A systematic dissection of sequence elements ...

A systematic dissection of sequence elements determining β-Klotho and FGF interaction and signaling

Sally Yu Shi, Ya-Wen Lu, Jason Richardson, Xiaoshan Min, Jennifer Weiszmann, William G. Richards, Zhulun Wang, Zhongqi Zhang, Jun Zhang, and Yang Li

WT 317F-A 318K-A 321Q-A 380K-A 381P-A 387K-A 388M-A 389G-A 391N-A 392V-A 394L-A 395N-A 420F-A 422D-A 430T-A 431T-A 434Y-A 435M-A 437K-A 523C-A 526S-A 527W-A 528G-A 530T-A 531E-A 751N-A 752P-A 753Y-A 755D-A 756S-A 757H-A 758W-A 759R-A 828T-A 847I-A 848Q-A 850L-A 852D-A 853I-A 855R-A 857S-A 858S-A 859P-A 861R-A 862L-A 877R-A 893D-A 894D-A 895Q-A 897L-A 898E-A 899D-A 901R-A 902L-A 904K-A 905Y-A 906Y-A 908G-A 909K-A 926K-A 936E-A 937K-A 938S-A 939K-A 941R-A

β-Klotho (µg/mL)

4

B 4

WT 317F-R 318K-R 321Q-R 380K-R 381P-R 386A-R 387K-R 388M-R 389G-R 391N-R 392V-R 394L-R 395N-R 420F-R 422D-R 430T-R 431T-R 434Y-R 435M-R 437K-R 523C-R 526S-R 527W-R 528G-R 530T-R 531E-R 750A-R 751N-R 752P-R 753Y-R 754A-R 755D-R 756S-R 757H-R 758W-R 828T-R 847I-R 848Q-R 850L-R 852D-R 853I-R 857S-R 858S-R 859P-R 862L-R 863A-R 893D-R 894D-R 895Q-R 896A-R 897L-R 898E-R 899D-R 902L-R 904K-R 905Y-R 906Y-R 908G-R 909K-R 926K-R 936E-R 937K-R 938S-R 939K-R

β-Klotho (µg/mL)

Supplementary Figure S1

A Ala mutation

3 WT Residues in KL1 Residues in KL2

2

1

0

Arg mutation

3

2

1

0

Supplementary Figure S1. Expression of β-Klotho (A) alanine and (B) arginine mutants in CM determined by ELISA. Dotted lines represent the cutoff point (0.3 µg/mL) for subsequent solid-phase binding assay.

WT 317F-R 318K-R 321Q-R 380K-R 381P-R 386A-R 387K-R 388M-R 389G-R 391N-R 394L-R 395N-R 422D-R 431T-R 434Y-R 435M-R 437K-R 523C-R 526S-R 752P-R 753Y-R 754A-R 755D-R 756S-R 758W-R 848Q-R 850L-R 852D-R 853I-R 857S-R 858S-R 859P-R 862L-R 863A-R 895Q-R 896A-R 897L-R 898E-R 904K-R 905Y-R 908G-R 909K-R 926K-R 936E-R 937K-R 938S-R 939K-R

Fold change in EC50

B WT 317F-A 318K-A 321Q-A 380K-A 381P-A 387K-A 388M-A 389G-A 391N-A 392V-A 422D-A 430T-A 431T-A 434Y-A 435M-A 437K-A 523C-A 526S-A 530T-A 531E-A 752P-A 753Y-A 755D-A 756S-A 757H-A 758W-A 759R-A 847I-A 848Q-A 850L-A 852D-A 853I-A 855R-A 857S-A 858S-A 859P-A 861R-A 862L-A 877R-A 893D-A 894D-A 895Q-A 897L-A 898E-A 901R-A 902L-A 904K-A 905Y-A 906Y-A 908G-A 909K-A 936E-A 937K-A 938S-A 939K-A 941R-A

Fold change in EC50

Supplementary Figure S2 A 2.0

2.5

Ala mutation

1.5

WT Residues in KL1 Residues in KL2

1.0

0.5

0.0

Arg mutation

2.0

1.5

1.0

0.5

0.0

Supplementary Figure S2. EC50 values determined from the solid-phase binding assay between β-Klotho and an anti-β-Klotho antibody, 39F7, for (A) alanine and (B) arginine mutants. EC50 values are expressed as fold change relative to WT CM.

Supplementary Figure S3 B Gene expression (Fold change over 293T)

1.5 1.0 0.5

KL B

KL

0.0 FG FR 1 FG FR 2 FG FR 3 FG FR 4

Gene expression (Fold change overFGFR1)

A

40000

KLB

30000 20000 10000 0

293T

293T + KLB WT

Supplementary Figure S3. (A) Expression of FGFR isoforms, α-Klotho and β-Klotho in 293T cells measured by quantitative RT-PCR. (B) Expression of β-Klotho in 293T cells transiently transfected with full-length WT β-Klotho construct measured by quantitative RT-PCR. Results are mean ± SD of two independent experiments.

Supplementary Figure S4

Average Possible Log (Protection Factor)

A

8 7 6 5 4 3 2 1 0

Free FGF19 Bound to β-Klotho

40

60

80

100

120

140

160

180

200

140

160

180

200

Residue Number

∆Log (Protection Factor)

B 1

0

-1 40

60

80

100

120 Residue Number

Supplementary Figure S4. Identification of potential β-Klotho interaction regions on FGF19 by HDX-MS. (A) Average possible protection factor plot and (B) differential protection factor plot of β-Klotho-bound and free FGF19.

Fold change in Kd

174P-R

184Q-R 185P-R 186P-R 187D-R 188V-R 189G-R 190S-R 191S-R 192D-R 193P-R 194L-R 195S-R 196M-R 197V-R

202G-R 204S-R 205P-R 206S-R 207Y-R 208A-R 209S-R

184Q-R 185P-R 186P-R 187D-R 188V-R 189G-R 190S-R 191S-R 192D-R 193P-R 194L-R 195S-R 196M-R 197V-R

202G-R 204S-R 205P-R 206S-R 207Y-R 208A-R 209S-R

0 174P-R

10 164F-R

20

164F-R

30 WT

B

WT

Fold change in Ka 1.5

* 209S-A

0 209S-A

203R-A 204S-A 205P-A 206S-A 207Y-A

*

203R-A 204S-A 205P-A 206S-A 207Y-A

186P-A 187D-A 188V-A 189G-A 190S-A 191S-A 192D-A 193P-A 194L-A 195S-A 196M-A 197V-A 198G-A

174P-A

164F-A

WT

Fold change in Ka

1.5

186P-A 187D-A 188V-A 189G-A 190S-A 191S-A 192D-A 193P-A 194L-A 195S-A 196M-A 197V-A 198G-A

174P-A

164F-A

WT

Fold change in Kd

Supplementary Figure S5 A Ala mutation

1.0

0.5

0.0 *

100

50

10 10

5

*

Arg mutation

1.0

0.5

0.0

Supplementary Figure S5. Analysis of β-Klotho binding kinetics of selected Fc-FGF21 (A) alanine and (B) arginine mutants by bio-layer interferometry. Top, association rate constant (Ka); bottom, dissociation rate constant (Kd). Values are expressed as fold change relative to WT. *, could not be estimated.

Supplementary Figure S6 B

Competition with FGF21

150

IC50 (nM) FGF21183-209 2.1 4.5 183P-A d.n.c 188V-A d.n.c 193P-A 6.0 198G-A 7.3 203R-A d.n.c 207Y-A

100 50 0

-10

-9 -8 -7 -6 Log[FGF21 peptide], M

Alpha signal (% β-Klotho binding)

Alpha signal (% β-Klotho binding)

A

Competition with FGF19

150

IC50 (nM) FGF21183-209 0.52 1.0 183P-A 5.2 188V-A 5.5 193P-A 2.6 198G-A 0.28 203R-A d.n.c 207Y-A

100 50 0

-10

-9 -8 -7 -6 Log[FGF21 peptide], M

Supplementary Figure S6. Inhibition of β-Klotho binding to (A) FGF21 and (B) FGF19 by FGF21 C-terminal mutant peptides measured using AlphaScreen. d.n.c., did not converge.

Supplementary Figure S7 Fold change in EC50 or IC50

100000

FGF19 FGF21

20 20 15 10 5 0 FGF19: S D M F S S P L E T D S M D P F G L V T G L E A V R S P S F E K FGF21: G I L A P Q P P D V G S S D P L S M V - G P S Q G R S P S Y A S

Supplementary Figure S7. IC50 of FGF19 mutant peptides and EC50 of Fc-FGF21 alanine mutants graphed along aligned C-terminal sequences. Conserved residues are colored in red.