Mapping the Binding Interface in a Ligand to Create Specificity in Target Affinities

Specificity in binding is the cause of many biological reactions. The binding depends on the position of certain amino acids and its property. Any change in each of the amino acids can affect target affinity. Therefore, identifying these positions is crucial for developing specific protein-protein interactions. Sorted protein libraries and their sequences obtained from Next Generation Sequencing (NGS) do not provide comparison or grading according to their target specificities, which makes it difficult to compare and quantify the absolute binding attribute of the various sequences. In addition, methods that include selective screening are difficult to perform when using on multiple targets simultaneously. In contrast, the approach described here allows us to scan multiple libraries against one target at a time, and compare multiple scans of different targets. In this method, gene libraries obtained from FACS underwent NGS and examined for mutation. Each variant containing a single mutation in the binding interface is scored by a based ER (Enrichment Ratio) method for affinity and selectivity. The method was tested using N-TIMP2 library screened against three MMP (Matrix Metalloproteinases) targets, having similar binding sites, a challenge for obtaining specific binders. Previous studies have identified certain positions in the binding interface of N-TIMP2 that have direct influence on its binding properties. Data obtained in our study showed that inhibitors that contained a mutation in those positions result in significant changes in N-TIMP2 binding affinity and selectivity towards the selected MMP targets. The method helps in engineering proteins with improved binding affinity and specificity.