Abstract
Acyl carrier proteins play a central role in metabolism by transporting substrates in a wide variety of pathways including the biosynthesis of fatty acids and polyketides. However, despite their importance, there is a paucity of direct structural information concerning the interaction of ACPs with enzymes in these pathways. Here we report the structure of an acyl-ACP substrate bound to the Escherichia coli fatty acid biosynthesis enoyl reductase enzyme (FabI), based on a combination of x-ray crystallography and molecular dynamics simulation. The structural data are in agreement with kinetic studies on wild-type and mutant FabIs, and reveal that the complex is primarily stabilized by interactions between acidic residues in the ACP helix alpha2 and a patch of basic residues adjacent to the FabI substrate-binding loop. Unexpectedly, the acyl-pantetheine thioester carbonyl is not hydrogen-bonded to Tyr(156), a conserved component of the short chain alcohol dehydrogenase/reductase superfamily active site triad. FabI is a proven target for drug discovery and the present structure provides insight into the molecular determinants that regulate the interaction of ACPs with target proteins.
Highlights
Saccharides [4], lipopolysaccharides [5, 6], and phospholipids [7]
Acyl carrier proteins (ACPs) that function in FASII-mediated biosynthesis are small, highly soluble, acidic proteins that vary in molecular mass from 7.5 kDa (Escherichia coli) to 13 kDa (Mycobacterium tuberculosis) (1, 8 –11)
Whereas the structures of ACPs from a variety of different species have been determined by x-ray crystallography [12] and NMR spectroscopy, only one structure has been determined of ACP in complex with another protein, the holo-ACP synthase (AcpS) [15], and no structural information is available for the interaction between ACP and enzymes of the fatty acid biosynthesis pathway
Summary
Preparation of Substrates and Enzymes—Plasmids for wildtype FabI and the Y156F mutant were available from a previous study [23]. AcpS was removed by centrifugation at 6000 rpm for 15 min and the supernatant was applied to a 1-ml Q-Sepharose column, equilibrated with 20 mM BisTris, 1 mM dithiothreitol, pH 6.5 (Buffer B), containing 50% isopropyl alcohol. Equilibration dynamics was performed on the minimized structure, with a constant temperature of 300 K maintained by coupling to a thermostat using the Langevin algorithm with the collision frequency set to 1 psϪ1. R.m.s. deviation values were calculated using the initial model structure (prior to equilibration) as a reference. We recently showed that this combination of force field and solvent model was able to accurately reproduce experimental data for large conformational changes in human immunodeficiency virus type 1 protease that occur upon addition or removal of an inhibitor [56, 57]. After the substrate was drawn into the FabI active site, the system was simulated for an additional 2500 ps
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