Fig 1.
FcRn allows maintenance of protein homeostasis.
The soluble extracellular domain of neonatal Fc receptor (FcRnECD, 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; FcRnECD, extracellular domain of the neonatal Fc receptor; HSA, Human Serum Albumin; IgG, Immunoglobulin G; PDB, Protein Data Bank.
Fig 2.
Crystal structure of the compound UCB-FcRn-303 (R enantiomer) bound to FcRnECD.
(A) The protein crystallized as a dimer composed of two β2m (dark grey and green) and two α-chain (light grey and blue) molecules. (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; FcRnECD, extracellular domain of the neonatal Fc receptor.
Fig 3.
Proton-detected MAS NMR on fully protonated FcRnECD.
(A) The soluble FcRnECD (42 kDa) was sedimented by ultracentrifugation at 100,000 x g directly into a 0.7 mm MAS NMR rotor using a home-made filling tool. (B) 2D 15N-1H correlation spectrum recorded at 100 kHz MAS of fully protonated [13C,15N]-labeled FcRnECD. (C) 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; FcRnECD, extracellular domain of the neonatal Fc receptor.
Fig 4.
Triple-resonance MAS NMR spectra enable assignments of chemical-shifts.
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 FcRnECD at 100 kHz MAS. As an example, the sequential connections from K41β2m to R45β2m in β2m are indicated by dashed lines. All assigned chemical-shifts can be found in S1 Table, S2 Data, and in the BMRB (accession number 27437). β2m, β2-microglobulin; BMRB, Biological Magnetic Resonance Data Bank; FcRnECD, extracellular domain of the neonatal Fc receptor; MAS, magic-angle-spinning.
Fig 5.
CSPs (Δδ) indicate structural changes in FcRnECD upon ligand binding.
(A) CSPs of all assigned amino acids in FcRnECD 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) CSPs upon UCB-FcRn-303 binding to FcRnECD in 2D planes of 3D (H)CANH spectra with (black) and without (blue) the ligand, both recorded in the presence of 3% DMSO. (C) Structural view of UCB-FcRn-303 (red) bound to FcRnECD 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). (D) Structural view of FcRnECD in complex with UCB-FcRn-303 (red) with the same color-coding of changes in chemical-shifts as in (C). All chemical-shifts can be found in S1 Table, S2 Data, and in the BMRB (accession number 27437). BMRB, Biological Magnetic Resonance Data Bank; CSP, chemical-shift perturbation; FcRn, neonatal Fc receptor; FcRnECD, extracellular domain of the neonatal Fc receptor.
Fig 6.
CSPs cluster at the potential FcRnECD diprotomer interface.
(A) CSPs in surface representation of the FcRnECD diprotomer crystal structure in complex with UCB-FcRn-303 (red), with the same color-coding as in Fig 5. (B) For orientation, the FcRnECD crystal structure is shown in cartoon representation with β2m in green and dark grey and the α-chain molecules in blue and light grey. (C) 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; FcRnECD, extracellular domain of the neonatal Fc receptor; HSA, Human Serum Albumin; IgG, Immunoglobulin G.