Unraveling Energy and Dynamics Determinants to Interpret Protein Functional Plasticity: The...

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 organization 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, CH55-LEH, and the two thermostable Re-LEH variants Re-LEH-F1b 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 tune protein stability and dynamic properties.