Evolutionary paths that link orthogonal pairs of binding proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Evolutionary Biology

Background:

• Protein-binding pairs can evolve extreme specificity, insulating them from similar proteins.
• Evolution typically involves accumulating single-point mutations selected for sufficient binding affinity.
• Discovering functional evolutionary paths between distantly related protein pairs was previously limited.

Purpose of the Study:

• To develop a computational framework for identifying low-molecular-strain, single-mutation paths between protein pairs.
• To apply this framework to bacterial endonuclease-immunity pairs with significant sequence divergence.
• To understand the evolutionary mechanisms driving functional divergence and specificity changes in proteins.

Main Methods:

• Development of a computational framework to predict mutation paths with minimal molecular strain.
• Application of the framework to orthogonal bacterial endonuclease-immunity pairs.
• In vivo validation of the predicted evolutionary pathways.

Main Results:

• A strain-free 19-mutation path was discovered between two orthogonal protein pairs.
• This path included mutations bridging identities not exchangeable by single-nucleotide changes.
• The functional shift in binding preference occurred abruptly, driven by single radical mutations in each partner.
• Specificity-switching mutations enhanced fitness, indicating positive Darwinian selection.

Conclusions:

• The computational framework enables the discovery of functional evolutionary pathways between distantly related protein pairs.
• Functional divergence and abrupt changes in protein specificity can be driven by positive selection.
• Understanding these evolutionary mechanisms provides insights into protein adaptation and design.