NMR structure of the first Ig module of mouse FGFR1

Abstract

Fibroblast growth factor (FGF) receptors (FGFRs) regulate a multitude of cellular processes during embryogenesis and in the adult. The extracellular part of the prototypical FGFR consists of three Ig modules (Ig1 – Ig3), in which Ig2 and Ig3 determine affinity and specificity for FGF and heparin, while the Ig1 module is thought to have a regulatory function. The crystal structures of the Ig2 and Ig3 modules alone and in complex with FGF have previously been reported. The structure of the Ig1 module is unknown, and very little is known about the structural determinants for the regulatory function of this module. We describe here the NMR structure of the Ig1 module of mouse FGFR1. The three-dimensional fold of the module belongs to the intermediate Ig subgroup and can be described as a β-barrel consisting of two β-sheets. One sheet is formed by A′, G, F, C, and C′, and the other by A, B, B′, E, and D β-strands. The overall strand topology of the Ig1 module is similar to that of the Ig2 and Ig3 modules. However, the A/A′ loop of the Ig1 module is much longer than that of the Ig2 and Ig3 modules. It contains eight extra residues compared to the Ig3 module, and five extra residues compared to Ig2.

Fibroblast growth factor receptors (FGFR1–FGFR4) are a family of transmembrane tyrosine kinases involved in signaling via interactions with the family of fibroblast growth factors (FGF1–FGF23) (McKeehan et al. 1998; Itoh and Ornitz 2004) and cell adhesion molecules such as NCAM, L1, and N-cadherin (Doherty and Walsh 1996; Kiselyov et al. 2003). FGFRs regulate a multitude of cellular processes during embryogenesis and in the adult (Bottcher and Niehrs 2005). The prototypical FGFR consists of three Ig modules (Ig1–Ig3), a trans-membrane domain, and a cytoplasmic tyrosine kinase domain. The linker region between the Ig1 and Ig2 modules is very long, consisting of 20–30 amino acid residues, including a stretch of acidic amino acids termed the “acid box.” FGFRs also bind heparin/heparan sulfate, which is required for the high-affinity FGF–FGFR interaction (Yayon et al. 1991; Ornitz et al. 1992). Binding studies of several FGFs to various FGFR fragments and crystal structures of several FGFs in complex with fragments of FGFRs consisting of the Ig2 and Ig3 modules indicate that these modules and the Ig2–Ig3 linker region are sufficient for the specific FGF–FGFR interaction (Wang et al. 1995a; Plotnikov et al. 1999; Pellegrini et al. 2000). FGF–FGFR binding results in dimerization of FGFR leading to autophosphorylation of the receptor tyrosine kinase domains. Based on crystal structures of the ternary FGF–FGFR–heparin complex, two competing models of FGFR dimerization have been proposed (Plotnikov et al. 1999, 2000; Pellegrini et al. 2000; Schlessinger et al. 2000). Regulation of the binding specificity of FGFs is primarily achieved by alternative splicing of FGFRs. Alternative splicing of exons encoding the C-terminal part of the Ig3 module in FGFR1–3 results in two isoforms (3b and 3c) possessing different FGF-binding specificity (Miki et al. 1992; Yayon et al. 1992). There are also FGFR isoforms lacking the Ig1 module (FGFR1 and 2), the Ig1 module combined with the Ig1–Ig2 linker sequence (FGFR2), or the Ig1–Ig2 linker alone (in FGFR3) (McKeehan et al. 1998; Shimizu et al. 2001).

The structure of the Ig1 module has so far not been determined, and the physiological significance of the module is not well elucidated. Although the Ig1 module is dispensable for FGF–FGFR binding, the triple Ig-module form of FGFR1 (FGFR1α) has an eightfold and threefold lower affinity for FGF1 and heparin, respectively, compared to the double Ig-module form (FGFR1β) (Wang et al. 1995b). Recently, the same effect of the Ig1 module has been demonstrated for FGFR3, and the module has been shown to bind an FGFR3 fragment consisting of the Ig2 and Ig3 modules with a dissociation constant (K_d) of 20 μM (Olsen et al. 2004).

In order to study the mechanism of the inhibitory effect of the Ig1 module, a structure study of the module was initiated, and in this article we describe the NMR structure of the Ig1 module of mouse FGFR1. The present work provides a structural basis for the further study of the autoinhibitory function of the Ig1 module by NMR and other biophysical methods.

Results

To study the structure of the FGFR1 Ig1 module, a recombinant protein corresponding to the Ig1 module of mouse FGFR1 was expressed in a yeast expression system of Pichia pastoris. A nearly complete assignment of the ¹H and ¹⁵N chemical shifts has previously been reported (Kiselyov et al. 2006) and deposited into BMBR with accession number 6885.

Structure determination

The X-PLOR program (Brünger 1992) was used for structure calculation. A total of 1360 nonredundant NOE distance constraints (734 long-range, 125 medium-range, 374 sequential, and 127 intraresidue NOE constraints) was used in the structure calculations together with 83 backbone dihedral angle constraints derived from the ³J_HNHα coupling constants and 94 hydrogen-bond constraints, which were applied in the later stages of the structure calculation. Hydrogen-bond constraints were identified using several methods, such as a D₂O exchange ¹⁵N HSQC experiment, analysis of the pattern of sequential and interstrand NOEs involving HN and C_αH protons, and analysis of the hydrogen-bond energies in the later stages of the structure calculation. A final set of 20 structures with the smallest energy values was selected out of 100 calculated structures. The final set contains no structures with an NOE constraint violation of >0.5 Å or an angle violation of >5°. The root mean square (RMS) violations for NOE and backbone dihedral angle constraints are 0.030 Å and 0.034°, respectively. All structures exhibit good covalent geometry, with the RMS deviations (RMSDs) from the idealized geometry for bonds, bond angles, and improper bond angles of 0.0029 Å, 0.44°, and 0.36°, respectively. Of the (φ, ψ) angle combinations of the entire ensemble, 98.8% fall into the allowed regions of the Ramachandran plot, and 70.8%, into the most favored region. The RMSDs for the final set of 20 structures from the average structure are 1.05 Å for the backbone atoms and 1.53 Å for all heavy atoms. A stereo view of the overlay of 20 superimposed structures for the backbone atoms is shown in Figure 1. A summary of structural statistics is given in Table 1.

Figure 1.

Structure of the FGFR1 Ig1 module. Stereo view of an overlay of the backbone atoms of 20 superimposed structures of the Ig1 module.

Table 1.

Structural statistics of the Ig1 module of FGFR1

Solution structure

The three-dimensional fold of the module belongs to the intermediate Ig subgroup and can be described as a β-barrel consisting of two β-sheets. One sheet is formed by A′ (L⁴³–V⁴⁵), G (G¹⁰⁸–V¹⁰⁷), F (S⁹⁶–S¹⁰⁵), C (S⁶³–R⁶⁸), and C′ (V⁷¹–L⁷³) β-strands, and the other by A (T²⁶–L²⁷), B (D⁴⁹–R⁵⁴), B′ (L⁵⁷–R⁵⁸), E (E⁸⁵–D⁹⁰), and D (T⁷⁹–T⁸²) strands. A ribbon representation of the structure demonstrating the elements of the secondary structure is shown in Figure 2A. The general strand topology of the Ig1 module is similar to that of the Ig2 and Ig3 modules (Plotnikov et al. 1999; Pellegrini et al. 2000). However, the A/A′ loop of the Ig1 module is much longer than that of the Ig2 and Ig3 modules. It contains eight extra residues compared to the Ig3 module, and five extra residues compared to Ig2. In contrast to the Ig2 and Ig3 modules, where the A/A′ loop is in parallel with the β-barrel, the A/A′ loop of the Ig1 module is situated perpendicularly to the β-barrel (Fig. 2B).

Figure 2.

Comparison of the secondary structures of the Ig1 and Ig3 modules of FGFR. (A) Ribbon representation of the structure of the Ig1 module. (B) Comparison of the structures of the A/A′ loop region of the Ig1 and Ig3 modules.

Discussion

We have here described the structure of the Ig1 module of mouse FGFR1. The structure of the module was found to be of the intermediate Ig subgroup, with the general strand topology being similar to that of the Ig2 and Ig3 modules. However, the A/A′ loop of the Ig1 module is much longer than that of the Ig2 and Ig3 modules of FGFR1 and of the Ig modules of the intermediate type found in other molecules. This indicates that this region of the Ig1 module of FGFR1 may play an important role in the function of the module. The function of the Ig1 module has so far not been well characterized. The Ig1 module is thought to inhibit the FGFR binding to its ligands such as FGF and heparin (Wang et al. 1995b), presumably through the intramolecular binding of the Ig1 module to the Ig2–Ig3 modules (Olsen et al. 2004). Thus, the A/A′ loop region of the Ig1 module can be hypothesized to play a role in the binding of the Ig1 module to the Ig2–Ig3 modules. However, since there is currently no structural information available about the intramolecular Ig1 binding to the Ig2–Ig3 modules, this subject requires further analysis.

The results presented in this report provide a structural basis for the further study of the autoinhibitory and other functions of the Ig1 module by NMR and other biophysical methods.

Materials and methods

Protein expression and purification

The Ig1 module of mouse FGFR1 consists of a His-tag, AGHHHHHHE, and amino acids 21–126 (SWISS-PROT p16092). The Ig1 module was expressed in the KM71 strain of yeast P. pastoris (Invitrogen) according to the manufacturer's instructions. All the proteins were purified as described (Kiselyov et al. 2006) by affinity chromatography using Ni²⁺-NTA resin (QIAGEN) and/or ion exchange chromatography and gel filtration.

NMR measurements

The following samples were used for recording of NMR spectra: 2 mM Ig1 or Ig2 modules (in H₂O or D₂O), 2 mM ¹⁵N-labeled Ig1 or Ig2 modules (in H₂O), 0.5 mM ¹⁵N, ¹³C (50%)-labeled Ig2 module (in H₂O). The buffer was 10 mM sodium phosphate (pH 7.4), 150 mM NaCl, except for the double-labeled sample, where 10 mM sodium phosphate (pH 7.4), 30 mM NaCl was used. The following NMR spectra were recorded and used for assignment of the Ig1 and Ig2 modules: TOCSY in H₂O or D₂O (45 and 70 msec mixing times), NOESY in H₂O or D₂O (80 and 200 msec mixing times), DQFCOSY, ¹⁵N-HSQC, ¹⁵N-TOCSY-HSQC (70 msec mixing time), and ¹⁵N-NOESY-HSQC (125 msec mixing time). For the assignment of the Ig2 module, HNCACB, CBCA(CO)NH, HNCO, HN(CA)CO, HNCA, and HN(CO)CA were also used. All spectra were recorded using the standard setup provided by ProteinPack. The spectra were processed by NMRPipe (Delaglio et al. 1995) and analyzed by Pronto3D (Kjaer et al. 1994). The NMR experiments were performed using Varian Unity Inova 750 and 800 MHz spectrometers. All spectra were recorded at 298 K.

Structure calculation

A simulated annealing protocol using the X-PLOR program (Brünger 1992) was used for structure calculation. A total of 1360 NOE restraints were derived from 80-/200-msec NOESY and 125-msec ¹⁵N-NOESY-HSQC spectra with upper bounds of 2.7, 3.3, and 6.0 Å increased by 0.5 Å if the restraint included a methyl group. Eighty-three ϕ angle restraints with bounds of −120° ± 40° and −57° ± 40° (derived from the ³J_HNHα coupling constants) were used. After inspection of hydrogen bond energies and the rate of hydrogen exchange, 94 hydrogen bond restraints were applied as NOE restraints (in the final structure calculations) with upper bounds of 2 Å and 3 Å for the NH–O and N–O distances, respectively. Of 100 structures calculated, 100 were accepted by X-PLOR, discriminating any structure with an NOE restraint violation >0.5 Å or an angle violation >5°. The structures were analyzed and checked using MOLMOL (Koradi et al. 1996) and PROCHECK_NMR (Laskowski et al. 1993) programs. From these 100 structures, 20 structures with the smallest energies were chosen to represent the structure of the Ig1 module of FGFR1 (PDB code 2ckn).