Unraveling Energy and Dynamics Determinants to Interpret Protein Functional Plasticity: The Limonene-1,2-epoxide-hydrolase Case Study

The balance between structural stability and functional plasticity in proteins that share common three-dimensional folds is the key factor that drives protein evolvability. The,ability, to distinguish the parts of homologous proteins that underlie common structural-nrganiZation patterns from the parts acting as regulatory modules that can sustain modifications in response to evolutionary pressure may provide fundamental insights for understanding sequence-structure- dynamics relationships. In applicative terms, this would help develop rational protein design methods. Herein, we.apply recently developed computational methods, validated by experimental tests, to address these questions in a set of homologous enzymes representative of the limonene-1,2-epoxide-hydrolase family (LEH) characterized by different stabilities, namely Rhodococcus erythropolis LEH (Re-LEH), Tomks-LEH, CHSS-LEH, and the two thermostable Re-LEH variants Re-LEH-Flb and Re-LEH-P. Our results show that these enzymes, despite significant sequence variations, exploit a few highly conserved stabilization determinants to guarantee structural stability linked to biological functionality. Multiple sequence analysis shows that these key elements are also shared' by a larger set of LEHs structural homologues, despite very low sequence identity and:functional diversity. In this framework, stabilizing elements that we `hypothesize to have an accessory role are characterized by a lower degree of sequence identity and higher mutability. We suggest that our approach can be successfully used to pinpoint the distinctive energy fingerprint of a class of proteins as well as to locate those modulators whose modification could be exploited to tuneprotein stability and dynamic properties.