1. Sequence and Structural Analysis of BMP9: Functional profiling, receptor binding, and stem cell differentiation.
Presented by: Karl Balch

2. Presentation Outline:
BMP family: Activity and mechanism of signalling
Receptors and SMADS
Common structure and assembly
Modulators
BMP9: Unique and common properties
Described function
Receptor activation

3. Presentation Outine -2 My Research:
Theoretical background on alignments and structural comparisons
Some background on ligand binding
Functional grouping based on microarray/gene expression data

4. Presentation Outline -3
My Research: BMP9
Type II receptor binding analysis
Type I receptor binding analysis
Modulator binding analysis

5. Presentation Outline -4
My Research: Other BMPs
Osteogenic Profile
Ligament/Joint/Tendon Profile
“Unique Function” BMP profile
BMP3 (inhibitory BMP) hypothesized mechanism of action.

6. Presentation Outline -5
Potential for application of structural/functional data:
Design of superactive BMPs
Design of targeted pathway ligands for tissue engineering
Design of inhibitory drugs
Disrupting tissue-specific growth in tumors derived from mesenchymal stem cells.

7. Transforming Growth Factor Beta (TGFB) family
Superfamily members active as disulfide linked homo and heterodimers.
Regulate stem cell proliferation, differentiation, and many other functions in cells
Picture from: Mazerbourg S, Hsueh AJ (2006)

8. Signal Transduction of TGFB family ligands
TGFB Ligands recruit and bind transmembrane serine/threonine receptors which form heteromeric dimers with cytosolic kinase activity.
Most TGFB family ligands have been shown to first bind a high affinity receptor which then participates through either direct or indirect mechanisms to recruit the low affinity receptor
Picture from: Sebald W, Nickel J, et al (2004)

9. TGFB Family Signalling
33 Unique ligands
7 Type I receptors
5 Type II receptors
Promiscuous Receptors!

10. Signal Transduction of TGFB family ligands
Type II receptor constitutively active. TII kinase phosphorylates (P) TI receptor to activate it.
Type I receptor (P) SMAD proteins which activates gene transcription downstream.
Picture from:

11. Receptor Roles Type II = Activator of receptor complex
Type I = Activator of cytosolic signal pathway.
Type I or II can be high affinity for ligand.
Type II underemphasized in signal propagation.
Picture from: Sebald W, Nickel J, et al (2004)

12. Type II Roles in Signal Propagation
Strength of kinase activity
Alignment with Type I activation regions
Which type I kinases become active and their cytosolic location
Picture from: Sebald W, Nickel J, et al (2004)

13. Bone Morphogenic Proteins (BMPs)
Comparison of BMP family based on sequence homology
Diverse function among BMP family
Structurally similar proteins show differing activity.
Picture from: Kang et al

14. BMPs in Neuronal Stem Cells
Picture from: Mishra et al (2005)

15. Overview of BMP function in Mesenchymal Stem Cells
Picture from: Mishra et al (2005)

16. BMP functions determined from mouse knockout studies
Picture from: Tseng YH, He TC (2007)

17. BMPs divided into 3 classes:
Osteogenic BMPs: B9,2,6,7,4, 3 (I)
Tendon/ Ligament/Joint forming: B14,13,12
“Unique Function” BMPs: B11,15,10

18. BMP common structural features.
Defining feature of BMP family is 3 paired intrachain cysteine bonds forming a “Cysteine Knot”.
Described “hand” with concave “palm side” and 2 parallel B-sheet form “fingers”
BMP9 Monomer

19. BMP: Common Structural Features
Mature dimer described as having two “fingers” F1 + F2 on each monomer which participate in binding TII receptors.
Alpha 3 region shown to be critical to binding TI receptors
Picture from: Brown MA, Zhao Q (2005)

20. Modulators of BMP function: In Vivo
BMP signalling is regulated by inhibitory factors to modulate regional signal propagation. There are 3 classes of inhibitory proteins which act via different mechanisms.
Noggin (Blocks TI + TII receptor regions) Bone and cartilage formation.
DAN family: DAN, Cerberus and Gremlin;
Chordin (binds BMP)
Noggin binding BMP7 dimer
Picture from: Groppe J, Greenwald J, et al (2002)

21. BMP9 (GDF2) Why BMP9? Relatively uncharacterized
Highest osteogenic activity in vitro
Highest osteogenic activity in vivo
Shown association with novel type I receptor, suggestive of parallel osteogenic pathway
BMP9 Crystal structure

22. BMP9: Activity Activity: Promotes cell proliferation and migration
Cholinergic (acetylcholine) neuron differentiation
Induces chondrogenic (cartilage) differentiation
Hepatocyte (liver cell) growth/development
Hematopoiesis (formation/dev blood cells)
Regulates protein kinase B signalling (cell survival/metabolism)

23. BMP9: Location In Vivo BMP9 found expressed in liver in:
Endothelial cells: have no basement membrane and are separated from the hepatocytes by the space of Disse which drains lymph into the portal tract lympatics. Kupffer cells are scattered between endothelial cells
Kupffer cells: specialized macrophages located in the liver that form part of the reticuloendothelial system and phagocytose spent erythrocytes.
Stellate cells: Stellate (Ito) cells store vitamin A and produce ECM and collagen. The stellate cell is the major cell type involved in liver fibrosis (formation of scar tissue in response to liver damage). The activated stellate cell is characterized by proliferation, contractility, and chemotaxis.

24. BMP9: Location In Vivo
Picture from:

25. BMP9 Receptors
Receptor binding and functional (ALP) assays have shown BMP9 to primarily use:
Type I:
Primary: ALK1
Secondary: BMPR1A
Type II:
Primary: ACTRIIB / ACTRIIA
Secondary: BMPRII

26. Receptor Comparison TI: ALK1 TII: ACTRIIB BMPRIA (ALK3) ACTRIIA
BMPRIIA
B14
Picture from: Mazerbourg S, Hsueh AJ (2006)

27. Unresolved Questions: BMP9
BMP3 (inhibitory to osteogenic BMPs) does not effect BMP9 activity. Suggestive of difference from other BMPs (alt. mech?, higher binding affinity?)
Role of ALK1 and mechanism of binding/recruitment.
Why is BMP9 more osteogenic than other BMPs?

28. Research Background: Sequence Alignment
Sequence alignment arranges the primary sequence of AA for several proteins to identify regions of similarity. AA are organized into groups of similar character and level of conservation can be compared. Regions with high similarity may be a consequence of functional, structural, or evolutionary relationships. AA Aligned as rows in a matrix with dashes indicating gaps to ensure alignment of proteins in columns.
Technique powerful for comparison of structurally similar BMPs as crystal structures have identified conserved receptor binding regions. Allows comparison of sequence variation across proteins with data from well defined BMPs and TGFB family proteins.

29. Research Background: Ligand/Receptor Binding
Types of bonding
Ionic bonds (A)
Hydrogen bonds (B)
MC-MC (1)
MC-SC (2)
SC-SC (3)
Hydrophobic interactions (C)
Disulfide bonds (D) #2 #1 #3
Picture from:

30. Research Background: Sequence Comparison
Compare AA type among binding regions.
Use published knowledge of crystal structure, mutational analysis, and binding literature to characterize the types of receptor binding critical to interaction regions.

31. Research Background: Sequence Comparison
Compare AA hydrophobicity among binding regions.
Important for altering hydrophobic interaction regions, binding pockets, steric hindrance, or the ease of contact between residues

32. Research Background: Sequence Comparison
Compare AA charge among binding regions.
Important for polar or nonpolar interaction regions. AA charge can be critical to effective binding or disrupting binding.

33. Research Background: Organizing Data:
BMPs with similar sequence but different function ideal for identifying critical regions determining downstream differentiation.
Pictures from: Kang et al

34. Research Background: BMP Alignment grouping
Order set to match microarray data, colored according to function, then added other family member alignments for additional information (TGFB1-3, Activin)

35. Research Background: TI Receptor Alignments
For binding comparison of data and for prediction of receptor binding among untested BMPs. Alignment of extracellular regions.
Compare: BMPR1a (ALK3), BMPR1B (ALK6), ALK1, ALK2, ALK4, TGFBR1, ALK7

36. Research Background: TII Receptor Alignment
When possible alignments checked against published mutations and confirmed with published alignments of proteins.
Compare: ACTIIRA, ACTIIRB, BMPRII, TGFR2

37. Sequence Analysis and Results

38. Results: B9 TII Analysis
B9- R333
Osteo: Y44 (b7) binds N65 ACTIIRA SC-SC
Activin: F17 hydrophobic contact w/ F82 ACTIIRB
Charge: Tendon, ligament, joint forming (B14,13,12) and osteo-inhibitory B3.

39. Results: B9 TII Analysis
Identified hydrophobic interactions between activin (F17) and ACTRIIB (F82). F82 of ACTRIIB has interaction w/ B9 E (H align activin). Sidechain interactions betweenin B7 (Y44) and ACTRIIA (N65) receptor.
B9 high charge (very basic) and less hydrophobicity. R very active in H bonding more than K (b3) or Y (Osteo).
Suggests Repulsion of BMPRII and TGFRII for B9 and attraction to acidic region of ACTIIR B2 B9
B14

40. Results B9 TII Analysis Important TII binding region.
B9- APKEYE ( )
Important TII binding region.
Higher variability than other TII regions with clustered similarity by function.
B9 KEYE unique alignment for BMPRII.
Balanced charge may allow ACTRII binding

41. B9 Analysis: KEYE region alignment
Charge B9 is better match for binding BMPRII than B3 may explain B9 higher affinity b3.
B2 does not show strong charge alignment BUT B2 is a TI high affinity receptor ligand. Suggestive that Cart BMPs may bind TII with higher affinity than most osteogenic BMPs.
Last E in KEYE is hydrophobic interaction region for NOGGIN (I) which suggests the H (b2) and E (b9) important for repelling inhibition in vivo.

42. B9 Analysis: TII Regions
B9 KEYE is a unique combination of K (TGFB) and EYE (Cart class). The K basic character and high hydrophilicity seperates it from Cart class BMPs which all have a highly hydrophobic residue in its place.
While other BMPs have the K or EYE type in this region the KE juxtaposition is unique which may serve an additional role to cancel much of the charge of the region. Can be stable bound or unbound. B2 B9
B14
Pictures from: Hart et al

43. B9 Analysis: TII Regions
Less Variable than previous region among different BMP subtypes.
All osteogenic BMPs except B9 have AIS (B9 = PIS) No major difference in binding from literature.
K B9 likely to disrupt binding ActIIRA (hydrophobic binding region) but improve ActIIRB hbonding

44. B9 Analysis TII Regions
K (b9) shown in 3d structures is highly hydrophilic and unique to B9. Causes that region of b9 to open wider than other BMPs that have hydrophobic residues in that region. B2 B9
B14

45. B9 Analysis: TII Regions
Consistent profile of non-osteogenic BMPs in final TII binding region.
L125 (b7) binds F42, W60 (ACTR2A), nonpolar-nonpolar, SC-SC interactions. Mutation shows disrupted binding. L125 -> A disrupts binding ACTRII and BMPRII. Tyrosine (Y) in region for non-osteogenic BMPs disrupts nonpolar interactions with receptor for TII binding.

46. B9 Analysis: TII Regions
Red arrows show interactions established from literature
B9 Y likely to participate in hydrophobic interaction with W on receptors
B9 has unique AA in YH region. H is strong candidate for binding ALK1. TGFR2 which binds ALK1 in TGFB has a H at same position. Also H is adjacent to perimeter of TI binding region.

47. B9 Analysis: TII Regions
B9 YH region protrudes further away from the core as it has greater hydrophobic character than B2 and B14.
Further extension may be important for contacting the TI region of ALK1 B2 B9
B14

48. B9 Analysis: TII Regions
Final isolated TII region identified in activin for ActRIIB.
Charged amino acids in all except B9 and B10. Both show disaligned glutamic acid (E) residue.
Significance not well characterized.

49. B9 Analysis of TII: Summary
Multiple regions where hbonding affinity is B9 > B3 > Osteogenic BMPs. Could explain lack of B3 inhibition of B9. B9 is able to out compete B3 for binding TII receptors.
B9 has greater repulsion of unwanted binding partners which increases likelihood of correct binding events.

50. B9 Analysis of TII: Summary
B9 has some regions suggestive of a higher binding affinity for BMPRII than other osteogenic BMPs which are TI high affinity ligands.
B9 has some AA that give it a greater resistance to inhibition from Noggin the endogenous inhibitor of osteogenic BMPs in vivo. Repulsion of Noggin is B9 > B2 > Other Osteogenic BMps.

51. B9 Analysis of TII: Summary
Some regions disruptive to ACTRIIA binding may explain experimental results of BMP9 binding ACTRIIB more readily.
Greater TII region hydrophilic character (protrusion) in border regions to T1 binding may serve as important contacts for ALK1 binding. Some of these extensions mimic border regions of TGFBRII receptor which is known to recruit ALK1 and are only found in B9 and B10.

52. B9 Analysis of TII: Summary
Disruption of some critical regions of TII binding may be critical for determining the fate of stem cells by modifying signal propagation. Different profiles between all osteogenic and non osteogenic BMPs

53. Exhausted Yet?

54. B9 Analysis: TI Binding Regions
TI binding regions far less defined in research literature.
Only data for B2 binding on BMPRIA (ALK3) exists.
First TI binding region mostly the same across osteogenic BMPs

55. B9 Analysis: TI Binding Regions
V of B2 connects S BMPR1A
W connects with D in BMPR1A.
BMP9 stronger and closer acidic residues may be important in binding the highly basic ALK1

56. B9 Analysis: TI Regions B2
Labeled acidic residues showing their proximity in the TI binding domain.
B14 shows some clustering of acidic residues but in ALL cart BMPs one of the leading acidic residues is bordered by a basic residue which would neutralize much of the charge. B9
B14

57. B9 Analysis: TI Regions
Major differences between osteogenic, chondrogenic, and unique function BMPs in this region. May indicate importance in determining function.
Acidic residues border the T1 region in all cart BMPs. Proximal F used to block H20 inside protein cleft. Acidic hydrophilic character proximal likely disrupts this. H20 binding in this region found in BMPR1A/B2 binding.

58. B9 Analysis: TI Regions
Leu (L) B2 "HOT SPOT" binding (MC) hbonds w/ (Q86) (SC) BMPR1A, mutation shows disrupted binding(>8000 fold increase KD). L is common to most BMPs and critical to proper binding of TI receptors.
Proximal R residue on Cart BMPs shown on mutation to improve binding of BMPR1 greater than 10 fold.
DD (B9), DH (B2), and SH (B14) show a distinction among the TI region between highly acidic (B9), more balanced charge (B2), and basic (b14). Asp (D) in B2 MC/MC hbonds w/ C77 BRIA mutation -> P (53X > KD) D53 also binds T55 which is minor contributor

59. B9 Analysis: TI Regions
1st D in osteogenic BMPs binds C in B2. Balanced charge of most osteogenic BMPs favors binding BMPR1A/B while larger acidic character favors binding ALK1 Strong basic region.

60. B9 Analysis: TI B2
Highlighted adjacent charge regions in the identified TI binding region. B2 region shows balanced charge, high acidity in B9 and basicity in B14. B9
B14

61. B9 Analysis: TI Regions
Much more similarity in this region among osteogenic BMPs.
Major Difference is K in osteogenic N region.
Literature identified N mutation disrupted binding BMPR1A.

62. B9 Analysis: TI B2
KH and NH occupy same region. Likely has an effect on BMPR1A binding.
Further research into function of region is needed. B9
B14

63. B9 Analysis: TI Regions
S69 (b2) binds MC-MC w/ Q94 BMPR1a, S69 (b2) binds SC-SC w/ R97. S69 ->R mutation BRIA 17 fold reduction binding. B9 K is basic like R and should significantly disrupt BMPR1A binding

64. B9 Analysis: TI Regions
N of B2,4 binds BMPR1A with higher affinity than H of B6,7 may explain higher osteogenic activity in B2,4.
Strong basic character of B9 (HLK) would repel the double RR of BMPR1A but attract regional E of ALK1.

65. B9 Analysis: TI B2
Differential charge in the area likely works to determine the efficacy of binding to the TI receptor. B9
B14

66. B9 Analysis: TI Regions H that mimics TGFBR2 borders this region.
Y (b2) binds SC-SC D84 BMPR1A hbond, mutation shows minor binding effect (7.8x increase Kd)

67. B9 Analysis: TI Regions
Proximal Acidic residue to Y would repel binding of D for BMPR1A but facilitate binding HR (highly basic region) of ALK1.
H identified earlier may be attracted by E in ALK1.

68. B9 Analysis: TI B2
Additional region of differential binding between BMPR1A and ALK1.
Cart BMPs highly acidic in region without H for binding acidic profile on ALK1. Likely disrupts TI binding for CART class BMPs. B9
B14

69. B9 Analysis of TI Regions: Summary
Binding of ALK1 undefined in literature but structural info for BMPR1A exists.
Several Regions of B9 show charge repulsion for BMPR1A/B but charge attraction to ALK1 which likely explains the differential binding affinity found in experiments.

70. B9 Analysis of TI Regions: Summary
Regional charge in binding areas seem to be strong effector of affinity. Differences in charge seen across levels of activity and type of cell fate.
For ex: Acidic region binds ALK1, Neutral charge binds BMPR1A sets osteogenic fate, Basic charge leads to downstream initiation of Cart pathway. Manipulation of charged areas crucial for determining activity and cell fate.

71. Additional Considerations: In Vivo

72. Inhibitory Analysis
Aligned binding regions of inhibitory proteins and osteogenic BMPs
May be critical to activity in vivo but is undetected in vitro due to lack of inhib reg.
BMP9 showing same charge repulsion to repel inhibitory proteins without disrupting distant binding regions and conserving overall skeletal structure to allow flexible receptor binding.

73. Differences in vivo
Prodomain reported to remain bound to BMP9 without disrupting its receptor binding. Could serve multiple roles:
Protection from degradation?
Structural framework that facilitates binding of ALK1?
Disruption of inhibitory modulation in vivo?

74. Differences In Vivo:
Synergy between ligand pairs could create new effects. BMP9 and Cart class BMPs higher recruitment of TII receptors. Osteogenic BMPs higher affinity for TI receptors. Combinations will create quicker osteogenic growth in vivo.

75. Differences In Vivo
Regional expression of BMPs different In Vivo so that some combinations may not naturally occur.
Thereaputic application of BMPs outside of the region they are normally expressed can yield interesting results. (BMP9)

76. Final Summary:
BMP9 is resistant to inhibition from BMP3 because it can outcompete BMP3 in binding the TII receptors and it uses a different primary TI receptor than the other osteogenic BMPs.

77. Final Summary: BMP9 also resistant to Noggin inhibition
Regions known to bind Noggin are different in BMP9 compared to all other osteogenic BMPs in the core and TII binding regions.

78. Final Summary:
BMP9 likely to have higher affinity for TII binding relative to other osteogenic BMPs with preferential binding of ACTRIIB over ACTRIIA.
Greater repulsion of unwanted binding events.

79. Final Summary:
Changes in TII and TI affinity and binding likely determine cell fate.
Regional character preserved across proteins in clusters similar to microarray expression.

80. Final Summary:
Unique changes in BMP9 confer greater binding affinity for ALK1 in TI and border TII regions.
Repulsion of BMPR1A also allows differential binding of BMP9.

81. Questions????