Mechanism based protein crosslinking of domains from the 6-deoxyerythronolide B synthase.
The critical role of protein-protein interactions in the chemistry of polyketide synthases is well established. However, the transient and weak nature of these interactions, in particular those involving the acyl carrier protein (ACP), has hindered efforts to structurally characterize these interactions. We describe a chemo-enzymatic approach that crosslinks the active sites of ACP and their cognate ketosynthase (KS) domains, resulting in the formation of a stable covalent adduct. This process is driven by specific protein-protein interactions between KS and ACP domains. Suitable manipulation of the reaction conditions enabled complete crosslinking of a representative KS and ACP, allowing isolation of a stable, conformationally constrained adduct suitable for high-resolution structural analysis.
Kapur
Shiven
S
Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
Worthington
Andrew
A
Tang
Yinyan
Y
Cane
David E
DE
Burkart
Michael D
MD
Khosla
Chaitan
C
D000213:Acyl Carrier Protein / Q000737:chemistry*; D015153:Blotting, Western; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D015394:Molecular Structure; D048630:Polyketide Synthases / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary
D000213:Acyl Carrier Protein; D003432:Cross-Linking Reagents; D048630:Polyketide Synthases