Despite recent methodological advances, the study of protein-protein interactions still presents multiple types of experimental challenges, particularly when membrane proteins are involved, due to their biochemical intractability and the highly transient nature that typifies many of their interactions.
“We developed a new approach to address the paucity of existing methods that existed for studying this class of protein-protein interactions,” says Gavin J. Wright, Ph.D., team leader at the cell surface signalling laboratory, the Wellcome Trust Sanger Institute.
To identify and study low-affinity interactions between membrane-embedded receptor proteins and their ligands, Dr. Wright and colleagues designed a method called AVEXIS (AVidity-based EXtracellular Interaction Screen). “We hope that by applying this technology, we will facilitate the identification of interactions involved in cellular recognition processes that are difficult to detect using other approaches,” explains Dr. Wright.
AVEXIS was created by generating libraries of proteins that consist of only the extracellular regions of the receptors, which are expressed as soluble recombinant proteins in mammalian cells.
“The idea was to take two cell types that are known to interact, make a protein library that represents the cell surface receptor repertoire of each cell type, and then screen within the two libraries to identify new interactions in a very systematic and unbiased way,” notes Dr. Wright.
To circumvent the challenges raised by the low affinity of the interactions, Dr. Wright and colleagues multimerized the protein by using a peptide from the cartilage oligomeric matrix protein, which induces formation of pentamers, increasing the local concentration of the protein of interest. This helps increase the half-life of the interaction, which is often less than a second, to tens of minutes or several hours, enabling the detection of interactions that would otherwise be challenging to capture.
“The appreciation that the interaction affinities can be weak is important, because regular biochemical purification methods aren’t likely to work for these very transient interactions,” explains Dr. Wright.
By using AVEXIS, researchers in Dr. Wright’s lab recently found that basigin, an erythrocyte surface receptor, binds RH5, a Plasmodium falciparum ligand, and that the basigin-RH5 interaction was essential for erythrocyte invasion by the parasite. This finding now holds promise for the development of an antimalarial blood-stage vaccine.
Dr. Wright and colleagues are also focusing on dissecting receptor-ligand interactions involved in platelet aggregation, an area that promises to unveil key molecular events in cardiovascular diseases and stroke, and on understanding molecular events involved in sperm-egg recognition.
“Understanding the molecular basis of how sperm and egg recognize each other and interact is a remarkably understudied area with important clinical applications, and represents a fundamental biological problem that has gone largely unaddressed due to the lack of technology,” explains Dr. Wright.