Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR

Abstract

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)–Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and β2-microglobulin (β2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to β2m, both of which participate in the FcRnECD–IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns.

Sedimentation Yields Long-Term Stable Protein Samples as Shown by Solid-State NMR

“…Sample preparation by sedimentation allowed for the investigation of proteins that are difficult (or even impossible) to crystallize. Theoretically, the molecular mass of the protein determines the success of sedimentation, and proteins, such as the RNA polymerase subunits Rpo4/7 * (the * indicates that the Rpo7 unit is uniformly 13 C/ 15 N labeled) with a molecular weight of 34 kDa (Torosyan et al, 2019), the pRN1 primase with 40 kDa (Boudet et al, 2019), the neonatal Fc receptor with 40 kDa (Stöppler et al, 2018) as well as the human superoxide dismutase with 32 kDa (Fragai et al, 2013), the bacterial helicase DnaB with 708 kDa (Gardiennet et al, 2012), the iron-storage protein ferritin with 480 kDa (Bertini et al, 2012), a variety of supramolecular assemblies (Lecoq et al, 2018;Gauto et al, 2019), and PEGylated proteins (Ravera et al, 2016), were shown to form sediments suitable for solid-state NMR. This is for some of these systems probably a consequence of oligomerization that has been induced by the high protein concentrations (Bertini et al, 2013;Fragai et al, 2013).…”

“…Protein-protein complexes were studied by co-sedimentation (Gardiennet et al, 2016;Xiang et al, 2018) and protein-ligand complexes by sedimentation with the respective ligand (e.g. nucleic acids) directly into the NMR rotor (Wiegand et al, 2016a;Kaur et al, 2018;Stöppler et al, 2018;Lacabanne et al, 2019b).…”

Nucleotide Binding Modes in a Motor Protein Revealed by 31P‐ and 1H‐Detected MAS Solid‐State NMR Spectroscopy

“…These interactions have been typicallys tudied in the past by high-resolution X-ray crystallography. [14][15][16][17][18][19][20][21][22][23] Proton chemical-shift values are highly sensitivet oh ydrogen bonding [24][25][26][27] as shown in theoretical, [26,27] buta lso in experimental studies. [8] And indeed, solid-state NMR spectroscopy has been used to identify residues at protein-RNA interfaces in smaller proteins.…”

“…[9] Intermolecular information can also be obtained from 31 P-detected, heteronuclear correlation experimentsp robingt he spatial proximity of nucleotide 31 Pa nd protein 15 No r 13 Cn uclei. [14][15][16][17][18][19][20][21][22][23] Proton chemical-shift values are highly sensitivet oh ydrogen bonding [24][25][26][27] as shown in theoretical, [26,27] buta lso in experimental studies. [14][15][16][17][18][19][20][21][22][23] Proton chemical-shift values are highly sensitivet oh ydrogen bonding [24][25][26][27] as shown in theoretical, [26,27] buta lso in experimental studies.…”

An Efficient, Robust New Scheme for Establishing Broadband Homonuclear Correlations in Biomolecular Solid State NMR

“…Continuous technological and conceptual developments have endowed contemporary high‐resolution solid‐state nuclear magnetic resonance (NMR) spectroscopy, with capabilities that rival those of solution‐state NMR when tackling structural and dynamic investigations of large biomolecules . When performing such studies the problem of spectral assignment and resolution nearly invariably arises, demanding the use of high‐dimensional NMR experiments correlating labeled 13 C‐ and/or 15 N sites in the biomolecule.…”