Supplemental Experimental procedures

Thermodynamic analysis - Thermodynamic experiments were performed using a Biacore T100

biosensor over a temperature range of 4°C to 40°C. Data were fitted to the integrated non-linear

form of van’t Hoff equation (Zhukov, A., & Karlsson, R. (2007). Statistical aspects of van't Hoff

analysis: a simulation study. J. Mol. Recognit. 20, 379-385) (Figure S1):

(1)

where R is the gas constant, T0 is a reference temperature, commonly 298 K, KA(T0) and ΔH°(T0)

- association binding constant and binding enthalpy at T0, and ΔC°p is a heat capacity increment.

In several cases, where the relationship lnKA vs. 1/T assumed strictly linear form, the heat

capacity contribution was virtually equal to zero and the relationship was described by a more

simple form of the same equation:

(2)

Confidence probability of 0.95 was used for fitting. The fitting was performed using Microcal

Origin 7 software.

Reporter gene assay - The effect of different ALK1 mutations on BMP9 and BMP10 signaling

was evaluated in human glioblastoma T98G cells. Briefly, TG98 cells were co-transfected with

pGL3-BRE reporter plasmid containing firefly luciferase gene controlled by SMAD1/5/8 and

pRL-CMV-luc plasmid containing Renilla luciferase controlled by a constitutively active CMV

promoter at a 20:1 ratio. Cells were incubated for 16 hours at 37°C with BMP9 (600 pg/mL) or

BMP10 (350 pg/mL) with or without ALK1 mutants. Cells were lysed, assayed using a Dual-

Luciferase reporter assay kit (Promega) and results expressed as a ratio of firefly over Renilla

luciferase activity in relative light units.

Cooperativity experiment performed by SPR – ALK1ECD-BMP9 complex was formed by mixing

BMP9 with ALK1ECD-hFc. Pre-assembled complex was captured on anti-hFC IgG Biacore chip.

Different concentrations of ActRIIBECD-mFC were injected over captured pre-assembled binary

ALK1-BMP9 complex. Kinetic parameters for ActRIIB binding to binary ALK1-BMP9 complex

were compared with kinetic parameters of ActRIIB binding to BMP9. In a separate experiment

assay was flipped and ActRIIBECD-BMP9 complex was formed by mixing BMP9 with

ActRIIBECD-mFC. Assembled binary complex was captured on anti-mFc IgG Biacore chip.

2

Different concentrations of ALK1ECD-hFc were injected over captured binary ActRIIB-BMP9

complex. Kinetic parameters for ALK1 binding to binary ActRIIB-BMP9 complex were

compared with kinetic parameters of ALK1 binding to BMP9.

Supplemental Figures:

Supplemental Figure 1. Van’t Hoff plots for (A) BMP9 and (B) BMP10 binding to

ALK1ECD-Fc, ActRIIBECD-Fc and ActRIIAECD-Fc.

Supplemental Figure 2. Higher order assembly in the ALK1ECD-BMP9-ActRIIBECD crystal

structure Asn24 on ActRIIB facilitates dimerization of two ternary complexes via the attached N-

acetyl-glucosamine (NAG).

Supplemental Figure 3. Comparison of ALK1 with ALK3 and ALK6 (A) Structure-based

sequence alignment of ALK1, ALK3 and ALK6, showing residues involved in ligand

recognition. Polar (red) and hydrophobic (grey) contacts are highlighted. (B) Peeled-away surface

of ALK1, ALK3 and ALK6 mapping residues involved in ligand recognition (colored as in A).

Supplemental Figure 4. Sequence alignment of BMP9 with select TGF-β ligands. BMP9

residues at the ALK1 and ActRIIB interface are highlighted. Asterisks denote key specificity

determinants.

Supplemental Figure 5. The ActRIIB/BMP9 interface. Interactions within (A) the conserved

ActRIIB hydrophobic binding pocket and (B) surrounding interface are highlighted.

Supplemental Figure 6. Binding of type I and type II receptor ECDs in ALK1-BMP9-

ActRIIB complex is independent. To measure possible cooperativity binding of ActRIIBECD to

pre-assembled ALK1ECD-BMP9 binary complex (A) was compared to binding of ActRIIBECD to

BMP9 (B). Assay was flipped and binding of ALK1 to pre-assembled ActRIIBECD-BMP9

complex (C) was compared to binding of ALK1ECD to BMP9 (D).

Supplemental Tables:

Supplemental Table 1: Crystallographic data collection and refinement statistics.

Supplemental Table 2: Interactions at the ALK1/BMP9 interface.

Supplemental Table 3: Interactions at the ActRIIB/BMP9 interface.

3.1 3.2 3.3 3.4 3.5 3.6 3.7

20

22

24

26

28

3.1 3.2 3.3 3.4 3.5 3.6 3.7

18

20

22

24

26

ln K

aln

Ka

ActRIIAECD-Fc/BMP10

ActRIIBECD-Fc/BMP10

ALK1ECD-Fc/BMP10

37oC

ActRIIAECD-Fc/BMP9

ActRIIBECD-Fc/BMP9

ALK1ECD-Fc/BMP9

A

25oC

B

1000/T, K-1

Supplemental Figure 1

Supplemental Figure 2

NAG

NAGNAG

NAG

NAG

NAG NAG

N24 N24

ActRIIBECD

ALK1ECD

BMP9BMP9

ActRIIBECD/ActRIIB ECD

Conserved residuesPolar contacts

Hydrophobic contacts

788869

- - P L V T C T C E S P H C K G - - - - P T C R G - AWC T V V L V R E E G R H P Q E H R G C G N - L H R - E L C RC C C C C C CHH EE E N H E RV RVH H L LT L P F L K C Y C S G - H C P D D A I N N T C I T N G H C F A I I E E D D Q G E T T L A S G C MK Y E G S D F Q C KC C C C C C CHHH C E D QT K GY F KN IFPCG D- - - - IL R C K C H H - H C P E D S V N N I C S T D G Y C F T M I E E D D S G MP V V T S G C L G L E G S D F Q C RC C C C C C CHH QDECSTHH L G F RT LC P D S D F M E

798970

103120100

G R P T E - - F V N H Y C C D - S H L C N H N V S L V L E - - - - - - -C C CR EG T F V HD S P K A Q L R R T I E C C R - T N L C N Q Y L Q P T L P P V V I G P FC C CQ RD K A L ID T P I P H Q R R S I E C C T E R N E C N K D L H P T L P P L - - - - -C C CPD T P I P Q R

303217

F1 loop F2 loop

F3 loop

Pre-helix loop

ALK1ALK3ALK6

ALK1ALK3ALK6

M

* * * * * * *

*

* ** * * *

ALK1

ALK3

ALK1

ALK3

* *

β1

β1

β2

β2

β3

β3

β4

β4

β5

β5

α1

α1

S

A

E58

E59

V56

V54H66

H40L72H73

E75R78

G79

R80T82

E83

F84

V85H87

L76

N71

ALK1

B

H23

H24S56

P26

D28

C25

S35T36

C58

T55

R78

P74I73 P72

T71 D70

R69

F66Q67 D65

E62

S64

D37

I80

Q76

M43

F41

E45

L61

L59

Q94

R97

Q86

E81

D84

C77T55

H43

K92P91

S90D89

K88F85

Y80

A93

I62F60

M78K79

C44P45

I99

G82

G42

D46

N57

ALK3 ALK6

Supplemental Figure 3

ECD ECD ECD

BMP9BMP10BMP2BMP4BMP6BMP7GDF5GDF6GDF7GDF8GDF11Activin AActivin B

BMP9BMP10BMP2BMP4BMP6BMP7GDF5GDF6GDF7GDF8GDF11Activin AActivin B

BMP9BMP10BMP2BMP4BMP6BMP7GDF5GDF6GDF7GDF8GDF11Activin AActivin B

1091618404021213017171313

- - - - - - - - - - - - - - - - - - - - S AG AG S H - - - - - - - - - - - - - - - - - - - - - - C Q K- - - - - - - - - - - - - - - - - - - - - NA KG NY - - - - - - - - - - - - - - - - - - - - - - C K R- - - - - - - - - - - - - - Q A K HKQR K R L K S - - - - - - - - - - - - - - - - - - - - - - S C K R- - - - - - - - - - - - - S P K HHS QR AR K K NK - - - - - - - - - - - - - - - - - - - - - NC R R- - - - - - - - - - - - S A S S R R R QQS R NR S T Q S QD V AR V S S A S D Y NS S E L K T AC R K- - - - - - - - - - - - S T G S KQR S QNR S K T P K NQE A L R MANV A E NS S S DQR QAC K KA P L A - T R - - - - - - - - QG K R P S K NL K AR - - - - - - - - - - - - - - - - - - - - - - C S RT A F A - S R - - - - - - - - HG K R HG K K S R L R - - - - - - - - - - - - - - - - - - - - - - C S KT A L AG T R T AQG S GGG AGR G HGR R GR S R - - - - - - - - - - - - - - - - - - - - - - C S R- - - - - - - - - - - - - - D F G L DC D E HS T E S - - - - - - - - - - - - - - - - - - - - - R C C R- - - - - - - - - - - - - - NL G L DC D E HS S E S - - - - - - - - - - - - - - - - - - - - - R C C R- - - - - - - - - - - - - - - - G L E C DG K V N - - - - - - - - - - - - - - - - - - - - - - - I C C K- - - - - - - - - - - - - - - - G L E C DGR T N - - - - - - - - - - - - - - - - - - - - - - - L C C R

58576466888869697863636565

T S L R V NF E D I GWD SW I I A P K E Y E A Y E C KGGC F F P L ADD V T P - - - - T K HA I VQT P L Y I D F K E I GWD SW I I A P P G Y E A Y E C R G VC NY P L A E HL T P - - - - T K HA I I QHP L Y VD F S D VGWNDW I V A P P G Y HA F Y C HG E C P F P L AD HL NS - - - - T NHA I VQHS L Y VD F S D VGWNDW I V A P P G YQA F Y C HGDC P F P L AD HL NS - - - - T NHA I VQHE L Y V S F QD L GWQDW I I A P KG Y AANYC DG E C S F P L NAHMNA - - - - T NHA I VQHE L Y V S F R D L GWQDW I I A P E G Y AA Y Y C E G E C A F P L NS YMNA - - - - T NHA I VQK A L HV NF KDMGWDDW I I A P L E Y E A F HC E G L C E F P L R S HL E P - - - - T NHAV I QK P L HV NF K E L GWDDW I I A P L E Y E A Y HC E G VC D F P L R S HL E P - - - - T NHA I I QK P L HVD F K E L GWDDW I I A P L D Y E A Y HC E G L C D F P L R S HL E P - - - - T NHA I I QY P L T VD F E A F GW - DW I I A P K R Y K ANYC S G E C E F V F L QK Y P - - - - - HT HL V HQY P L T VD F E A F GW - DW I I A P K R Y K ANYC S GQC E YMF MQK Y P - - - - - HT HL VQQKQF F V S F KD I GWNDW I I A P S G Y HANYC E G E C P S H I AG T S G S S L S F HS T V I NHQQF F I D F R L I GWNDW I I A P T G Y YG NYC E G S C P A Y L AG V P G S A S S F HT A V V NQ

110108114116139139120120129109109116115

T L V HL K F P T K VG K AC C V P T K L S P I S V L Y KDDMG V P T L K Y HY E GMS V A E C GC RA L V HL K NS QK A S K AC C V P T K L E P I S I L Y L D - K G V V T Y K F K Y E GMAV S E C GC RT L V NS V N - S K I P K AC C V P T E L S A I S ML Y L D E NE K V V L K - NYQDMV V E GC GC RT L V NS V N - S S I P K AC C V P T E L S A I S ML Y L D E YD K V V L K - NYQ E MV V E GC GC RT L V HL MNP E Y V P K P C C A P T K L NA I S V L Y F DDNS NV I L K - K Y R NMV VR AC GC HT L V HF I NP E T V P K P C C A P T Q L NA I S V L Y F DD S S NV I L K - K Y R NMV VR AC GC HT L MNS MD P E S T P P T C C V P T R L S P I S I L F I D S ANNV V Y K - Q Y E DMV V E S C GC RT L MNS MD PG S T P P S C C V P T K L T P I S I L Y I D AG NNV V Y K - Q Y E DMV V E S C GC RT L L NS MAPD AA P A S C C V P AR L S P I S I L Y I D AANNV V Y K - Q Y E DMV V E AC GC RANP R G S AG - - - - - P C C T P T KMS P I NML Y F NG K E Q I I Y G - K I P AMV VDR C GC SANP R G S AG - - - - - P C C T P T KMS P I NML Y F ND KQQ I I Y G - K I P GMV VDR C GC SY R MR G HS P F ANL K S C C V P T K L R PMS ML Y YDDGQN I I K K - D I QNM I V E E C GC SY R MR G L NP - G T V NS C C I P T K L S T MS ML Y F DD E Y N I V K R - D V P NM I V E E C GC A

Supplemental Figure 4

*

*

*

ActRIIB contacts ALK1 contacts (BMP9)ALK1 contacts (BMP9)

β1 β2 β3 α1

β4 β5

β1

α1

3101

BMP9

BMP9

BMP9β6 β7

*

*

*

Supplemental Figure 5

A

B

S83P81

S80K78

H98

L95

G91

P93

V92

Y97

E34

E32

L61

W60Y42

K56 N17V55

R46

BMP9

ActRIIBECDβ1

β2

β3

β4

β5

β4β3

β6

β4

β3

β5

β2

β1

Q80

Q81 E76 F83

F64

N65

D63

K87R14

E31

P29A28L85

I27

D C

ka = 2.24 x 106 (M-1s-1)

kd = 1.29 x 10-4 (s-1)

KD = 58 pM

ka = 7.35 x 105 (M-1s -1)

kd =1.28 x 10-4 (s-1)

KD = 175 pM

B A

ka = 4.27 x 106 (M-1s-1)

kd = 2.10 x 10-4 (s-1)

KD = 49 pM

ka = 3.74 x 106 (M-1s -1)

kd =4.42 x 10-4 (s-1)

KD = 118 pM

Supplemental Figure 6

Supplemental Table 1: Crystallographic Data

Data collection Statistics

Space group P3112

Cell dimensions

a, b, c (Å) 216.45, 216.45, 216.95

α, β, γ (°)

Wavelength (Å)

90.00, 90.00, 120.00

1.54

Resolution (Å) 26.8- 3.35 (3.41-3.35)

Rmerge 0.137 (0.372)

I / σI 20.9 (4.7)

Completeness (%) 99.5 (99.4)

Redundancy 5.8 (3.9)

Refinement Statistics

Resolution (Å)

No. of Ref lections

Rwork/Rfree

Average B (Å2)

Rmsd bond lengths (Å)

Rmsd bond angles (°)

26.8 -3.35

69520

21.9/26.1

78

0.014

1.2

Numbers in parentheses correspond to highest resolution shellRmerge = Σ |I - <I>| / Σ <I>Rwork = Σ||Fobs| - |Fcalc||/Σ|Fobs| where Fobs and Fcalc are the observed and calculated structure factors respectively. Rfree was calculated from a subset of reflections (5%) not used for refinement.

Supplemental Table 2: Interactions (d ≤ 4.5 Å) at the BMP9/ALK1 interface

BMP9 Residues highlighted in bold are involved in polar contacts

BMP9/ALK1 Site I BMP9/ALK1 Site II

ALK1 BMP9 ALK1 BMP9

His40 His7, Ala46 Arg78 Ser24, Trp25, Lys87, Asp88, Asp89

Val54 Phe43, Pro44 Gly79 Gly21

Val56 Phe43, Leu63 Arg80 Glu18, Gly21, Asp23

Arg57 Leu63 Thr82 Asp19, Gly21

Glu58 Phe42, Phe43, His62

Leu63

Glu83 Asp19, Lys64

Glu59 Phe42,His62, Leu63,

Lys71Phe84 Asp19, Lys64

His66 Pro44 BMP9/ALK1 Site IIIAsn71 Asp47 ALK1 BMP9

Val80 Phe43 Leu72 Asp47, Pro51

His87 Phe43, Pro44 His73 Pro51, Lys53, Ile56

Glu75 Lys53, Trp22, Trp25

Tyr99

Leu76 Phe43, Ile56, Leu60

Trp22

BMP9/ActRIIB BMP9/ActRIIB

ActRIIB BMP9 ActRIIB BMP9

Asn17 Val92 Lys56 Pro93, Thr94, Leu95

Glu21 Thr94 Cys59 Leu95, Tyr97

Cys31 Tyr97 Trp60 Ala28, Pro29, Ser83,

Val84, Leu85, Leu95

Glu32 Tyr97, His98 Leu61 Pro81, Ile82,Ser83,

Tyr97, His98

Glu34 Lys78, Ser80, Pro81,

Ala105

Asp63 Pro29

Lys37 Ser80, Pro81, Ser103 Phe64 Arg14, Glu31,Tyr32,

Glu33

Leu39 Tyr97 Asn65 Pro29, Glu31

Tyr42 Leu85, Leu95 Glu76 Lys30

Ser44 Leu85 Gln80 Ile27, Lys30, Lys87

Arg46 Gly91, Pro93 Val81 Ile27, Pro93

Val55 Pro93 Phe83 Ile27, Ala28, Leu85

BMP9 Residues highlighted in bold are involved in polar contacts

Supplemental Table 3: Interactions (d ≤ 4.5 Å) at the BMP9/ActRIIB interface