Conformational Rigidity within Plasticity Promotes Differential Target Recognition of Nerve Growth Factor

Nerve Growth Factor (NGF), the prototype of the neurotrophin family, is essential for maintenance and growth of different neuronal populations. The X-ray crystal structure of NGF has been known since the early '90s and shows a β-sandwich fold with extensive loops that are involved in the interaction with its binding partners. Understanding the dynamical properties of these loops is thus important for molecular recognition. We present here a combined solution NMR/molecular dynamics study which addresses the question of whether and how much the long loops of NGF are flexible and describes the N-terminal intrinsic conformational tendency of the unbound NGF molecule. NMR titration experiments allowed identification of a previously undetected epitope of the anti-NGF antagonist antibody αD11 which will be of crucial importance for future drug lead discovery. The present study thus recapitulates all the available structural information and unveils the conformational versatility of the relatively rigid NGF loops upon functional ligand binding.

Side chain aliphatic proton and carbon assignments were achieved by a combination of 3D 15 N-edited NOESY-HSQC, 13 C NOESY-HSQC, CBCACONH and HCCH-TOCSY.
(Hβ)Cβ(CγCδ)Hδ and (Hβ)Cβ(CγCδ)Hε experiments were used in combination with 13 C-HSQC, 13 C-NOESY-HSQC and HCCH-TOCSY tuned for the aromatic resonances for the assignment of the large number of the aromatic side chains.
All spectra were processed using NMRPipe/NMR-Draw (Delaglio et al., 1995) and analyzed using CARA (Keller, 2004). Intra-molecular proton distance restraints were derived from 15 N-and 13 C-NOESY-HSQC spectra (mixing time 100 ms). A second set of 15 N-and 13 C-NOESY-HSQC spectra was recorded at 35°C. Backbone assignment was guided by the identification of the expected set of connectivities within and inter-strands, while side chains assignment was complicated by the severe overlap of resonances from Leu, Ile, Lys, Arg residues which are highly abundant. Several of these resonances correspond to residues which cluster at the homodimeric interface. Assignment of the aromatic side chains could be obtained almost to completeness, although limited ambiguity remains for the side chains of some phenylalanines (Phe7, Phe12, Phe49).

Structure Determination
Automated NOESY cross-peak assignments and structure determination were performed using the ARIA 2.3 software (Rieping et al., 2007) based on an almost complete assignment of NGF and on a large number of intra-and inter-molecular NOEs. The ARIA input used to generate the final structures consisted of NGF intra-molecular NOE cross peaks from 15 N-and 13 C-NOESY-HSQC spectra (at 30°C and 35°C), along with a set of φ and ψ backbone dihedral restraints derived by TALOS+ (Shen et al., 2009). The NMR structural bundle was generated by ARIA using ambiguous and unambiguous intraprotomer distance restraints (both manually and automatically assigned) derived from 15 Nand 13 C-NOESY-HSQC (aliphatic and aromatic) experiments at 30°C and 35°C, in addition to a set of manually assigned unambiguous inter-protomer restraints. The total number of unambiguous intramolecular restraints used in the calculations was 2327 (per protomer) plus 33 intermolecular (per protomer). Dihedral restraints derived by TALOS+ (55 phi/psi couples/protomer) and secondary structure-derived H-bonds (46/protomer) were also used as restraints, the latter only being introduced when present in 50% the preliminary calculations. In the last ARIA run, 200 structures were generated in the final iteration. After refinement of the 60 lowest global energy structures by molecular dynamics simulation in water, 20 structures were selected as representative of the structure and used for statistical analysis. Structure quality was evaluated with PROCHECK-NMR (Laskowski et al., 1996).

Molecular Dynamics Simulations and Analysis
A molecular model of the full length mNGF encompassing residues (2-118) was built (Covaceuszach et al., 2015) from the crystal structures of murine bis-des-octa -NGF (PDB ID 1BTG, protomers B,C). The missing N-term region (Ser 2 -Met 9) was extended by molecular modeling on the basis of the crystallographic structure of hNGF from hNGF-TrkA complex (PDB ID 2IFG, protomers E,F). Molecular dynamics simulation (MD) was performed using the GROMACS software package (version 5.1.2) (Hess et al., 2008) conjugated with the Amber99SB force field. The protein was immersed in a cubic box with periodic boundary conditions and was solvated with explicit Single Point Charge waters (Berendsen et al., 1981). The protonation state of the ionisable groups of the protein was set according to pH 7.0 and the overall charge of the system was neutralized by adding an appropriate number of counter ions. The box dimensions (9.7 nm × 9.7 nm × 9.7 nm) were set to allow at least 1.2nm between the protein and the box faces on each side. The final system consisted of 3642 protein atoms surrounded by 11,000 water molecules. Before starting the production run, the system (protein and water) was energy minimized by using the steepest descent method until convergence (1000 iterations). Next, temperature and pressure were pre-equilibrated by short 50 ps canonical NVT ensemble and 1 ns isothermal-isobaric NPT ensemble runs with restraints on the solute. A 200 ns production MD was performed by coupling the system to an external temperature bath at 300 K and an external isotropic pressure bath at 1 bar, using a time step of 0.002 ps and the trajectories were saved at each 2 ps.
All the bond lengths were constrained to their equilibrium values using the LINCS algorithm (Hess et al., 1997) for the protein. Long-range electrostatic forces were treated using the Fast Particle-Mesh Ewald method (PME) (Essmann et al., 1995). Van der Waals forces were treated using a cut-off of 1.0 nm.