A dynamic model for the structure of acyl carrier protein in solution

Kim, Yangmee; Prestegard, James H.

doi:10.1021/bi00448a017

Cited by 137 publications

(117 citation statements)

References 20 publications

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“…(29 -31), and the results (Fig. 5A) agreed with the published proton assignment of E. coli ACP (32). We performed protein NMR spectroscopy experiments to monitor the chemical shift perturbations in the ACP structure when it is bound to the FabG to identify the ACP residues that undergo conformational/environment changes upon formation of the FabG⅐ACP complex.…”

Section: Materials-amershamsupporting

confidence: 71%

“…ACP functions to sequester the growing acyl chain attached to the prosthetic group from solvent as it shuttles the intermediates between enzymes. Our work, and the protein NMR analysis of other ACPs (32,(35)(36)(37), shows that the prosthetic group exists in two states, one is exposed to solvent and the other is bound to the protein. Our protein NMR experiments indicate the prosthetic group interacts with ACP along the interior face of helix ␣2 2 pointing the active site sulfhydryl toward residues in loop-2 between helices ␣2 and ␣3.…”

Section: Discussionmentioning

confidence: 99%

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Key Residues Responsible for Acyl Carrier Protein and β-Ketoacyl-Acyl Carrier Protein Reductase (FabG) Interaction

Zhang¹,

Zheng

et al. 2003

Journal of Biological Chemistry

133

148

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We tested this hypothesis by mutating two surface residues, Arg-129 and Arg-172, located in a hydrophobic patch adjacent to the active site entrance on ␤-ketoacyl-ACP reductase (FabG). Enzymatic analysis showed that the mutant enzymes were compromised in their ability to utilize ACP thioester substrates but were fully active in assays with a substrate analog. Direct binding assays and competitive inhibition experiments showed that the FabG mutant proteins had reduced affinities for ACP. Chemical shift perturbation protein NMR experiments showed that FabG-ACP interactions occurred along the length of ACP helix ␣2 and extended into the adjacent loop-2 region to involve Ile-54. These data confirm a role for the highly conserved electronegative/hydrophobic residues along ACP helix ␣2 in recognizing a constellation of Arg residues embedded in a hydrophobic patch on the surface of its partner enzymes, and reveal a role for the loop-2 region in the conformational change associated with ACP binding. The specific FabG-ACP interactions involve the most conserved ACP residues, which accounts for the ability of ACPs and the type II proteins from different species to function interchangeably.Fatty acid biosynthesis in bacteria and plants is carried out by a series of enzymes that are collectively known as the type II fatty-acid synthase and is most extensively studied in the Escherichia coli system (1, 2). ACP 1 is a small, acidic protein that functions as the central acyl group carrier in type II fatty-acid synthase systems. The ACPs are rod-shaped proteins with a preponderance of acidic residues organized into four ␣-helices (3-7). The acyl intermediates are attached to the terminal sulfhydryl of the 4Ј-phosphopantetheine prosthetic group (8), which is attached via a phosphodiester linkage to Ser-36 located at the beginning of the second helical segment. The primary gene product is an apoprotein that is converted to ACP by the transfer of the 4Ј-phosphopantetheine moiety of CoA to Ser-36 by [ACP]synthase (AcpS) (9, 10). ACP performs two functions. First, it sequesters the growing acyl chain from the aqueous environment, and second, upon binding to one of the type II proteins, it releases its grip on the fatty acid, which is inserted into the active site cavity of the enzyme. ACPs are widely distributed in nature and are easily recognized by their significant primary sequence identity, particularly at the prosthetic group attachment site and extending along helix ␣2 (11). These similarities are thought to underlie the observation that ACPs from virtually any source are substrates for AcpS and function with the fatty acid biosynthetic enzymes of E. coli (11). However, the individual enzymes of type II fatty acid synthesis do not share a primary structure motif that would define the presence of a common ACP-binding motif.The molecular details that govern the specific interactions between ACP and the type II enzymes are poorly known. The analysis of the binding of ACP to FabH points to ionic interactions playing a determinant ...

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Section: Materials-amershamsupporting

confidence: 71%

Section: Discussionmentioning

confidence: 99%

Key Residues Responsible for Acyl Carrier Protein and β-Ketoacyl-Acyl Carrier Protein Reductase (FabG) Interaction

Zhang¹,

Zheng

et al. 2003

Journal of Biological Chemistry

133

148

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“…To illustrate and test the ensemble reweighting formalisms described above, we consider a simple, analytically tractable problem. Consider a one-dimensional configuration coordinate x with a Gaussian reference distribution p 0 (x) = 2πs 2 −1/2 e −x 2 /2s 2 (31) and the mean Y = x as observable, with uncertainty σ, such that…”

Section: Resultsmentioning

confidence: 99%

“…15,[31][32][33][34] In the replica simulations, N copies (replicas) x α of a molecular system are simulated in parallel using the same energy function U (x α ), subject in addition to a biasing potential that attempts to match the observables obtained by averaging over the N copies to the experimental measurements.…”

Section: Bayesian Ensemble Refinement In Configuration Space: the mentioning

confidence: 99%

Bayesian ensemble refinement by replica simulations and reweighting

Hummer

Köfinger

2015

The Journal of Chemical Physics

221

394

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We describe different Bayesian ensemble refinement methods, examine their interrelation, and discuss their practical application. With ensemble refinement, the properties of dynamic and partially disordered (bio)molecular structures can be characterized by integrating a wide range of experimental data, including measurements of ensemble-averaged observables. We start from a Bayesian formulation in which the posterior is a functional that ranks different configuration space distributions. By maximizing this posterior, we derive an optimal Bayesian ensemble distribution. For discrete configurations, this optimal distribution is identical to that obtained by the maximum entropy "ensemble refinement of SAXS" (EROS) formulation.Bayesian replica ensemble refinement enhances the sampling of relevant configurations by imposing restraints on averages of observables in coupled replica molecular dynamics simulations. We show that the strength of the restraint should scale linearly with the number of replicas to ensure convergence to the optimal Bayesian result in the limit of infinitely many replicas. In the "Bayesian inference of ensembles" (BioEn) method, we combine the replica and EROS approaches to accelerate the convergence. An adaptive algorithm can be used to sample directly from the optimal ensemble, without replicas. We discuss the incorporation of singlemolecule measurements and dynamic observables such as relaxation parameters. The theoretical analysis of different Bayesian ensemble refinement approaches provides a basis for practical applications and a starting point for further investigations.

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“…ACP is small acidic protein, present in a dynamic equilibrium of two or more conformers (50). This equilibrium is governed in part by the concentration of divalent cations, which bind to acidic residues on ACP (37,38).…”

Section: Discussionmentioning

confidence: 99%

Steady-State Kinetics and Mechanism of LpxD, the N-Acyltransferase of Lipid A Biosynthesis

2008

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LpxD catalyzes the third step of lipid A biosynthesis, the R-3-hydroxymyristoyl-acyl carrier protein (R-3-OHC 14 -ACP)-dependent N-acylation of UDP-3-O-(R-3-hydroxymyristoyl)-α-D-glucosamine [UDP-3-O-(R-3-OHC 14 )-GlcN]. We have now over-expressed and purified E. coli LpxD to homogeneity. Steady state kinetics suggest a compulsory ordered mechanism in which R-3-OHC 14 -ACP binds prior to UDP-3-O-(R-3-OHC 14 )-GlcN. The product, UDP-2,3-diacylglucosamine, dissociates prior to ACP; the latter is a competitive inhibitor against R-3-OHC 14 -ACP and a noncompetitive inhibitor against UDP-3-O-(R-3-OHC 14 )-GlcN. UDP-2-N-(R-3-hydroxymyristoyl)-α-D-glucosamine, obtained by mild base hydrolysis of UDP-2,3-diacylglucosamine, is a noncompetitive inhibitor against both substrates. Synthetic R-3-hydroxylauroylmethylphosphopantetheine is an uncompetitive inhibitor against R-3-OHC 14 -ACP and a competitive inhibitor against UDP-3-O-(R-3-OHC 14 )-GlcN, but R-3-hydroxylauroyl-methylphosphopantetheine is also a very poor substrate. A compulsory ordered mechanism is consistent with the fact that R-3-OHC 14 -ACP has a high binding affinity for free LpxD, whereas UDP-3-O-(R-3-OHC 14 )-GlcN does not. Divalent cations inhibit R-3-OHC 14 -ACP-dependent acylation but not R-3-hydroxylauroylmethylphosphopantetheine-dependent acylation, indicating that the acidic recognition helix of R-3-OHC 14 -ACP contributes to binding. The F41A mutation increases the K M for UDP-3-O-(R-3-OHC 14 )-GlcN 30-fold, consistent with aromatic stacking of the corresponding F43 side chain against the uracil moiety of bound UDP-GlcNAc in the x-ray structure of Chlamydia trachomatis LpxD. Mutagenesis implicates E. coli H239 but excludes H276 as the catalytic base, and neither residue is likely to stabilize the oxyanion intermediate.Lipid A is the hydrophobic moiety of lipopolysaccharide (LPS) 1 , which constitutes the outer leaflet of the outer membrane of most Gram-negative bacteria (1-3). The lipid A moiety of LPS is usually required for bacterial growth (3,4) and is a potent activator of the mammalian innate immune system via the TLR4/MD-2 complex (5,6). Over-production of cytokines due to excessive stimulation of TLR4/MD-2 may occur during severe Gram-negative infections and may contribute to the life-threatening complications of septic shock (7,8).The Kdo 2 -lipid A substructure of Escherichia coli LPS is synthesized by a conserved system of nine constitutive enzymes (Fig. 1A) (3). LpxD catalyzes the third reaction in this scheme, the R-3-hydroxymyristoyl-acyl carrier protein (R-3-OHC 14 (Fig. 1A). Although essential for growth and an excellent target for the design of new antibiotics (9), LpxD is one of the least characterized enzymes in the pathway. Kelly and Raetz identified *Author to whom correspondence should be addressed: C. R. H. Raetz at (919) 684-3384; Fax (919) 684-8885; raetz@biochem.duke.edu. 1 The abbreviations are: ACP, acyl carrier protein; Bis-Tris, 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol; CMC, critical micelle concentration;...

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A dynamic model for the structure of acyl carrier protein in solution

Cited by 137 publications

References 20 publications

Key Residues Responsible for Acyl Carrier Protein and β-Ketoacyl-Acyl Carrier Protein Reductase (FabG) Interaction

Key Residues Responsible for Acyl Carrier Protein and β-Ketoacyl-Acyl Carrier Protein Reductase (FabG) Interaction

Bayesian ensemble refinement by replica simulations and reweighting

Steady-State Kinetics and Mechanism of LpxD, the N-Acyltransferase of Lipid A Biosynthesis

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