P121: Structural stability of the tandem acyl carrier protein arrangement in the polyunsaturated fatty acid synthase

Acyl carrier protein (ACP) is a highly conserved protein domain that participates in the biosynthesis of fatty acids and polyketides. The polyunsaturated fatty acid (PUFA) synthase complex responsible for the production of ω-3 fatty acids in deep-sea bacteria usually contains multiple ACP domains in tandem. Although it has been established that multiple ACPs result in higher yields of fatty acid production, no satisfying explanation exists for why this unprecedented arrangement has been selected. In this work we hypothesized that multiple ACP domains are required to: (i) recognize and bind different enzyme domains or (ii) to provide structural protein stabilization. We expressed ACP domains in E. coli and purified them by chromatographic methods. Tandem and individual ACP were assayed for binding to the dehydratase domains, (DH) using Surface Plasmon Resonance and Isothermal Titration Calorimetry. However, no protein interactions were detected. Alternatively, we studied the structural stabilization of the multi-ACP arrangement using Circular Dichroism, Differential Scanning Calorimetry, and Size Exclusion Chromatography. Results show that ACP domains are very stable proteins that tend to form quaternary structures, thus suggesting stabilization through domain-domain interactions. Analysis of multiple ACP sequences reveals the presence of a cluster of proline residues in the region corresponding to the IIIα-helix bundle. We propose that the multi-ACP arrangement is in response to the destabilization of the helix of ACPs by the presence of the proline cluster.  The results from this work help to understand how tandem ACP domains have been selected to result in the functional optimization in ω-3 biosynthesis.