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

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

Crystal structure of the compound UCB-FcRn-303 (R enantiomer) bound to FcRn_ECD.

<p><b>(A)</b> The protein crystallized as a dimer composed of two β2m (dark grey and green) and two α-chain (light grey and blue) molecules. <b>(B)</b> At the interface of β2m and the α-chain, UCB-FcRn-303 (grey) occupies a binding pocket with Glycine, Cysteine, hydrophobic (Leucine), charged (Histidine, Aspartate), and polar uncharged (Serine, Glutamine) residues. β2m, β2-microglobulin; FcRn, neonatal Fc receptor; FcRn_ECD, extracellular domain of the neonatal Fc receptor.</p

CSPs cluster at the potential FcRn_ECD diprotomer interface.

<p><b>(A)</b> CSPs in surface representation of the FcRn_ECD diprotomer crystal structure in complex with UCB-FcRn-303 (red), with the same color-coding as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006192#pbio.2006192.g005" target="_blank">Fig 5</a>. <b>(B)</b> For orientation, the FcRn_ECD crystal structure is shown in cartoon representation with β2m in green and dark grey and the α-chain molecules in blue and light grey. <b>(C)</b> The IgG and HSA interaction sites are depicted in purple and orange, respectively. The highlighted residues are discussed in the text. CSP, chemical-shift perturbation; FcRn, neonatal Fc receptor; FcRn_ECD, extracellular domain of the neonatal Fc receptor; HSA, Human Serum Albumin; IgG, Immunoglobulin G.</p

Proton-detected MAS NMR on fully protonated FcRn_ECD.

<p><b>(A)</b> The soluble FcRn_ECD (42 kDa) was sedimented by ultracentrifugation at 100,000 x <i>g</i> directly into a 0.7 mm MAS NMR rotor using a home-made filling tool. <b>(B)</b> 2D ¹⁵N-¹H correlation spectrum recorded at 100 kHz MAS of fully protonated [¹³C,¹⁵N]-labeled FcRn_ECD. <b>(C)</b> Typical linewidths of ¹H (1) and ¹⁵N (2) at full-width-half-maximum (FWHM) of a selected cross peak from the ¹⁵N-¹H spectrum. β2m, β2-microglobulin; FcRn, neonatal Fc receptor; FcRn_ECD, extracellular domain of the neonatal Fc receptor.</p

CSPs (Δδ) indicate structural changes in FcRn_ECD upon ligand binding.

<p><b>(A)</b> CSPs of all assigned amino acids in FcRn_ECD upon binding to UCB-FcRn-303, calculated with Δδ = MIN{SQRT[(Δδ(¹H))² + (Δδ(¹³C)/10)² + (Δδ(¹⁵N)/5)²]} (standard deviation = 0.015 ppm). The changes were measured in 3D (H)CANH MAS NMR spectra. <b>(B)</b> CSPs upon UCB-FcRn-303 binding to FcRn_ECD in 2D planes of 3D (H)CANH spectra with (black) and without (blue) the ligand, both recorded in the presence of 3% DMSO. <b>(C)</b> Structural view of UCB-FcRn-303 (red) bound to FcRn_ECD with assigned residues in stick representation color-coded according to their CSP (Δδ < 0.02, white; 0.02 < Δδ < 0.03, cyan; 0.03 < Δδ < 0.04, marine; 0.04 < Δδ, dark blue). <b>(D)</b> Structural view of FcRn_ECD in complex with UCB-FcRn-303 (<i>red</i>) with the same color-coding of changes in chemical-shifts as in (C). All chemical-shifts can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006192#pbio.2006192.s016" target="_blank">S1 Table</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006192#pbio.2006192.s015" target="_blank">S2 Data</a>, and in the BMRB (accession number 27437). BMRB, Biological Magnetic Resonance Data Bank; CSP, chemical-shift perturbation; FcRn, neonatal Fc receptor; FcRn_ECD, extracellular domain of the neonatal Fc receptor.</p

FcRn allows maintenance of protein homeostasis.

<p>The soluble extracellular domain of neonatal Fc receptor (FcRn_ECD, PDB code 1EXU) is a heterodimer composed of β2m (green) and α-chain (blue) with a cavity at the interface between the two proteins. FcRn is involved in the regulation of HSA (orange) and IgG (red) levels. The binding of both HSA and IgG to FcRn is pH dependent, which provides a mechanism for protein homeostasis through endosomal trafficking. β2m, β2-microglobulin; FcRn, neonatal Fc receptor; FcRn_ECD, extracellular domain of the neonatal Fc receptor; HSA, Human Serum Albumin; IgG, Immunoglobulin G; PDB, Protein Data Bank.</p

Triple-resonance MAS NMR spectra enable assignments of chemical-shifts.

<p>Sequential resonance assignments using the experiments (H)CANH (blue), (H)CA(CO)NH (red), and (H)CBCANH (green) recorded on fully protonated [¹³C,¹⁵N]-labeled FcRn_ECD at 100 kHz MAS. As an example, the sequential connections from K41_β2m to R45_β2m in β2m are indicated by dashed lines. All assigned chemical-shifts can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006192#pbio.2006192.s016" target="_blank">S1 Table</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006192#pbio.2006192.s015" target="_blank">S2 Data</a>, and in the BMRB (accession number 27437). β2m, β2-microglobulin; BMRB, Biological Magnetic Resonance Data Bank; FcRn_ECD, extracellular domain of the neonatal Fc receptor; MAS, magic-angle-spinning.</p