Decoding complement: A multi-technique approach to complex interactions

Biology is governed by the dynamic interactions of a multitude of small molecules, proteins, cells, and their aggregates. Describing these processes is a fundamental challenge, and one which is highly relevant to medicine and pharmacology. A prime example for complex interactions involved in human health and disease can be found in the complement system. One of its modes of activation, the classical pathway, proceeds via the formation of immunoglobulin (IgG) hexamers on the surface of pathogenic cells. It is known that these hexamers are held together by Fc:Fc interactions, but the molecular events involved in IgG binding, oligomerization, and subsequent complement activation are poorly understood. The present doctoral thesis introduces a combination of single molecule force spectroscopy, quartz crystal microbalance, and high-speed atomic force microscopy designed to characterize this vital process. Initially, experimental results were analyzed and interpreted individually: Single molecule force spectroscopy revealed the kinetic rates governing the Fc:Fc interaction of two IgGs in solution, and demonstrated that these bonds dissociate quickly after being formed. Thus, oligomerization is effectively precluded in the absence of antigenic surfaces. Quartz crystal microbalance provided ensemble kinetics, and showed that the investigated monovalent Fab molecules bind epitope-membranes only transiently. Functionally monovalent bispecific IgGs, on the other hand, are capable of long-term opsonization due to their ability to oligomerize via their Fc regions. Fc:Fc interactions thus rescue these molecules from dissociation by establishing multivalent configurations, i.e. IgG oligomers. High-speed AFM provided direct visualization of IgG association, hexamer formation, as well as the subsequent binding of C1q, the first component of complement. These experiments also confirmed the geometry of IgG hexamers under physiological conditions, determined their structural stability, and gauged the impact of point mutations on the oligomerization process. Finally, the techniques were combined to build a model describing the molecular details of the classical complement activation, from initial antigen recognition, to the formation of nucleation sites for recruitment of IgGs from solution, to complete hexamerization. Findings from one technique inspired experiments using another, and a complete picture including kinetic rate constants and interaction energies emerged.