A single residue switch reveals principles of antibody domain integrity

Abstract

Despite their importance for antibody architecture and design, the principles governing antibody domain stability are still not understood in sufficient detail. Here, to address this question, we chose a domain from the invariant part of IgG, the C_H2 domain. We found that compared with other Ig domains, the isolated C_H2 domain is a surprisingly unstable monomer, exhibiting a melting temperature of ∼44 °C. We further show that the presence of an additional C-terminal lysine in a C_H2 variant substantially increases the melting temperature by ∼14 °C relative to C_H2 WT. To explore the molecular mechanism of this effect, we employed biophysical approaches to probe structural features of C_H2. The results revealed that Lys¹⁰¹ is key for the formation of three secondary structure elements: the very C-terminal β-strand and two adjacent α-helices. We also noted that a dipole interaction between Lys¹⁰¹ and the nearby α-helix, is important for stabilizing the C_H2 architecture by protecting the hydrophobic core. Interestingly, this interaction between the α-helix and C-terminal charged residues is highly conserved in antibody domains, suggesting that it represents a general mechanism for maintaining their integrity. We conclude that the observed interactions involving terminal residues have practical applications for defining domain boundaries in the development of antibody therapeutics and diagnostics.

Keywords: CH2 domain; antibody; antibody constant domain; antibody engineering; biophysics; immunoglobulin fold; molecular dynamics; nuclear magnetic resonance (NMR); protein aggregation; protein folding; protein stability.

Figure 1.

Secondary, tertiary and quaternary structure analysis of C_H2 variants. A, scheme of C_H2 variants. B, cartoon representation of the MAK33 C_H2-SKTK isolated from the Fc fragment (PDB 3HKF, includes C_H2 and C_H3) with indicated termini, intramolecular disulfide bond (yellow), tryptophan residues (orange), and C-terminal residues (Ser¹⁰⁰, red; Lys¹⁰¹, green; Thr¹⁰² and Lys¹⁰³, blue). Far (C) and near (D) UV CD spectra of C_H2 variants. All variants share the same WT-like secondary structure. The tertiary structure, however, exhibits significant differences between the C_H2 variants with maximal distinction between 275 and 295 nm, which indicates changes in the tertiary structure especially for Trp residues. E, SEC-MALS experiments exhibit monomers as the prevalent species for all C_H2 variants. Only a small oligomer fraction (<7%) was found. Hence, terminal extensions of the C_H2 domains do not alter the quaternary structure.

Figure 2.

Stability of C_H2 variants. A, thermal, and B, chemical equilibrium unfolding transitions of C_H2 variants reveal a significant impact of the C-terminal Lys¹⁰¹ on the conformational stability. For chemical denaturation, aliquots of the C_H2 variants were incubated for at least 12 h with increasing concentrations of GdmCl and monitored by intrinsic tryptophan fluorescence.

Figure 3.

Conformational differences of C_H2 variants. A, intrinsic tryptophan fluorescence of both, Trp⁴⁰ and Trp⁷⁶, probes conformational changes. All folded C_H2 variants (solid lines) display different Trp fluorescence intensities at the same concentration (7 μm). However, variants extended by Lys¹⁰¹ feature altered emission maxima with a significant blue shift of ∼8 nm compared with C_H2 WT and C_H2-S. For comparison, all variants show identical spectra when unfolded using 3.8 m GdmCl (dotted lines). B, Stern-Volmer plots show different acrylamide quenching profiles for C_H2 variants. The introduction of the C-terminal Lys¹⁰¹ (C_H2-SK and C_H2-SKTK) leads to a significantly lower ability of acrylamide to quench intrinsic Trp fluorescence. Thus, Lys¹⁰¹ alters the accessibility of the Trp residues.

Figure 4.

HDX experiments to probe protein dynamics. A, relative fractional uptake ratio for C_H2/C_H2-SKTK reveals pronounced altered HDX levels up to 2.3-fold for distinct regions. Data represent the mean of two individual experiments for each variant; shadows indicate standard deviation. B, relative uptake ratio for C_H2/C_H2-SKTK displayed on the crystal structure of C_H2 (PDB 3HKF). Color gradient from blue (no difference) to red (2.3-fold decrease in exchange relative to C_H2) indicates structural and dynamical alteration between C_H2 and C_H2-SKTK. Four distinct regions display pronounced differences caused by the presence of SKTK at the C terminus. Black: no HDX data available for these residues. HDX experiments for the shown data were quenched after 10 s of exchange.

Figure 5.

NMR chemical shift changes for C_H2 and EV-C_H2-SK. A, ¹H,¹⁵N-chemical shift differences observed for C_H2 and EV-C_H2-SK. The largest changes in chemical shifts occur in the C-terminal part of the protein, whereas the α-helical region involving residues 10–14 and 72–78 could not be assigned for C_H2 WT. Residue numbering was according to the C_H2 WT sequence. Asterisks indicate unassigned residues. B, ¹³Cα chemical shift differences δ(¹³C, experimental) − δ(¹³C, random coil). Random coil chemical shifts were taken from Wishart and Sykes (28). Positive values indicate propensity for an α-helix, negative values for a β-strand. The top panel shows the chemical shift differences for WT C_H2, the bottom panel for EV-C_H2-SK. Blue and red bars at the bottom of the lower panel show the secondary structure as indicated in the crystal structure (PDB 3HKF), full height shaded areas highlight the major changes in secondary structure propensity. The secondary chemical shifts of assignable residues in the extended variant indicate the presence of two stable α-helices in the regions between residues 10–14 and 73–80 in EV-C_H2-SK. These residues probably show dynamics on a time scale that makes the residues unresolvable by NMR in the WT. In addition, the C-terminal region of EV-C_H2-SK shows propensity to form a β-strand, whereas the same region in the WT appears more likely to occupy a random coil conformation. Asterisks indicate unassigned residues.

Figure 6.

Mean solvent accessibility during MD simulations. The ratio in mean SASA of residues in C_H2 WT relative to C_H2-SK (green) and relative to C_H2-SKTK (blue) was calculated for each residue over a simulation period of 1 μs at 300 K. A SASA ratio >1 indicates increased mean accessibility relative to C_H2-SK or relative to C_H2-SKTK, respectively (obtained as sliding window average over 10 consecutive residues).

Figure 7.

Calculated free energy change upon dissociation of the C-terminal β-strand from its placement in the folded structure to a solvated unfolded state. The free energy change (PMF) was calculated along a reaction coordinate that corresponds to the center of mass distance of backbone atoms of the C-terminal segment (delimited by residues 94 to 98) and the backbone atoms of the protein (delimited by residues 1 to 91). Representative snapshots taken in the folded, intermediate, and dissociated states of the C-terminal segment are indicated (color-code of the cartoon representation corresponds to the line color in the plot). The C-terminal Lys residue in C_H2-SK is indicated as van der Waals sphere representation and still interacts with the helical segment (residues 72–81) in the intermediate state (at which the C terminus of the C_H2 WT is already fully dissociated).