Human Fibroblast Growth  Factor 9 (FGF 9) Dimer

by

Larry P. Taylor, Ph. D.

 

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Email: lpt

Molecular & Behavioral Neuroscience Institute

The University of Michigan

Ann Arbor, MI

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FGF Site: FGF Intro     Nomenclature     Notes     References      FGF Sequences     FGFR Sequences  

Human Fibroblast Growth Factor  9 (FGF 9) Dimer

 

FGF 9 was originally described as glia-activating factor (GAF). It shares about 30% sequence identity with FGF 2. FGF 9 activates proliferation of glia cells without being mitogenic for endothial cells. The specificity of biological activity is most likely related to its differential receptor binding profile. Its highest binding affinity is with FGFR 3 with slightly lesser binding to FGFR 2 and very little binding affinity for FGFR 1.

 

The unit cell for FGF 9 (Kinemage 1) shows a dimer of dimers. FGF 9 is unique in that in solution it forms a dimer ( Kinemage 2  & Kinemage 3  ) that occludes areas of the molecule that in other FGF species are associated with heparin and receptor binding. An overlay of the individual chains of the unit cell (Kinemage 4) shows that the core of the FGF 9 molecule is not altered upon dimerization. (The FGF 9  monomer is available as a separate Kinemage) The characteristics of the unit cell for this structure are summarized at pdbsum.

Comparison of FGF 9 with FGF 2 (Kinemage 4 )  shows some obvious differences in the beta trefoil core between FGF 9 and FGF 2. At FGF 9 position Thr-70 to Gly-71 (Kinemage 4, view 2 ... an extra glycine here in FGF 2 adds a one residue length to the FGF 2 loop; bottom, left of center) the FGF 9 loop is one residue shorter than the corresponding area in FGF 2. The FGF loop at Asp-88 to Ser-90 shows an altered orientation (,Kinemage 4 view 3: center) compared to FGF 2. A major divergence between FGF 2 and FGF 9 occurs in the vicinity of Tyr-153 to Arg-161 (the low affinity loop, Kinemage 4, view 3; bottom left). Since this is a primary contact point between FGF ligands and receptors, this divergence in structure is most likely a prime component in FGF 9 receptor selectivity. Lastly, the N and C terminal regions (Kinemage 4, view 1 & 5) of FGF 9 show an extension, relative to FGF 2, with alpha helix secondary structure. This is a major departure from most FGF molecules and can be considered as a separate entity from the FGF beta trefoil core.

FGF 9, perhaps more than any FGF moiety, shows a tendency to self-dimerize under physiological conditions. Since other FGF molecules do not have the well-ordered alpha helix C and N terminal extensions, these extensions appear to be a significant contributor to the tendency to form dimers in the absence of heparin. (Other FGF species typically show structures that are X-ray unresolved at the extensions. The inability to resolve the extensions is associated with a lack of organized structure,) These interactions are highlighted in kinemages Kinemage 5  ,  Kinemage 6 ,  and Kinemage 7.

Although the FGF 9 dimer interface is hall marked by hydrophobic interactions, there is some stability to dimerization added by 4 hydrogen bonds and 2 salt bridges. There are hydrogen bonds between each of the C and N terminal helix extensions at Arg-64 to the backbone carbonyl of Val-197 and Tyr-67 to Asn-143. There is a salt bridge between Arg-62 and Asp-193. These are shown in Kinemage 5.

The primary driving force for dimerization is the exclusion of solvent via multiple hydrophobic contacts of the alpha helical terminal extension region. These involve residues Leu-54, Leu-57, Ile-60, Leu-61, Pro-194, Val-197 and Leu-200 of both chains involved in the dimerization. These are shown in Kinemage 6.

There is also a less extensive hydrophobic patch involving both chain residues Trp-144, Tyr-145, Pro-189, the aliphatic portion of the side chain of Arg-190, and Pro-191. These are shown in Kinemage 7.

The beta turn between sheets 8 and 9 of FGF 9 is different from the loop found in FGF 1 and 2. The distortion of this loop (compared to FGF 2) is maintained by a network of hydrogen bonds from residues Glu-141, Glu-142, His-181, and His-186. There is also a salt bridge connecting residues Arg-69 with Glu-142. Since this loop is implicated in binding to the D2 domain of FGF receptors, the shape of this loop is a critical component of ligand-receptor specificity. This loop is shown in  Kinemage 8.

Many of the residues implicated by analogy to FGF 1 and 2 as "high affinity" binding sites are buried within the FGF 9 dimer structure and thus unavailable for binding to a FGF receptor. The primary dimer-buried residues involved are Ile-60, Tyr-67, Tyr-145, Leu-180, His-186 and Leu-188. These are shown in Kinemage 9.  Since these residues are buried within the dimer conformation, it is necessary that the dimer quaternary structure be broken for the monomer to approach the ligand binding domain of a FGF receptor. The mechanism for this is currently unknown.

Finally, the "Basic Canyon" of potential heparin binding sites is somewhat obscured in the dimer formation. Potential heparin binding sites include Arg-69, Arg-137, Tyr-153, Lys-154, Arg-161, Tyr-163, Arg-173, Arg-177, Arg-180, and Arg-184. A Phe residue (Phe-184) partially obscures some of the "basic canyon" and, in the dimer state, would show a different face to heparin than the corresponding regions of the FGF 2 monomer. These sites are shown in Kinemage 10.


The Kinemages

 

The real-time visualization using KiNG of the structures on this site requires a java-enabled (JRE from Java) browser. 

 

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A single click on the KiNG logo will launch the appropriate kinemage.

Kinemage 1: The Unit Cell  for FGF 9 (1G82)

 



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62 K

Click on KiNG to see Unit Cell: A Dimer of Dimers

 

Kinemage 2: FGF 9 Dimer

 



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531 K

Click on KiNG to see The FGF 9 Dimer


Kinemage 3: FGF 9 Dimer (Cartoon Rendering)

 



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439 K

Click on KiNG to see FGF 9 Dimer:  Secondary Structure Cartoon


Kinemage 4: FGF 9 Chains A, B, C, & D Superimposed upon FGF 2

 

The individual FGF chains were extracted from the unit cell (IG82) with SYBYL and then superimposed upon FGF 2 (1BFF) with Deep View "Magic Fit." Non-protein material from the 1BFF was included to maintain orientation during image manipulation.


View 1   the overlay
View 2   smaller loop at FGF 9 Arg-69 thru Gly-71
View 3   alteration at FGF 9 Asp-88 thru Ser-90 loop
View 4   low affinity loop
View 5  Glu-142
View 6  N & C terminal junction



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289 K

Click on KiNG to see FGF 9 Chains Superimposed on FGF 2

 

Kinemage 5: FGF 9 Dimer Interface: Polar Interactions

Although the FGF 9 dimer interface is hall marked by hydrophobic interactions, there is some stability to dimerization added by 4 hydrogen bonds and 2 salt bridges. There are hydrogen bonds between each of the C and N terminal helix extensions at Arg-64 to the backbone carbonyl of Val-197 and Tyr-67 to Asn-143. There is a salt bridge between Arg-62 and Asp-193.

 

View 1  the dimer
View 2  H bonding at Arg-64 of chain A to the backbone carbonyl of chain D Val-197.
View 3  H bonding at Tyr-67 of chain A to Asn-143 of Chain D.
View 4  one salt bridge at Arg-62 of chain A to Asp-193 of chain D.
View 5  H bonding at Arg-64 of chain D to the backbone carbonyl of chain A Val-197.
View 6  H bonding at Tyr-67 of chain D to Asn-143 of chain A.
View 7  salt bridge at Arg-62 of chain D to Asp-193 of chain A.
View 8  interface between the individual members of the dimer complex

 



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461 K

Click on KiNG to see Dimer Interface Polar Interactions

 

Kinemage 6: FGF 9 Dimer Interface: Primary Hydrophobic Interactions

Hydrophobic interactions at the dimer interface involve residues Leu-54, Leu-57, Ile-60, Leu-61, Pro-194, Val-197 and Leu-200.


View 1  the Complex
View 2  an arbitrary top
View 2  corresponding side 

 



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458 K

Click on KiNG to see  FGF 9 Dimer: Primary Hydrophobic Interactions

 

Kinemage 7: FGF 9 Dimer Interface: Secondary Hydrophobic Interactions

The hydrophobic interactions at the dimer interface involves residues  Trp-144, Tyr-145, Pro-189, the aliphatic portion of the side chain of Arg-190, and Pro-191


View 1  the Complex
View 2  hydrophobic interactions 1
View 3  hydrophobic interactions 2

 



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459 K

 

Click on KiNG to see  FGF 9 Dimer: Secondary Hydrophobic Interactions

 

Kinemage 8: FGF 9 Dimer: Turn At beta 8/beta 9 (Residues 139-146)

The residues involved in hydrogen binding are Glu-141, Glu-142, His-181, and His-186. There is also a salt bridge connecting residues Arg-69 with Glu-142.


View 1  the Complex
View 2  the turn, chain A
View 3  the turn, chain D

 



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472 K

Click on KiNG to see Turn at beta 8/9

 

Kinemage 9: FGF 9 Dimer: Potential Receptor Binding Sites

Potential FGF 9-Receptor binding sites are Ile-60, Tyr-67, Tyr-145, Leu-180, His-186 and Leu-188.

(Because these residues are buried, they are best visualized by selectively toggling various chains, side chains and ribbons to the off position.)

 



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458 K

Click on KiNG to see Buried Potential Receptor Binding Sites

 

Kinemage 10: FGF 9 Dimer: Potential Heparin Binding Sites

Potential Heparin binding sites includes residues Arg-69, Arg-137, Tyr-153, Lys-154, Arg-161, Tyr-163, Arg-173, Arg-177, Arg-180, and Arg-184.  Phe-184 partially obscures some of the "basic canyon."


View 1  the dimer
View 2  close up of the "basic canyon" region of chain A
View 3  close up of the "basic canyon" region of chain D

 



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463 K

Click on KiNG to see Potential Heparin Binding Sites
 

Sequence: Corresponds to residues 52 to 208 of Human FGF 9)

Unresolved N-Terminal: DHLGQSEAGGLPRGPAV

X-Ray Resolved: 
TDLDHLKGILRRRQLYCRTGFHLEIFPNGTIQGTRKDHSRFGI
LEFISIAVGLVSIRGVDSGLYLGMNEKGELYGSEKLTQECVFREQFEENWYNTYSSNLYK
HVDTGRRYYVALNKDGTPREGTRTKRHQKFTHFLPRPVDPDKVPELYKDILSQS


Source:

cDNA corresponding to the full-length of human FGF 9 was isolated as BamHI/blunt fragment from pET vector and subcloned into the vector pBacPAK9. Material was transfected into Sf9 cells. Structural coordinates were taken from the Brookhaven Data Base File1G82.

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FGF Site: FGF Intro     Nomenclature     Notes     References     FGF Sequences     FGFR Sequences 

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Copyright 2005-2020 by Larry P. Taylor
Molecular & Behavioral Neuroscience Institute
The University of Michigan

All Rights Reserved

Supported by the Pritzker Neuropsychiatric Disorders Research Consortium, and by NIH Grant 5 P01 MH42251, Conte Center Grant #L99MH60398, RO1 DA13386 and the Office of Naval Research (ONR) N00014-02-1-0879 to Huda Akil & Stanley J. Watson. at the Molecular & Behavioral Neuroscience Institute.