Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate^,

Abstract

Acyl carrier protein (ACP) is a cofactor in a variety of biosynthetic pathways, including fatty acid metabolism. Thus it is of interest to determine structures of physiologically relevant ACP-fatty acid complexes. We report here the NMR solution structures of spinach ACP with decanoate (10:0-ACP) and stearate (18:0-ACP) attached to the 4′ phosphopantetheine prosthetic group. The protein in the fatty acid complexes adopts a single conformer, unlike apo-and holo-ACP, which interconvert in solution between two major conformers. The protein component of both 10:0-and 18:0-ACP adopts the four-helix bundle topology characteristic of ACP, and a fatty acid binding cavity was identified in both structures. Portions of the protein close in space to the fatty acid and the 4′ phosphopantetheine were identified using filtered/edited NOESY experiments. A docking protocol was used to generate protein structures containing bound fatty acid for 10:0-and 18:0-ACP. In both cases, the predominant structure contained fatty acid bound down the center of the helical bundle, in agreement with the location of the fatty acid binding pockets. These structures demonstrate the conformational flexibility of spinach-ACP and suggest how the protein changes to accommodate its myriad binding partners.Acyl carrier proteins participate in a wide variety of biosynthetic processes, including the synthesis of fatty acids (1), toxins (2), oligosaccharides (3,4), polyketides (5,6), biotin (7), and depsipeptides (8). They ferry small molecules between enzymes involved in biosynthesis by covalent linkage as a thioester to the thiol group of 4′-phosphopantetheine ( Figure 1). This prosthetic group is attached by a post-translational modification to a serine (S38 in spinach ACP 1 ) in the middle a conserved Asp-Ser-Leu (DSL) sequence. Whereas the sequences of different acyl carrier proteins are divergent, they share a similar four-helix bundle topology and often can be interchanged with full activity in reactions in vitro. This prompts the question: are ACPs simply passive molecules that constantly present their cargo to enzymes, or do protein-protein and protein-ligand interactions affect the utility of ACP as an enzyme substrate?One well-studied biosynthetic process involving ACP is the desaturation of the fatty acid stearate by the enzyme stearoyl-ACP Δ 9 -desaturase (Δ9D). This enzyme, in a reaction dependent on O 2 and reducing-equivalent, acts on ACP with an attached stearate (18:0-ACP) † This work was supported by the National Institutes of Health Grants R01 GM-50853 to B.G.F. and R01 GM-58667 to J.L.M. NMR data were collected at the National Magnetic Resonance Facility at Madison (NMRFAM), which is supported by grants from the NIH Center for Research Resources Biomedical Research Technology Program (P41 RR-02301) and the National Institutes of General Medical Sciences (P41 GM-GM66326) with additional instrumentation purchased with funds from the University of Wisconsin, the NSF Biological Instrumentation Program, the NIH Shared Instrumentat...

Electrospray ionization mass spectra of acyl carrier protein are insensitive to its solution phase conformation

“…harveyi ACP° [23].°This°stabilization°occurs°as°the acyl chain is enclosed within the relatively hydrophobic interior of the three parallel ␣-helices of folded ACP, as indicated°by°both°NMR° [21]°and°X-ray°crystallography [20].°As°shown°in°Figure°5a,°the°CSD°of°myristoyl-ACP was°very°similar°to°that°of°apo-rACP°(Figure°2a)°at neutral pH (average net charge 7.6), although only the former would actually be in a folded helical conformation°in°solution°under°these°conditions° (Figure°1d).…”

“…The NMR solution structure of several ACPs reveal a highly conserved folded conformation consisting of three parallel ␣-helices; in acyl-ACP, these helices enclose a fatty acyl group that is covalently attached to the 4=-phosphopantetheine prosthetic group [20,21]. Based on circular dichroism (CD) and hydrodynamic approaches, ACP from some bacterial species such as Vibrio harveyi is largely unfolded in solution at neutral pH, and its folded conformation can be stabilized by either fatty acylation or divalent cation binding to the acidic Helix II [22][23][24].…”

Structure and function of eukaryotic fatty acid synthases

“…Several structures of type II ACPs with bound acyl chains of different length showed that the insoluble fatty acid was buried deep within a hydrophobic pocket of the ACP, which might represent a transport form for substrate shuttling between the individual enzymes (Roujeinikova et al 2007 ;Zornetzer et al 2006). Based on these observations, Leibundgut et al proposed a switchblade-like mechanism, in which the acyl chain remains buried within the fungal ACP during inter-domain-shuttling in a similar manner as observed in type II FAS, whereas it is flipped out into the hydrophobic reaction pockets for catalysis .…”

Intein-mediated Cyclization of Bacterial Acyl Carrier Protein Stabilizes Its Folded Conformation but Does Not Abolish Function

“…The coding regions of the C-and N-terminal splicing domains of the Ssp GyrB intein were amplified from pDSG 3 with primer pairs IC-for/IC-rev and IN-for/INHIS-rev, respectively; the products were fused by anneal-extension PCR and subsequently amplified using primers IC-for/INHIS-rev. The complete product was cloned into pT (a derivative of the pTWIN1 vector from New England Biolabs) 4 using SacI and PstI, creating pTI C (NS)I N H. The coding regions of the V. harveyi ACP L46W and F50A mutants were amplified from pGEX-L46W (15) and pGEX-F50A (20), respectively, using primers ACP-for/ACP-rev and cloned into pTI C (NS)I N H using NheI and SapI, resulting in pTCYC-L46W and pTCYC-F50A.…”

“…Over two dozen nuclear magnetic resonance (NMR) and x-ray crystal structures have revealed a conserved "ACP fold" consisting of a four-helix bundle (1). Fatty acids covalently attached to the phosphopantetheine prosthetic group at the N-terminal end of helix II are enclosed within the hydrophobic interior of this bundle, interacting predominantly with residues on helices II-IV (2)(3)(4)(5). Further computational (6,7), crystallographic (8,9), and mutagenic (10 -13) analyses have implicated the acidic central helix II as a "recognition helix" for interaction with most of the ACP enzyme partners.…”