Supplementary Information

Structural basis of pharmacological chaperoning for human β-galactosidase

Hironori Suzuki1, Umeharu Ohto

1, Katsumi Higaki

2, Teresa Mena-Barragán

3, Matilde

Aguilar-Moncayo3, Carmen Ortiz Mellet

3, Eiji Nanba

2, Jose M. Garcia Fernandez

4, Yoshiyuki

Suzuki5, and Toshiyuki Shimizu

1,6,

1 Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,

Tokyo 113-0033, Japan.

2 Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori

University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan.

3 Department of Organic Chemistry, Faculty of Chemistry, University of Seville, Profesor García

González 1, E-41012, Seville, Spain

4 Institute for Chemical Research (IIQ), CSIC – University of Sevilla, Americo Vespucio 49, Isla de la

Cartuja, E-41092 Sevilla, Spain

5 International University of Health and Welfare Graduate School, Kita Kanemaru, Otawara, Tochigi

324-8501, Japan. (Present address: Tokyo Metropolitan Institute of Medical Science, 2-1-6

Kami-Kitazawa, Setagaya-ku, Tokyo 156-8506, Japan)

6 CREST, JST

Running head: Structural insights into pharmacological chaperones

Figure S1-S5

Supplementary Figure legends

Fig. S1 ITC analysis for the PC, NOEV or 6S-NBI-DGJ, to WT or mutant β-Gals.

Raw ITC data. Top panel: 20 injections of PC, NOEV or 6S-NBI-DGJ, solution were titrated

into WT or two mutants of β-Gal solution in ITC cell. The area of each injection peak

corresponds to the total heat released for that injection. Bottom panel: the binding isotherm

for these PCs and β-Gals interaction. The dissociation constants (KD) and stoichiometry (N)

were determined by curve fitting, whose data injected 6S-NBI-DGJ were fit with a fixed

stoichiometry of 1.

Fig. S2 Effects of PC compounds on the stability of the WT and two mutants of β-Gals at pH

4.8

The purified WT and two mutants of β-Gals were incubated in 0.1 M sodium citrate pH 4.8

with or without galactose, DGJ, NOEV and 6S-NBI-DGJ at 48°C for the incubated time.

Then, enzyme assay was performed in the same way as Figure 3.

Fig. S3 Overall structure of human β-Gal.

Structure of human β-Gal:NOEV complex. TIM barrel domain, green; TIM-β1 loop, purple;

β-domain 1, cyan; β-domain 2, orange. The NOEV are shown in space-filling representation.

Fig. S4 Sugar configuration of the PC compounds

Top panel: 6S-NBI-DGJ and 6S-NBI-GJ molecules are viewed form C1-C2 bond after

superimposing by those of 6-membered ring. The angle between 6-memberd and 5-membered

ring is shown.

Bottom panel: Only 6-memberd and 5-membered ring are shown. Substituents are omitted for

clarity.

Fig. S5. CD spectra of WT and two mutant β-Gals.

Far-UV CD spectra of WT and two mutants β-Gals with or without ligand in Na acetate buffer

(pH 5.0). The average of 16 scans are shown

Following pages are Summary Validation Reports provided by PDB.

3WEZ (β-GalWT

-NOEV)

3WF0 (β-GalWT

-6S-NBI-DGJ)

3WF1 (β-GalWT

-6S-NBI-GJ)

3WF2 (β-GalWT

-NBT-DGJ)

3WF3 (β-GalI51T

-galactose)

3WF4 (β-GalI51T

-6S-NBI-DGJ)

Page 2 Preliminary wwPDB X-ray Structure Validation Report 3THC

1 Overall quality at a glance iO

The reported resolution of this entry is 2.30 A.

Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown inthe following graphic. The table shows the number of entries on which the scores are based.

MetricWhole archive

(#Entries)Similar resolution

(#Entries, resolution range(A))

Rfree 65580 5027 (2.36-2.24)Clashscore 76988 5057 (2.34-2.26)

Ramachandran outliers 75395 5827 (2.36-2.24)Sidechain outliers 75377 5827 (2.36-2.24)

RSRZ outliers 65576 5027 (2.36-2.24)

The table below summarises the geometric issues observed across the polymeric chains and their fitto the electron density. The red, orange, yellow and green segments on the lower bar indicate thefraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria.The upper red bar (where present) indicates the fraction of residues that have poor fit to theelectron density.

Mol Chain Length Quality of chain

1 A 6051 B 6052 C 6032 D 603

Page 2 Preliminary wwPDB X-ray Structure Validation Report 3THC

1 Overall quality at a glance iO

The reported resolution of this entry is 2.15 A.

Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown inthe following graphic. The table shows the number of entries on which the scores are based.

MetricWhole archive

(#Entries)Similar resolution

(#Entries, resolution range(A))

Rfree 65580 7452 (2.22-2.10)Clashscore 76988 5113 (2.20-2.12)

Ramachandran outliers 75395 5015 (2.20-2.12)Sidechain outliers 75377 5016 (2.20-2.12)

RSRZ outliers 65576 7451 (2.22-2.10)

The table below summarises the geometric issues observed across the polymeric chains and their fitto the electron density. The red, orange, yellow and green segments on the lower bar indicate thefraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria.The upper red bar (where present) indicates the fraction of residues that have poor fit to theelectron density.

Mol Chain Length Quality of chain

1 A 6051 B 6052 C 6032 D 603

Page 2 Preliminary wwPDB X-ray Structure Validation Report 3THC

1 Overall quality at a glance iO

The reported resolution of this entry is 2.30 A.

Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown inthe following graphic. The table shows the number of entries on which the scores are based.

MetricWhole archive

(#Entries)Similar resolution

(#Entries, resolution range(A))

Rfree 65580 5027 (2.36-2.24)Clashscore 76988 5057 (2.34-2.26)

Ramachandran outliers 75395 5827 (2.36-2.24)Sidechain outliers 75377 5827 (2.36-2.24)

RSRZ outliers 65576 5027 (2.36-2.24)

The table below summarises the geometric issues observed across the polymeric chains and their fitto the electron density. The red, orange, yellow and green segments on the lower bar indicate thefraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria.The upper red bar (where present) indicates the fraction of residues that have poor fit to theelectron density.

Mol Chain Length Quality of chain

1 A 6051 B 6052 C 6032 D 603

Page 2 Preliminary wwPDB X-ray Structure Validation Report 3THC

1 Overall quality at a glance iO

The reported resolution of this entry is 2.00 A.

Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown inthe following graphic. The table shows the number of entries on which the scores are based.

MetricWhole archive

(#Entries)Similar resolution

(#Entries, resolution range(A))

Rfree 65580 5535 (2.02-1.98)Clashscore 76988 6016 (2.00-2.00)

Ramachandran outliers 75395 5929 (2.00-2.00)Sidechain outliers 75377 5931 (2.00-2.00)

RSRZ outliers 65576 5534 (2.02-1.98)

The table below summarises the geometric issues observed across the polymeric chains and their fitto the electron density. The red, orange, yellow and green segments on the lower bar indicate thefraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria.The upper red bar (where present) indicates the fraction of residues that have poor fit to theelectron density.

Mol Chain Length Quality of chain

1 A 6051 B 6052 C 6032 D 603

Page 2 Preliminary wwPDB X-ray Structure Validation Report 3THC

1 Overall quality at a glance iO

The reported resolution of this entry is 2.20 A.

Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown inthe following graphic. The table shows the number of entries on which the scores are based.

MetricWhole archive

(#Entries)Similar resolution

(#Entries, resolution range(A))

Rfree 65580 5143 (2.26-2.14)Clashscore 76988 5129 (2.24-2.16)

Ramachandran outliers 75395 5031 (2.24-2.16)Sidechain outliers 75377 5032 (2.24-2.16)

RSRZ outliers 65576 5143 (2.26-2.14)

The table below summarises the geometric issues observed across the polymeric chains and their fitto the electron density. The red, orange, yellow and green segments on the lower bar indicate thefraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria.The upper red bar (where present) indicates the fraction of residues that have poor fit to theelectron density.

Mol Chain Length Quality of chain

1 A 6051 B 6052 C 6032 D 603

Page 2 Preliminary wwPDB X-ray Structure Validation Report 3THC

1 Overall quality at a glance iO

The reported resolution of this entry is 2.11 A.

Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown inthe following graphic. The table shows the number of entries on which the scores are based.

MetricWhole archive

(#Entries)Similar resolution

(#Entries, resolution range(A))

Rfree 65580 8442 (2.20-2.04)Clashscore 76988 5415 (2.18-2.06)

Ramachandran outliers 75395 5348 (2.18-2.06)Sidechain outliers 75377 5349 (2.18-2.06)

RSRZ outliers 65576 8441 (2.20-2.04)

The table below summarises the geometric issues observed across the polymeric chains and their fitto the electron density. The red, orange, yellow and green segments on the lower bar indicate thefraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria.The upper red bar (where present) indicates the fraction of residues that have poor fit to theelectron density.

Mol Chain Length Quality of chain

1 A 6051 B 6052 C 6033 D 602