Quantifying the Impact of Membrane Microtopology on Effective Two-dimensional Affinity

Abstract

Just as interactions of soluble proteins are affected by the solvent, membrane protein binding is influenced by the surface environment. This is particularly true for adhesion receptors because their function requires tightly apposed membranes. We sought to demonstrate, and further, to quantify the possible scale of this phenomenon by comparing the effective affinity and kinetic rates of an adhesion receptor (CD16b) placed in three distinct environments: red blood cells (RBCs), detached Chinese hamster ovary (CHO) cells, and K562 cells. Effective affinity reflects both the intrinsic receptor-ligand kinetics and the effectiveness of their presentation by the host membranes. Expression of CD16b, a low affinity Fc␥ receptor, was established by either transfection or spontaneous insertion via its glycosylphosphatidylinositol anchor. Binding to IgG-coated RBCs, measured using a micropipette method, indicated a 50-fold increase in effective affinity for receptors on RBCs over CHO and K562 cells, whereas the off rates were similar for all three. Electron microscopy confirmed that specific tight contacts were broad in RBC-RBC conjugates but sparse in CHO-RBC conjugates. We suggest that through modulation of surface roughness the cytoskeleton can greatly impact the effectiveness of adhesion molecules, even those with no cytoplasmic structures. Implications for locomotion and static adhesion are discussed.Cell-cell bond formation must be preceded by the creation of a contact zone, a region in which surface-bound receptors and ligands are able to bridge narrow gaps, properly aligning by lateral and rotational diffusion (1). Rough cells initially may form isolated point contacts that over time might be broadened and connected through active membrane processes (2). Alternatively, the cells may simply possess inherently smooth surfaces capable of broad initial contacts. A smooth membrane will not enhance binding to a soluble ligand because the soluble protein can diffuse freely into the membrane folds of rough cells. However, in adhering to immobilized ligand, the ability to form an expansive tight contact area is a distinct advantage. Therefore, cell-cell adhesion depends not only on the intrinsic kinetic rates of the receptor-ligand interaction but also on how effectively the host membranes present these molecules (3).In this study we quantified the effects of various microtopological presentations on cell adhesion by comparing the effective affinity of the same receptor (CD16b) in three distinct host environments: erythrocytes (RBCs), 1 Chinese hamster ovary (CHO) cells, and erythroleukemic K562 cells. Although all other Fc receptors have intracellular domains, CD16b (Fc␥RIIIb) terminates at the lipid bilayer with a glycosylphosphatidylinositol (GPI) moiety and so has no direct cytoskeletal interface. Currently there are no data to suggest the existence of multiple activation states in CD16b. These simplifications permit a more focused investigation into the role of the membrane environment in the functionality of this rec...

Kinetics of MHC-CD8 Interaction at the T Cell Membrane

“…These interactions can be impacted by the cell surface environment, as previously shown for membrane microtopology and stiffness (33,34). Another aspect of the membrane environment may be membrane rafts into which CD8 can partition (4).…”

“…More importantly, the two interacting cells have to be brought together because membranes separated by a distance greater than the molecular size would physically preclude binding. It has been demonstrated that the molecular length and orientation (32) as well as cell surface microtopology and stiffness can significantly affect 2D (but not 3D) affinity (33,34). Although the reverse-rate k r has the same unit (in s Ϫ1 ) for 2D and 3D interaction, only in the 2D (but not 3D) case can k r be regulated by force applied to the molecular bonds-they would be disrupted by the separation of the cells (31).…”

T cells like a firm molecular handshake

“…For example, although having no impact to 3D affinity, reducing flexibility by shortening the length and randomizing the orientation of some receptor-ligand molecules has been found to decrease 2D affinity by reducing 2D on-rate but not 2D off-rate (17). Disrupting the smoothness and continuity of the confinement zone by surface roughness has been shown to reduce the effective 2D affinity (18). Conformational changes in the binding site have been shown to result in different changes in the 2D and 3D on-rates and off-rates (19).…”

A Response Calculus for Immobilized T Cell Receptor Ligands

“…Also, a recent study has demonstrated that membrane roughness can modulate the effective affinity of membrane proteins (33). Taken together with the unexpectedly high values for antibodies and SEC3 variants on surface A, it appeared possible that chemical changes of the ligand-presenting surface could enhance the 2D-affinity of immobilized ligands.…”