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. 2013 Nov 5;21(11):2087-93.
doi: 10.1016/j.str.2013.08.026. Epub 2013 Oct 10.

Structure of FGFR3 Transmembrane Domain Dimer: Implications for Signaling and Human Pathologies

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Structure of FGFR3 Transmembrane Domain Dimer: Implications for Signaling and Human Pathologies

Eduard V Bocharov et al. Structure. .
Free PMC article


Fibroblast growth factor receptor 3 (FGFR3) transduces biochemical signals via lateral dimerization in the plasma membrane, and plays an important role in human development and disease. Eight different pathogenic mutations, implicated in cancers and growth disorders, have been identified in the FGFR3 transmembrane segment. Here, we describe the dimerization of the FGFR3 transmembrane domain in membrane-mimicking DPC/SDS (9/1) micelles. In the solved NMR structure, the two transmembrane helices pack into a symmetric left-handed dimer, with intermolecular stacking interactions occurring in the dimer central region. Some pathogenic mutations fall within the helix-helix interface, whereas others are located within a putative alternative interface. This implies that although the observed dimer structure is important for FGFR3 signaling, the mechanism of FGFR3-mediated transduction across the membrane is complex. We propose an FGFR3 signaling mechanism that is based on the solved structure, available structures of isolated soluble FGFR domains, and published biochemical and biophysical data.


Figure 1
Figure 1. NMR spectra of FGFR3tm in a membrane-mimicking environment
Heteronuclear 1H/15N-TROSY NMR spectra of FGFR3tm in mixed DPC/SDS (9/1) micelles at D/P of 140 (A) and 65 (B), 40 °C and pH 5.7. The 1H-15N backbone and side-chain resonance assignments are shown. The TM region 367-399 undergoes a slow monomer-dimer transition, as proved by the comparison of the 1H/15N-TROSY (appearance of signal doubling) and 15N,13C-F1-filtered/F3-edited-NOESY (registration of inter-monomeric NOE) spectra acquired at D/P of 140 and 65. (C) Generalized chemical shift changes, Δδ(1H15N)d-m, for the FGFR3tm amide groups are calculated as the geometrical distance (with weighting of 1H shifts by a factor of 5 compared to 15N shifts) between the amide cross-peaks assigned to the dimeric and monomeric FGFR3tm states in the 1H-15N TROSY spectrum acquired at D/P of 65. The measurement uncertainty is shown in the upper right corner. Bottom, pattern of unambiguous inter-monomeric NOE connectivities (shown with solid and dashed lines), identified between the subunits (underlined) of the symmetric FGFR3tm dimer.
Figure 2
Figure 2. Spatial structure of the FGFR3tm dimer
(A) The 25 NMR structures of the FGFR3tm dimer are superimposed on backbone atoms of the TM helical regions (Tyr373-Leu398)2. Heavy atom bonds are shown only and painted in black and magenta for different dimer subunits. (B) Ribbon structure of the FGFR3tm dimer. The negative charged, positive charged, aromatic, large hydrophobic and small side-chains are shown in red, blue, cyan, light yellow and green, respectively. The approximate position of the membrane borders is highlighted by the yellow strips. Pathogenic mutations are shown by arrows. (C) Properties of the FGFR3tm dimer interface. Left: hydrophobic and hydrophilic (polar) surfaces of the TM helix subunit are colored in yellow and green, respectively. The complementary subunit is shown in a stick representation. Right: hydrophobicity map of the molecular surface of the TM helix with the isolines encircling hydrophobic regions with high molecular hydrophobicity potential (MHP) values. The map, constructed as described in Supplemental experimental procedures, is presented in cylindrical coordinates associated with the TM helix. The observed helix packing interface of FGFR3tm is indicated with magenta dots. An alternative dimerization interface, rich in consensus helix packing GG4-like motifs, is encircled by the light green oval. Noteworthy, similar dimerization interfaces were predicted for the FGFR3 TM domain by molecular modeling (Li et al., 2006; Volynsky et al., 2013). Residues which harbor pathogenic mutations (Li and Hristova, 2006) are highlighted in yellow with amino acid substitution marked additionally in red.
Figure 3
Figure 3. “String-puppet” mechanism of FGFR3 activation under the assumption that two alternative dimer structures exist in the presence and absence of ligand
(A) Left: The FGFR3 TM domain dimer is formed via the heptad motif shown in Figure 2. The small TM helix-helix crossing angle keeps the receptor cytoplasmic domains in a symmetric configuration, resulting in a basal phosphorylation state, which is stabilized by the homodimeric adaptor protein Grb2. The autophosphorylation sites are schematically presented by open and filled orange circles. The disposition of the EC domains in the unliganded FGFR3 dimer is not known. Right: FGFR3 activation requires asymmetric configuration of the kinase domains, which is easier to achieve when the C-termini of the TM FGFR3 domains are spaced apart (by ~20 Å). This configuration corresponds to an alternative dimerization mode of the TM domain, utilizing the N-terminal tetrad GG4-like motif. Ligand-binding (FGF and heparin/heparan) induces a conformational change in the EC domain and pushes the D3 subdomains (i.e. the C-termini of the EC domains) away from each other (by ~50 Å). This structural change imposes spatial restraints via the short extracellular JM regions (“strings”) on the configuration of the entire receptor dimer, inducing motions in the receptor dimer, Grb2 release and receptor activation. The TM domain dimer switches into the high crossing angle structure, and the kinase domains adopt the fully active asymmetric configuration. (B) Schematic top view of the FGFR3 TM domain dimer in its putative basal phosphorylation (left) and fully active (right) conformations mediated by the heptad and tetrad motifs, respectively. Analogous TM helix packing diversity was recently observed for other RTKs (Bocharov et al., 2010b), see also Figure S9. Ligand-binding followed by structural rearrangements of the EC domains and extension of the extracellular JM regions likely induces unfolding of the N-terminal turn A369GSV372 and rotational movements of the TM helices, which increases the distance between their C-termini and allows for the formation of the asymmetric kinase dimer.

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