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.