Abstract
Human immunoglobulin G, subclass 2 (hIgG2), plays an important role in immunity to bacterial pathogens and in numerous pathological conditions. However, there is a lack of information regarding the three-dimensional (3D) structure of the hIgG2 molecule. We used electron microscopy (EM), differential scanning microcalorimetry (DSC) and fluorescence for structural analysis of the hIgG2. DSC and fluorescence indicated two types of interaction between CH1 domain of Fab (antigen-binding fragment/subunit) and CH2 domain of Fc (complement fixation fragment/subunit) simultaneously present in the sample: close interaction, which increases the thermostability of both, CH1 and CH2 domains, and weak (or no) interaction, which is typical for most IgGs but not hIgG2. Thermodynamics could not determine if both types of interactions are present within a single molecule. To address this question, EM was used. We employed a single-particle reconstruction and negative staining approach to reveal the three-dimensional structure of the hIgG2. A three-dimensional model of hIgG2 was created at 1.78 nm resolution. The hIgG2 is asymmetrical: one Fab subunit is in close proximity to the upper portion of the Fc subunit (CH2 domain) and the other Fab is distant from Fc. The plane of Fab subunits is nearly perpendicular to Fc. EM structure of the hIgG2 is in good agreement with thermodynamic data: a Fab distant from Fc should exhibit a lower melting temperature while a Fab interacting with Fc should exhibit a higher melting temperature. Both types of Fab subunits exist within one molecule resembling an A/B hIgG2 isoform introduced earlier on physicochemical level by Dillon et al. (2008). In such an arrangement, the access to the upper portion of Fc subunit is partially blocked by a Fab subunit. That might explain for instance why hIgG2 mildly activates complement and binds poorly to Fc receptors. Understanding of the three-dimensional structure of the hIgG2 should lead to better design of antibody-based therapeutics.
Highlights
The immunoglobulin G (IgG) molecule is composed of two Fab subunits that are linked to Fc subunit via a hinge region
Different Human IgG (hIgG) subclasses have different abilities to activate the classic complement cascade, which are mediated by the structural properties of the hinge region, and/or by the structure of the C1q binding site located in CH2 domains [1,2,3,4]
At acidic pH values, all IgG and isolated Fc fragments studied exhibit a low-temperature peak of heat absorption, which corresponds to melting of the CH2 domains [20,21,22,23,24,25]
Summary
The immunoglobulin G (IgG) molecule is composed of two Fab subunits that are linked to Fc subunit via a hinge region. Human IgG (hIgG) subclasses exhibit a tremendous variety of functions while in general, the structure of Fab and Fc is quite conservative. Different hIgG subclasses have different abilities to activate the classic complement cascade, which are mediated by the structural properties of the hinge region, and/or by the structure of the C1q binding site located in CH2 domains [1,2,3,4]. A strong modulating effect of the lower hinge region of hIgG1 on C1q binding may be mediated via a change in the predominant shape of an hIgG1 molecule [5], i.e. the binding site may be opened or closed for ligand binding according to the reciprocal arrangement of Fab and Fc subunits
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