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Structure of the tandem fibronectin type 3 domains of neural cell adhesion molecule.

Carafoli F, Saffell JL, Hohenester E - J. Mol. Biol. (2008)

Bottom Line: The two putative FGFR1-binding segments, one in each NCAM FN3 domain, are situated close to the domain interface.They form a contiguous patch in the more severely bent conformation but become separated upon straightening of the FN3 tandem, suggesting that conformational changes within NCAM may modulate FGFR1 activation.Thus, the NCAM-FGFR1 interaction at the cell surface is likely to depend upon avidity effects due to receptor clustering.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Biophysics Section, Blackett Laboratory, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
Activation of the fibroblast growth factor receptor (FGFR) by neural cell adhesion molecule (NCAM) is essential for NCAM-mediated neurite outgrowth. Previous peptide studies have identified two regions in the fibronectin type 3 (FN3)-like domains of NCAM as being important for these activities. Here we report the crystal structure of the NCAM FN3 domain tandem, which reveals an acutely bent domain arrangement. Mutation of a non-conserved surface residue (M610R) led to a second crystal form showing a substantially different conformation. Thus, the FN3 domain linker is highly flexible, suggesting that it corresponds to the hinge seen in electron micrographs of NCAM. The two putative FGFR1-binding segments, one in each NCAM FN3 domain, are situated close to the domain interface. They form a contiguous patch in the more severely bent conformation but become separated upon straightening of the FN3 tandem, suggesting that conformational changes within NCAM may modulate FGFR1 activation. Surface plasmon resonance experiments demonstrated only a very weak interaction between the NCAM FN3 tandem and soluble FGFR1 proteins expressed in mammalian cells (dissociation constant >100 muM). Thus, the NCAM-FGFR1 interaction at the cell surface is likely to depend upon avidity effects due to receptor clustering.

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Structure of the NCAM FN3 tandem. (a) Cartoon drawing of the wild-type 1FN3–2FN3 structure. Two loops implicated in FGFR1 binding (see the text) are labelled. (b) Cartoon drawing of the 1FN3–2FN3 dimer viewed along the 2-fold non-crystallographic symmetry axis. The 1FN3 and 2FN3 domains are shown in yellow and brown, respectively. The side chain of M610 in the dimer interface (see the text) is shown as a ball-and-stick model and is labelled. (c) Close-up view of the domain interface in wild-type 1FN3–2FN3: 1FN3 is shown in yellow; 2FN3, in brown. Selected residues are shown as ball-and-stick models. Hydrogen bonds are indicated by dashed lines. (d) Sequence alignment of the FN3 tandem of selected vertebrate NCAMs. Conserved residues are shaded pink. The numbering scheme and secondary structure elements of human NCAM are indicated above the alignment. The alternative splice inserts in the 1FN3–2FN3 linker (see the text) are indicated by black boxing. Two sequences implicated in FGFR1 binding (see the text) are underlined in blue. Residues involved in forming the dimer shown in (b) are indicated by filled circles, with the number of circles being proportional to the accessible surface area buried in the dimer: red circles indicate dimer contact between the α-helix of 1FN3 and the GFCD sheet of 2FN3; cyan circles, dimer contact between β-strands A and G of 2FN3.
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fig2: Structure of the NCAM FN3 tandem. (a) Cartoon drawing of the wild-type 1FN3–2FN3 structure. Two loops implicated in FGFR1 binding (see the text) are labelled. (b) Cartoon drawing of the 1FN3–2FN3 dimer viewed along the 2-fold non-crystallographic symmetry axis. The 1FN3 and 2FN3 domains are shown in yellow and brown, respectively. The side chain of M610 in the dimer interface (see the text) is shown as a ball-and-stick model and is labelled. (c) Close-up view of the domain interface in wild-type 1FN3–2FN3: 1FN3 is shown in yellow; 2FN3, in brown. Selected residues are shown as ball-and-stick models. Hydrogen bonds are indicated by dashed lines. (d) Sequence alignment of the FN3 tandem of selected vertebrate NCAMs. Conserved residues are shaded pink. The numbering scheme and secondary structure elements of human NCAM are indicated above the alignment. The alternative splice inserts in the 1FN3–2FN3 linker (see the text) are indicated by black boxing. Two sequences implicated in FGFR1 binding (see the text) are underlined in blue. Residues involved in forming the dimer shown in (b) are indicated by filled circles, with the number of circles being proportional to the accessible surface area buried in the dimer: red circles indicate dimer contact between the α-helix of 1FN3 and the GFCD sheet of 2FN3; cyan circles, dimer contact between β-strands A and G of 2FN3.

Mentions: To obtain insight into the relative orientation of the two FN3 domains of NCAM, we determined the crystal structure of 1FN3–2FN3 at 2.3-Å resolution (Table 1). Both 1FN3 and 2FN3 adopt the typical β-sandwich fold of all FN3 domains consisting of seven strands arranged in two antiparallel sheets (ABE and GFCD) (Fig. 2a and b). Preceding strand A in both domains are short proline-rich segments that are integrated into the FN3 fold, with the proline tetrahydropyrrole rings pointing into the hydrophobic core (Pro500 and Pro503 in 1FN3; Pro601 and Pro604 in 2FN3). A similar feature has been observed in other FN3 domains (e.g., in gp13033 and titin34). 1FN3 contains an unusual α-helix situated between strands D and E, as reported previously.311FN3 in our FN3 tandem structure matches the crystal structure of 1FN3 in isolation,31 with an r.m.s.d. of 0.50 Å for 100 Cα atoms. 2FN3 in our FN3 tandem matches the solution structure of 2FN3 in isolation,28 with an r.m.s.d. of 1.3 Å for 92 Cα atoms (the main differences are concentrated in the B–C and C–D loops).


Structure of the tandem fibronectin type 3 domains of neural cell adhesion molecule.

Carafoli F, Saffell JL, Hohenester E - J. Mol. Biol. (2008)

Structure of the NCAM FN3 tandem. (a) Cartoon drawing of the wild-type 1FN3–2FN3 structure. Two loops implicated in FGFR1 binding (see the text) are labelled. (b) Cartoon drawing of the 1FN3–2FN3 dimer viewed along the 2-fold non-crystallographic symmetry axis. The 1FN3 and 2FN3 domains are shown in yellow and brown, respectively. The side chain of M610 in the dimer interface (see the text) is shown as a ball-and-stick model and is labelled. (c) Close-up view of the domain interface in wild-type 1FN3–2FN3: 1FN3 is shown in yellow; 2FN3, in brown. Selected residues are shown as ball-and-stick models. Hydrogen bonds are indicated by dashed lines. (d) Sequence alignment of the FN3 tandem of selected vertebrate NCAMs. Conserved residues are shaded pink. The numbering scheme and secondary structure elements of human NCAM are indicated above the alignment. The alternative splice inserts in the 1FN3–2FN3 linker (see the text) are indicated by black boxing. Two sequences implicated in FGFR1 binding (see the text) are underlined in blue. Residues involved in forming the dimer shown in (b) are indicated by filled circles, with the number of circles being proportional to the accessible surface area buried in the dimer: red circles indicate dimer contact between the α-helix of 1FN3 and the GFCD sheet of 2FN3; cyan circles, dimer contact between β-strands A and G of 2FN3.
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fig2: Structure of the NCAM FN3 tandem. (a) Cartoon drawing of the wild-type 1FN3–2FN3 structure. Two loops implicated in FGFR1 binding (see the text) are labelled. (b) Cartoon drawing of the 1FN3–2FN3 dimer viewed along the 2-fold non-crystallographic symmetry axis. The 1FN3 and 2FN3 domains are shown in yellow and brown, respectively. The side chain of M610 in the dimer interface (see the text) is shown as a ball-and-stick model and is labelled. (c) Close-up view of the domain interface in wild-type 1FN3–2FN3: 1FN3 is shown in yellow; 2FN3, in brown. Selected residues are shown as ball-and-stick models. Hydrogen bonds are indicated by dashed lines. (d) Sequence alignment of the FN3 tandem of selected vertebrate NCAMs. Conserved residues are shaded pink. The numbering scheme and secondary structure elements of human NCAM are indicated above the alignment. The alternative splice inserts in the 1FN3–2FN3 linker (see the text) are indicated by black boxing. Two sequences implicated in FGFR1 binding (see the text) are underlined in blue. Residues involved in forming the dimer shown in (b) are indicated by filled circles, with the number of circles being proportional to the accessible surface area buried in the dimer: red circles indicate dimer contact between the α-helix of 1FN3 and the GFCD sheet of 2FN3; cyan circles, dimer contact between β-strands A and G of 2FN3.
Mentions: To obtain insight into the relative orientation of the two FN3 domains of NCAM, we determined the crystal structure of 1FN3–2FN3 at 2.3-Å resolution (Table 1). Both 1FN3 and 2FN3 adopt the typical β-sandwich fold of all FN3 domains consisting of seven strands arranged in two antiparallel sheets (ABE and GFCD) (Fig. 2a and b). Preceding strand A in both domains are short proline-rich segments that are integrated into the FN3 fold, with the proline tetrahydropyrrole rings pointing into the hydrophobic core (Pro500 and Pro503 in 1FN3; Pro601 and Pro604 in 2FN3). A similar feature has been observed in other FN3 domains (e.g., in gp13033 and titin34). 1FN3 contains an unusual α-helix situated between strands D and E, as reported previously.311FN3 in our FN3 tandem structure matches the crystal structure of 1FN3 in isolation,31 with an r.m.s.d. of 0.50 Å for 100 Cα atoms. 2FN3 in our FN3 tandem matches the solution structure of 2FN3 in isolation,28 with an r.m.s.d. of 1.3 Å for 92 Cα atoms (the main differences are concentrated in the B–C and C–D loops).

Bottom Line: The two putative FGFR1-binding segments, one in each NCAM FN3 domain, are situated close to the domain interface.They form a contiguous patch in the more severely bent conformation but become separated upon straightening of the FN3 tandem, suggesting that conformational changes within NCAM may modulate FGFR1 activation.Thus, the NCAM-FGFR1 interaction at the cell surface is likely to depend upon avidity effects due to receptor clustering.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Biophysics Section, Blackett Laboratory, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
Activation of the fibroblast growth factor receptor (FGFR) by neural cell adhesion molecule (NCAM) is essential for NCAM-mediated neurite outgrowth. Previous peptide studies have identified two regions in the fibronectin type 3 (FN3)-like domains of NCAM as being important for these activities. Here we report the crystal structure of the NCAM FN3 domain tandem, which reveals an acutely bent domain arrangement. Mutation of a non-conserved surface residue (M610R) led to a second crystal form showing a substantially different conformation. Thus, the FN3 domain linker is highly flexible, suggesting that it corresponds to the hinge seen in electron micrographs of NCAM. The two putative FGFR1-binding segments, one in each NCAM FN3 domain, are situated close to the domain interface. They form a contiguous patch in the more severely bent conformation but become separated upon straightening of the FN3 tandem, suggesting that conformational changes within NCAM may modulate FGFR1 activation. Surface plasmon resonance experiments demonstrated only a very weak interaction between the NCAM FN3 tandem and soluble FGFR1 proteins expressed in mammalian cells (dissociation constant >100 muM). Thus, the NCAM-FGFR1 interaction at the cell surface is likely to depend upon avidity effects due to receptor clustering.

Show MeSH
Related in: MedlinePlus