Mapping the binding interface in a ligand to create selectivity 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 contract, the approach described here allows 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 undergo NGS and examined for mutation. Each inhibitor containing a mutation at a selected position is scored by a based ER (Enrichment Ratio) method for affinity and selectivity, and compared to the presorted library.

The method was tested using N-TIMP2 library screened against target MMPs (Matrix Metalloproteinases) having similar binding site, a challenge for obtaining specific binders. Previous studies have identified certain positions in the binding interface of N-TIMP2s that have direct influence on its binding properties. Data obtained in our study showed that inhibitors that contained a mutation in the positions 4, 35, 38, 68, 71, 97 and 99, result in significant changes in N-TIMP2 affinity and selectivity binding towards MMPs. The method can be used on different proteins and compare many different sequences, allowing us to discover a sequence containing specific binding properties.