C.Horton, The Royal Society of Chemistry, Cambridge 90-125

5. Gouet, P., Courcelle, E., Stuart, D. I., and Metoz, F. (1999) Bioinformatics. 15, 305-

308

6. Langen, H., Takacs, B., Evers, S., Berndt, P., Lahm, H. W., Wipf, B., Gray, C., and

Fountoulakis, M. (2000) Electrophoresis 21, 411-429