Identification of substrate-specific allosteric fingerprints in matrix metalloprotease-1 at the single molecule level for precision control of function.

Abstract

MMPs, a family of 23 human enzymes, interact with and degrade many biomolecules. Any drug targeting the active site of MMPs inhibits intended and unintended functions and results in adverse side effects. If we identify substrate-specific allosteric locations (distant from the active site), we may change one role of MMPs without affecting their other functions for fewer side effects. We attached fluorescent dyes at two specific sites on the two domains of MMP1 to measure inter-domain dynamics on four water-insoluble substrates: collagen fibril, fibrin, alpha-synuclein (aSyn) aggregates, and amyloid-beta (AB) aggregates. We calculated the distance between the two locations from the single molecule Forster Resonance Energy Transfer (smFRET) between the two dyes, measured using a Total Internal Reflection Fluorescence (TIRF) microscope. Low FRET (open) MMP1 conformations with well-separated domains are relevant for collagen, fibrin, and aSyn. In contrast, high FRET (closed) conformations apply to AB aggregates. Functionally relevant conformations are present in active MMP1 but significantly absent in catalytically inactive MMP1. We found that each substrate has unique fingerprints (residues) in MMP1 and leads to changes at the catalytic and distant allosteric sites. Screening drugs against substrate-specific allosteric sites predicted substrate-specific drugs for targeting MMP1 based on single molecule insights.