Ancestral sequence reconstruction as a tool for structural analysis of modular polyketide synthases

Area of Science:

• Structural Biology
• Biochemistry
• Bioinformatics

Background:

• Modular polyketide synthases (PKSs) are crucial for producing polyketide antibiotics but are challenging to study structurally.
• Limited structural data hinders a complete mechanistic understanding of these complex enzymes.

Purpose of the Study:

• To explore the utility of ancestral sequence reconstruction (ASR) for overcoming structural analysis challenges in modular PKSs.
• To investigate the potential of ASR in enabling cryo-electron microscopy (cryo-EM) single-particle analysis for difficult protein targets.

Main Methods:

• Focused on the FD-891 PKS loading module (ketosynthase-like decarboxylase (KS_Q), acyltransferase (AT), and acyl carrier protein (ACP) domains).
• Constructed a chimeric didomain (KS_QAncAT) by replacing the native AT with an ancestrally reconstructed AT (AncAT) via ASR.
• Determined high-resolution crystal structure of the chimeric didomain and cryo-EM structures of the KS_Q-ACP complex.

Main Results:

• The KS_QAncAT chimeric didomain exhibited similar enzymatic function to the native KS_QAT didomain.
• Successfully obtained high-resolution crystal and cryo-EM structures of the chimeric didomain and KS_Q-ACP complex.
• Achieved cryo-EM structures that were not possible for the native protein, demonstrating ASR's utility for cryo-EM.

Conclusions:

• Integrating ASR with structural analysis provides deeper mechanistic insights into modular PKSs.
• ASR is a valuable tool for enabling structural studies, particularly cryo-EM, of challenging multi-domain proteins.
• Applying ASR to partial regions of proteins offers a flexible framework for investigating diverse multi-domain protein structures and functions.