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): ܭሺሻ ൌ ܭሺ ሻቂ ுሺ బ ሻା బ ோ ቃൈቀ ଵ െ ଵ బ ቁ ோ ൈ ቀ బ ቁ (1) where R is the gas constant, T 0 is a reference temperature, commonly 298 K, K A (T 0 ) and ΔH°(T 0 ) - association binding constant and binding enthalpy at T 0 , and ΔC° p is a heat capacity increment. In several cases, where the relationship lnK A 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: ܭሺሻ ൌ ܭሺ ሻቂ ுሺ బ ሻ ோ ቃൈቀ ଵ െ ଵ బ ቁ (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 – ALK1 ECD -BMP9 complex was formed by mixing BMP9 with ALK1 ECD -hFc. Pre-assembled complex was captured on anti-hFC IgG Biacore chip. Different concentrations of ActRIIB ECD -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 ActRIIB ECD -BMP9 complex was formed by mixing BMP9 with ActRIIB ECD -mFC. Assembled binary complex was captured on anti-mFc IgG Biacore chip.
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
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
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