Binding Site Vectors Enable Mapping of Cytochrome P450 Functional Landscapes

Understanding similarities between protein binding sites has long been of great interest, as such comparisons can reveal functional relationships that transcend sequence or fold. However, systematic comparison remains challenging due to the difficulty of defining active sites consistently and developing descriptors that are both general and discriminative. We present <i>binding site vectors</i>, a computational framework for a high-resolution comparison of macromolecular binding sites that integrates both structural and electrostatic properties. The vectors extend spherically from the center of the pocket, terminating at its surface to capture shape and electrostatic features in a multidimensional manner. Geometrically anchored, they enable a systematic comparison of binding sites across diverse systems. We applied this approach to cytochrome P450 (CYP) enzymes, analyzing over 600 human and plant CYP structures and a subset of 23 extensive structural ensembles obtained through molecular dynamics (MD) simulation. Comparisons based on binding site vectors reveal structural-functional relationships missed by sequence- or backbone-based groupings, particularly when full conformational ensembles are included. This demonstrates that binding site vectors provide a robust framework for both functional classification and deep mechanistic insights into macromolecular systems.