Figure 1. Structural characteristics of the C_H1 domain and its assembly mechanism with the C_L domain.

(A) shows a schematic representation of the IgG1 molecule. The IgG molecule is composed of two heavy chains (blue) and two light chains (green). The heavy chain consists of the V_H, C_H1, C_H2 and C_H3 domain and the light chain of the V_L and the C_L domain (listing from N- to C-terminus of each chain). The C_H2 domains of the heavy chains are glycosylated with complex biantennary oligosaccharides (depicted in grey). Each domain possesses an internal disulfide bridge (omitted for clarity) and additional disulfide bonds link the two heavy chains in the flexible hinge region. A single disulfide bond covalently connects C_H1 with the C_L domain. The two identical antigen binding sites (paratopes) are made up by the two variable domains V_H and V_L. The overall IgG molecule can be divided into two Fab fragments (composed of V_H, C_H1, V_L and C_L) and one Fc fragment (composed of two C_H2 and two C_H3 domains). (B) The isolated C_L domain (cyan) displays a typical all-β far-UV CD spectrum whereas the isolated C_H1 domain (blue) shows a random coil spectrum. To assess if C_L induces structural changes in C_H1, the spectrum of co-incubated C_L and C_H1 was recorded (green). From this spectrum and the far-UV CD spectrum of the isolated C_L domain, the spectrum of the C_H1 domain in the presence of C_L was calculated (red) which shows the characteristics of β-sheet secondary structure. (C) The affinity between C_H1 and C_L was determined by the change in the intrinsic fluorescence upon C_L induced folding of the C_H1 domain, recorded before and after a 4 h equilibration step. A one-site binding model was used to fit the data. The inset shows a representative single exponential trace observed after the addition of 1 µM C_L to 2 µM C_H1. A single exponential reaction with a very similar rate was observed by far-UV CD spectroscopy (D, red trace). The folding reaction could be accelerated by the PPIase CypB (red trace: 10 µM C_L and 10 µM C_H1 alone, blue trace: in the presence of 0.75 µM CypB). The inset shows the dependence of the slow reaction on CypB concentration. The observed rate in the absence of CypB is denoted as k₀, in the presence of CypB as k_cat. If 1µM CypB was inhibited by 2 µM cyclosporine A, no acceleration was observed (black cross). (E) Association of the C_H1 domain with a lucifer yellow labeled C_L domain was followed by the change in the lucifer yellow anisotropy signal. The observed rate constants were fitted with a linear function to yield the k_on value and the k_off value of 0.007 ±0.0002 µM⁻¹ min⁻¹ respectively 0.1 ±0.01 min⁻¹. The inset shows individual single exponential traces after the addition of 0 µM (black), 5 µM (blue), 10 µM (green) and 20 µM (red) C_H1 to 1 µM labeled C_L. (F) To assess the formation of the C_L/C_H1 interchain disulfide bridge, non-reducing SDS-PAGE experiments were carried out and the dimer band intensity was quantified. 25 µM of each domain were used. In the absence of CypB (red), a time constant of τ = 63 ±7 min was observed for the formation of covalent C_L/C_H1 dimers. In the presence of 5 µM CypB, a time constant of τ = 31 ±5 min was obtained. In (G), the overall C_H1/C_L assembly mechanism is shown. Only after formation of the internal disulfide bridge in the C_H1 domain (blue), the fast formation of a dimeric intermediate with the C_L domain (green) is observed. Subsequently, prolyl isomerization limits complete folding and formation of the interchain disulfide bridge. All measurements were carried out at 25°C in PBS.