Biomolecular NMR Assignments

, Volume 4, Issue 1, pp 83–85

Backbone chemical shift assignments of the acyl-acyl carrier protein intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum

Authors

  • Santosh Kumar Upadhyay
    • National Institute of Immunology
  • Ashish Misra
    • Molecular Biophysics UnitIndian Institute of Science
  • Namita Surolia
    • Molecular Biology and Genetics UnitJawaharlal Nehru Center for Advanced Scientific Research
  • Avadhesha Surolia
    • National Institute of Immunology
    • Molecular Biophysics UnitIndian Institute of Science
    • National Institute of Immunology
Article

DOI: 10.1007/s12104-010-9212-2

Cite this article as:
Upadhyay, S.K., Misra, A., Surolia, N. et al. Biomol NMR Assign (2010) 4: 83. doi:10.1007/s12104-010-9212-2
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Abstract

We report the backbone chemical shift assignments of the acyl-acyl carrier protein (ACP) intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum. The acyl-ACP intermediates butyryl (C4), -octanoyl (C8), -decanoyl (C10), -dodecanoyl (C12) and -tetradecanoyl (C14)-ACPs display marked changes in backbone HN, Cα and Cβ chemical shifts as a result of acyl chain insertion into the hydrophobic core. Chemical shift changes cast light on the mechanism of expansion of the acyl carrier protein core.

Keywords

Backbone assignmentsAcyl-ACPsPlasmodium falciparumFatty acid biosynthesis

Introduction

Malaria is one of the major health concerns in tropical countries, liable for high rates of mortality. Fatty acid biosynthesis pathway of Plasmodium sp. has been considered as a drug target against this disease, owing to the differences in fatty acid biosynthesis pathway proteins between the host and the parasite (Lu et al. 2005; Lu and Tonge 2008). In Plasmodium, fatty acids are synthesized by the type II pathway, multiple enzymes catalyzing various functions, while in the host, fatty acid biosynthesis is brought about by one single multidomain, fatty acid synthase, performing multiple functions (type I pathway). The structural differences between the two fatty acid pathways offer unique opportunities for drug design, targeting the protozoal pathway exclusively. A common cofactor, indispensable to both the pathways is the acyl carrier protein, which serves to shuttle the lengthening acyl chains to the catalytic site of FAS enzymes.

Acyl carrier protein is a domain of fatty acid synthase (FAS) in type I pathway, while in type II pathway, it is an independent protein. Acyl carrier protein is expressed as an apo protein (inactive), and converted to holo-ACP (active) by the transfer of a 4′-PP moiety from coenzyme A to a conserved serine residue, with ACP synthase acting as a catalyst. The acyl chains gets covalently tethered to the terminal sulfhydryl group of 4-phosphopentetheine (4′-PP) moiety of ACP, which in turn transfers the acyl chain to the active site of FAS enzymes. The mechanism by which the ACP molecule carries the acyl chain and transfers to the active site of FAS enzymes remains unknown. Numerous structural studies have been carried out by different groups to understand the mechanism of formation of acyl-ACP intermediates (Wu et al. 2009; Roujeinikova et al. 2002, 2007; Zornetzer et al. 2006; Sharma et al. 2006). In most of the acyl-ACP intermediates (with the exception of rat), the hydrophobic core increases in size to accommodate the growing acyl chain. The molecular basis of ACP expansion upon acyl chain insertion has recently been disclosed by our NMR studies on P. falciparum acyl-ACP intermediates (Upadhyay et al. 2009), where the electrostatic repulsion in conjunction with thrust due to the growing acyl chain favors expansion of the hydrophobic core of the ACP molecule, resulting in large chemical shift changes. In this paper, we report the backbone chemical shift assignments of the acyl-ACPs of P. falciparum viz. butyryl, octanoyl, decanoyl, dodecanoyl, and tetradecanoyl-ACP.

Materials and methods

Cloning, overexpression and purification of 15N-labeled PfACP

Proteins were expressed by cloning, overexpression and purification of P. falciparum holo and acyl-ACPs in E. coli as described (Upadhyay et al. 2009).

NMR experiments

NMR samples comprised of uniformly labeled [1H, 15N, and 13C] protein, 50 mM sodium phosphate buffer, pH 6.5, 100 mM NaCl, 2 mM DTT, 0.5% Sodium azide (NaN3), 90% H2O in 10% D2O. Concentration of protein was kept at 1 mM during all the NMR experiments. Two and three dimensional NMR experiments, viz. 1H15N HSQC, 1H15N TOCSY, HNCACB, CBCAcoNH, CCcoNH, HNcoCA, 13C15N-filtered experiments were acquired on a Varian Inova 500, installed at the National Institute of Immunology, New Delhi, India, equipped with a triple resonance, Z pulsed field gradient probe. Experiments were performed at 300 K. NMR data was processed on a workstation running Red Hat Enterprize Linux 5.0, using NMRPipe/NMRDraw (Delaglio et al. 1995) and analyzed using Sparky (Goddard and Kneller 2001). The data was multiplied by a phase shifted sinebell apodization function in all dimensions.

1H15N HSQC spectra were acquired using 1,024 data points in t2 dimension and 512 data points in t1 dimension. 1H15N HSQC-TOCSY experiments were collected with 1,024 (t3) × 72 (t1) × 48 (t2) data points and a mixing time of 150 ms. CBCAcoNH, HNCACB, CCcoNH experiments were collected with 1,024 (t3) × 36 (t1) × 24 (t2). Data was linear predicted in the forward direction for up to half the number of experimental points in the indirect dimensions. The temperature was maintained at 300 K during all experiments. 1 mM Sodium 4,4-dimethyl-4-silapentanesulfonate (DSS) was used as a chemical shift standard.

Assignments and data deposition

Figure 1a shows the 1H15N HSQC spectra of the holo-ACP (colored red) and tetradecanoyl-ACP molecule (colored blue). Figure 1b, c display regions of the spectrum in which all the acyl-ACPs we studied have been overlayed, viz. butyryl-, octanoyl-, decanoyl-, dodecanoyl-, tetradecanoyl-ACPs. As shown in the spectra, changes in backbone chemical shifts are remarkable as the acyl chain elongates. The assignments of HN, Cα and Cβ carbons of acyl intermediates were done using triple resonance experiments HNCACB, CBCAcoNH, 1H15N HSQC-TOCSY, 1H15N HSQC NOESY and HNcoCA. Chemical shift assignments for the backbone and side-chain resonances for the acyl-intermediates (butyryl- 91% HN, 91% Cα, 83% Cβ; octanoyl- 91% HN, 91% Cα, 88% Cβ; decanoyl- 91% HN, 88% Cα, 79% Cβ; dodecanoyl- 91% HN, 90% Cα, 87% Cβ; tetradecanoyl- 91% HN, 90% Cα, 78% Cβ) PfACP have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu/) under the accession numbers 16506, 16529, 16530, 16531, 16532, respectively.
https://static-content.springer.com/image/art%3A10.1007%2Fs12104-010-9212-2/MediaObjects/12104_2010_9212_Fig1_HTML.gif
Fig. 1

Overlayed 1H15N HSQC spectra of holo-ACP and the acyl-ACP intermediates of P. falciparum. a An overlayed spectra of holo-ACP (red) with tetradecanoyl-ACP (blue). Residues have been labeled for peaks corresponding to tetradecanoyl-ACP (colored blue). Side-chain amide resonances of asparagines and glutamine are shown by lines connecting the two side chain amines and indicated by residue name and number. b, c Regions of the spectra displaying changes in chemical shift for holo-ACP (red), butyryl- (green), octanoyl- (cyan), decanoyl- (yellow), dodecanoyl- (purple) and tetradecanoyl-ACP (blue). These regions correspond to dotted squares in Fig. 1a. The data was processed using NMRPipe (Delaglio et al. 1995) and analyzed using Sparky (Goddard and Kneller 2001)

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© Springer Science+Business Media B.V. 2010