FGF 12 (FHF2) Kinemages

Human Fibroblast Growth Factor 12 (FHF 1)

Larry P. Taylor, Ph. D.

Feedback appreciated; please send comments to:

Email: lpt

Molecular & Behavioral Neuroscience Institute

The University of Michigan

Ann Arbor, MI

FGF-11, FGF-12, FGF-13, and FGF-14, also called FGF homologous factors FHF-3, FHF-1, FHF-2, and FHF-4, respectively, form a sub-group within the FGF family. Although this FGF sub-group does bind heparin (about the same magnitude as FGF 10, but significantly less than FGF 1 or FGF 2), the FHF group fails to bind to FGF receptors. There appears to be no competition between FGF and FHF proteins for binding to the FGF receptor. Instead, the FHF family binds directly to other proteins, specifically, an intra-cellular kinase scaffold protein, islet brain-2 (IB2).

Since the FHF family of protein ligands do not bind FGF receptors, the FHF sub-family of FGF ligands was recently removed from the FGF family. This change in "status" of the FHF sub-family of FGF ligands means that the FGF protein family is now composed of at least 18 homologous proteins that bind FGF receptors.

The unit cell for FGF 12 is shown in Kinemage 1. The characteristics of the unit cell for this structure are summarized at pdbsum.

The crystal structure shows four bound sulfate ions. (Kinemage 2 ) Three of these are similar to other FGF heparin binding sites and most likely account for the fact that FGF 12 binds heparin. The probable residues involved in sulfate ion binding are Tyr-72, Gln-19, Lys-123, Arg-126, and Ser-133. A fourth sulfate resides in the vicinity of Arg-52, Lys-86 and Ser-88. Although not consistent with the strong heparin binding associated with the "basic canyon" of FGF 1 and FGF 2, this fourth site is similar to the sulfate site found in FGF 10.

Since the FHF family binds heparin, but does not bind to FGF receptors, it seems reasonable to assume there are surface features of the FHF family that interfere with binding to the FGF receptors. There are three likely locations for steric interference which would limit the approach of FHF proteins to the FGF receptors. (see Kinemage 3) The first area of divergence between FGF and FHF is the N-terminal region of FGF 12 (poorly resolved in the crystal). Based on an analogy to FGF 2 binding to FGFR 2, the FHF residues 6-9 (PQLK) would interfere with the approach of the FHF protein to the D3 region of the FGF receptor.

The second possible conflict between FGF 12 and the FGF receptor centers around FGF 12 residue Tyr-93 in the area corresponding to the FGF low affinity loop (formed by beta sheets 8 and 9). If FGF 12 were to approach the FGF receptor in the same manner as FGF 1 and 2, then this Tyr-93 would extend into the D3 region of the receptor. This contact clash limits binding possibilities. Other possible conflicts in this region are Arg-52 (Gly in FGF) and Val-95 (Asn in FGF). The Val in place of Asn is particularly disrupting because the Asn residue in FGF is part of a network of hydrogen bonds between the FGF ligand and the FGF receptor. The side chains of Valine do not form hydrogen bonds and thus, would be unable to participate in a hydrogen bonding network.

The third possible area of conflict upon binding is the loop made beta sheets 9 and 10. The loop is 2 residues longer in FHF than FGF. This would also interfere with approach of the protein to the D3 region of the FGF receptor.

The differences between FGF 12 and other FGF species is highlighted by superimposing the structure upon the universal FGF ligand FGF 2 (Kinemage 4).

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The probable residues involved in sulfate ion binding are Tyr-72, Gln-19, Lys-123, Arg-126, and Ser-133. A fourth sulfate resides in the vicinity of Arg-52, Lys-86 and Ser-88.

View 1 the 4 sulfate ions of the crystal
View 2 sites analogous to the "Basic Canyon"
View 3 sulfate binding region that is different from FGF 2

Image requires a Java enabled browser 194 K
Click on KiNG to see	Residues Binding Sulfates

Based on an analogy to FGF 2 binding to FGFR 2, the FHF residues 6-9 (PQLK) would interfere with the approach of the FHF protein to the D3 region of the FGF receptor.

The second possible conflict between FGF 12 and the FGF receptor centers around FGF 12 residue Tyr-93 in the area corresponding to the FGF low affinity loop (formed by beta sheets 8 and 9). If FGF 12 were to approach the FGF receptor in the same manner as FGF 1 and 2, then this Tyr-93 would extend into the D3 region of the receptor. This contact clash limits binding possibilities. Other possible conflicts in this region are Arg-52 (Gly in FGF) and Val-95 (Asn in FGF). The Val in place of Asn is particularly disrupting because the Asn residue in FGF is part of a network of hydrogen bonds between the FGF ligand and the FGF receptor. The side chains of Valine do not form hydrogen bonds and thus, would be unable to participate in a hydrogen bonding network.

The third possible area of conflict upon binding is the loop made beta sheets 9 and 10. The loop is 2 residues longer in FHF than FGF. This would also interfere with approach of the protein to the D3 region of the FGF receptor.

View 1 regions of difference between FGF and FHF
View 2 loop from beta sheets 9 and 10
View 3 low affinity loop region (Tyr-93)

Image requires a Java enabled browser 192 K
Click on KiNG to see	Residues Prohibiting Binding To FGF Receptors

View 1 the overlay
View 2 loop area created by beta sheets 9 and 10 ... note the extended size of FGF 12 relative to FGF 2.
View 3 Tyr-93 (toggle side chains on to see the Tyr residue ... note how it forms an extension not found in FGF 2)

Image requires a Java enabled browser 291 K
Click on KiNG to see	FGF 12 Superimposed on FGF 2

Unresolved N-Terminal: RRRPE

X-Ray Resolved: PQLKGIVTRLFSQQGYFLQMHPDGTIDGTKDENSDYTLFNLIPVGLRVVAIQGVKASLYVAMNGEGYLYSSDVFTPEC
KFKESVFENYYVIYSSTLYRQQESGRAWFLGLNKEGQIMKGNRVKKTKPSSHFVPKPIEV

The DNA fragment encoding residues 1-181 was sub-cloned into pET-3 bacterial expression vector and expressed in E. coli. The structural coordinates are from Brookhaven Database file1Q1U. Cys-144 of the human sequence was mutated to Ala to prevent disulfide oligiomerization.

Image requires a Java enabled browser 224 K
Click on KiNG to see	FGF 12