Bioluminescence-Based Complementation Assay to Correlate Conformational Changes in Membrane-Bound Complexes with Enzymatic Function.

The proteomics field has undergone tremendous development with the introduction of many innovative methods for the identification and characterization of protein-protein interactions (PPIs). Sensitive and quantitative protein association-based techniques represent a versatile tool to probe the architecture of receptor complexes and receptor-ligand interactions and expand the drug discovery toolbox by facilitating high-throughput screening (HTS) approaches. These novel methodologies will be highly enabling for interrogation of structural determinants required for the activity of multimeric membrane-bound enzymes with unresolved crystal structure and for HTS assay development focused on unique characteristics of complex assembly instead of common catalytic features, thereby increasing specificity. We describe here an example of a binary luciferase reporter assay (NanoBiT®) to quantitatively assess the heterodimerization of the catalytically active NADPH oxidase 4 (NOX4) enzyme complex. The catalytic subunit NOX4 requires association with the protein p22phox for stabilization and enzymatic activity, but the precise manner by which these two membrane-bound proteins interact to facilitate hydrogen peroxide (H2O2) generation is currently unknown. The NanoBiT complementation reporter quantitatively determined the accurate, reduced, or failed complex assembly, which can then be confirmed by determining H2O2 release, protein expression, and heterodimer trafficking. Multimeric complex formation differs between NOX enzyme isoforms, facilitating isoform-specific, PPI-based drug screening in the future.