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.