Structural studies of fatty acyl-(acyl carrier protein) thioesters reveal a hydrophobic binding cavity that can expand to fit longer substrates

doi:10.1016/j.jmb.2006.09.049 J. Mol. Biol. (2007) 365, 135–145

Structural Studies of Fatty Acyl-(Acyl Carrier Protein)Thioesters Reveal a Hydrophobic Binding Cavity thatCan Expand to Fit Longer Substrates

Anna Roujeinikova1, William J. Simon2, John Gilroy2, David W. Rice1

John B. Rafferty1 and Antoni R. Slabas2⁎

Present address: A. Roujeinikova,Sciences, The University of Manches88, Manchester, M60 1QD, UK.Abbreviations used: ACP, acyl carr

acid synthetases; rmsd, root mean sE-mail address of the correspondi

[email protected]

0022-2836/$ - see front matter © 2006 E

A knowledge of the structures of acyl chain loaded species of the acyl carrierprotein (ACP) as used in fatty acid biosynthesis and a range of othermetabolic events, is essential for a full understanding of the molecularrecognition at the heart of these processes. To date the only crystal structureof an acylated species of ACP is that of a butyryl derivative of Escherichia coliACP. We have now determined the structures of a family of acylated E. coliACPs of varying acyl chain length. The acyl moiety is attached via athioester bond to a phosphopantetheine linker that is in turn bound to aserine residue in ACP. The growing acyl chain can be accommodated withina central cavity in the ACP for transport during the elongation stages oflipid synthesis through changes in the conformation of a four α-helixbundle. The results not only clarify the means by which a substrate ofvarying size and complexity is transported in the cell but also suggest amechanism by which interacting enzymes can recognize the loaded ACPthrough recognition of surface features including the conformation of thephosphopantetheine linker.

© 2006 Elsevier Ltd. All rights reserved.

Keywords: acyl carrier protein; fatty acid biosynthesis; acyl chain binding;hydrophobic binding pocket; conformational changes

Introduction

Acyl carrier protein (ACP) is an essential cofactorin the biosynthesis of fatty acids.1–3 In bacteria andplants, ACP is a small (Mr<10 kDa) abundantmonomeric protein that is post-translationally mod-ified by the transfer of 4′-phosphosphopanthetheinefrom CoA to a conserved serine residue on ACP byholo-ACP synthase (ACPS). The acyl intermediatesof fatty acid biosynthesis are covalently attached tothe phosphopantetheine moiety of ACP as thio-esters, and shuttled from one enzyme to another in asequential manner in the cycles of fatty acid chainelongation. Recently, Escherichia coli ACP has beenidentified as a cellular target for the antibacterial

Faculty of Lifeter, The Mill, PO Box

ier protein; FAS, fattyquare deviation.ng author:

lsevier Ltd. All rights reserve

action of the pantothenamides.4 The mechanism ofinhibition of fatty acid biosynthesis by the pantothe-namides has been shown to involve formation of aninactive analogue of ACP that lacks the sulphydrylgroup for acyl chain attachment. In addition to fattyacid synthetases (FAS), ACPs are now known also tobe associated with a myriad of pathways thatinvolve acyl transfer reactions among which aresynthesis of polyketide antibiotics,5 lipid A,6 biotinprecursor,7 rhizobial lipochitooligosaccharide nodu-lation factors,8 membrane-derived oligosaccha-rides,9 acyl homoserine lactones,10 the aldehydesubstrate of luciferase,11 acyl transfer to glycerol-3-phosphate and monoacylglycerol-3-phosphate,12

activation of toxins,13 and lipoylation of pyruvateand α-ketoglutarate dehydrogenase complexes.14

Previous X-ray crystallographic and NMR studieson bacterial and plant FAS ACPs15–24 had shownthat they share a similar overall fold involving foursequentially connected α helices arranged in anapproximately 4-fold symmetrical bundle. Theapolar residues of the helices form a hydrophobiccleft or cavity which, according to previous stu-dies on acyl-ACPs,16,24–28 may accommodate the

d.

136 Crystallographic Analysis of Acyl-ACPs

thioester-bound fatty acyl chain. Prior to this study,the only reported crystal structure of an acylatedACP was that of butyryl-ACP.22,29 That studypresented the structure of acylated ACP in twodifferent crystal forms (A and B) and revealed aflexible hydrophobic cavity in the protein that couldform a putative acyl chain binding site.22,29 Incrystal form A (hereafter referred to as unligandedform), the hydrophobic cavity was contractedwhereas in form B, the cavity was found to be inan expanded state and was apparently occupied bythe butyryl and β-mercaptoethylamine moieties ofthe acylated 4′-phosphopantetheine group attachedto ACP. Unfortunately, the remaining part of the 4′-phosphopantetheine group was not resolved in theelectron density maps due to high B-factors and thusprecluded unambiguous determination of the pre-cise nature of the lipophilic ligand bound in thehydrophobic cavity of ACP. This work emphasizedthe necessity for further biochemical and crystal-lographic studies of different acylated species ofACP.Here, we present an analysis of the high-resolu-

tion X-ray structures of hexanoyl(C6)-, heptanoyl(C7)- and decanoyl(C10)-ACP (Figure 1(a)) fromE. coli, which provide an insight into the mannerof binding of a growing fatty acyl chain duringthe fatty acid synthesis cycle. In all our structures,the fatty acyl chain is found embedded in thetunnel-like hydrophobic cavity, which exhibitshigh plasticity and expands with an increase inthe length of the acyl chain. Two different bindingmodes for the acyl chain tethered to the phos-phopantetheine group are identified in thesestructures, which result in greater or lesser burialof the phosphopantetheine arm and consequentchanges in the surface of ACP. The possibleimplications for conformational diversity of differ-ent acyl-ACP intermediates in the fatty acid syn-thesis are discussed.

Results

Overall structure

The crystal structures of hexanoyl(C6)-, heptanoyl(C7)- and decanoyl(C10)-ACP have been deter-mined using a molecular replacement approachwith the coordinates of the high-resolution (1.2 Å)structure of butyryl-ACP I62M as a search model.The asymmetric unit of each crystal contains twoindependent monomers. The packing arrangement

Figure 1. (a) Structural formula for the acylated 4′-phosphand decanoyl- (n=8) ACP. (b) The overall fold of ACP using heand helical turns are shown as coils and the polypeptide chainN to C terminus. The phosphopantetheine group attached tocarbon, oxygen, nitrogen, sulphur and phosphorus atoms colouStereoviews of the electron density for the acylated 4′-phosphohexanoyl-ACP subunit A, (d) decanoyl-ACP subunit B, (e) heThemaps were calculated using sigmaA-weighted coefficientsprepared using PyMOL).41

of these two molecules is different from the crystalpacking observed previously in either form A orform B crystals of butyryl-ACP.22 The structures ofeach acyl chain loaded protein species share a com-mon protein fold, which is composed of four helices,α1 (residues 3–15), α2 (36–50), α3 (56–61) and α4(65–75) linked by loops L1-2 (residues 16–35), L2-3(51–55) and L3-4 (62–64) and is very similar to thatof butyryl-ACP (see Figure 1(b)).22

Superposition of the structures of the two mono-mers in the asymmetric unit of the hexanoyl-ACPcrystals showed an overall rmsd of 0.34 Å for 77 Cα

atoms, indicating that the protein moieties of thetwo molecules have a very similar structure. Ins-pection of the electron density maps for thesecrystals revealed clear continuous electron densityfor the full length of the prosthetic group linked tothe hexanoyl chain (Figure 1(c)) in both subunits, theshape of which suggested a single dominantconformation for this moiety. The fact that theaverage refined B-factor for the atoms of the acy-lated prosthetic group (31 Å2) is only slightly higherthan that for the protein atoms (24 Å2) is alsoconsistent with a single prevalent conformation.Superposition of the structures of the two mono-

mers in the asymmetric unit of the decanoyl-ACPcrystals showed an overall rmsd of 0.54 Å for Cα

atoms. In the crystal structure of decanoyl-ACP, theelectron density for the decanoyl, β-mercaptoethy-lamine, and phosphate moieties of the acylated 4′-phosphopantetheine group attached to ACP isobserved in one of the two monomers in the asy-mmetric unit (Figure 1(d)). These moieties werefitted into the corresponding density using sigmaAweighted (2mFo–DFc) and (mFo–DFc) maps,whereas the rest of the 4′-phosphopantetheinegroup was modelled in a stereochemically reason-able manner and omitted from refinement. Theshape of the electron density for the acyl chainsuggested that in addition to the major modelledconformation for the decanoyl chain, there aremultiple lower-occupancy conformations present.However, due to resolution limitations and theconformational disorder of part of the phosphopan-tetheinemoiety, these low-occupancy conformationscould not be included in the refinement. Thissomewhat compromises the completeness of thefinal model, which is reflected in higher but stillacceptable values for the crystallographic R-factorsfor the decanoyl-ACP model (Table 1).The structures of the two monomers in the

asymmetric unit of the crystal of heptanoyl-ACP areessentially identical and the corresponding Cα atoms

opantetheine group in hexanoyl- (n=4), heptanoyl- (n=5)ptanoyl-ACP as an example. The α-helices (labelled α1–α4)is coloured from blue through to red for the transition fromSer36 and the heptanoyl chain are shown as sticks withred grey, red, blue, yellow and orange, respectively. (c)–(f)pantetheine group bound in the hydrophobic pocket of (c)ptanoyl-ACP subunit A and (f) heptanoyl-ACP subunit B.(2mFo–DFc)

40 and contoured at 1.0 σ level. (The Figure was

137Crystallographic Analysis of Acyl-ACPs

can be superimposed with an overall rmsd of 0.26 Å.However, in contrast to the crystal of the hexanoyl-ACP, the electron density maps suggested that thebinding mode of the prosthetic group with the

Figure 1 (legend o

heptanoyl moiety attached to it is different in thetwomolecules in the asymmetric unit (Figure 1(e) and(f)) and that there is more than one conformation forthe heptanoyl moiety present. In the interpretation of

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Table 1. Crystallographic data

Hexanoyl-ACP Heptanoyl-ACP Decanoyl-ACP

Resolution range (Å) 30.00–1.76 (1.82–1.76) 15–1.6 (1.66–1.60) 15–1.55 (1.61–1.55)Completeness (%) 92 (95) 80 (74) 86 (67)Observed reflections 57,630 44,661 57,224Unique reflections 13,699 16,351 18,488Percentage of data with I/σ(I) >3 81 (49) 78(29) 83(40)Rmerge

a (%) 0.091 (0.202) 0.056 (0.400) 0.065 (0.311)

Refinement statisticsResolution range (Å) 10.00–1.76 10–1.6 10.00–1.55Completeness (%) 92 79 84R-factorb 0.184 0.209 0.214Free R-factorc 0.241 0.256 0.269Bond-length deviation from ideality (Å) 0.01 0.01 0.01Bond-angle deviation from ideality (°) 1.8 2.0 2.3SigmaA coordinate error estimate (Å)40 0.19 0.22 0.15No. of protein atoms 1194 1194 1194No. of water molecules 224 191 237No. of metal ions 11 9 8Ave. B (protein atoms) (Å2) 24 20 16.5Ave. B (water molecules) (Å2) 35 29 25Ave. B (acylated prosthetic group) (Å2)d 31 35 39

Numbers in parentheses indicate values for the highest resolution shell.a Rmerge=∑hkl(Ii–Im)/ΣhklIm, where Im is the mean intensity of the reflection.b R=∑|(|Fobs|–|Fcalc|)|/∑(|Fobs|).c The free R-factor was calculated on 5% of the data omitted at random.d Higher values of the B-factor of the prosthetic group reflect partial occupancy (see the text).

138 Crystallographic Analysis of Acyl-ACPs

the electron density maps, the strongest local peak inthe difference mapswas taken to indicate the positionof the sulphur atom of the β-mercaptoethylaminemoiety. The quality of the electron density maps didnot permit multi-conformer refinement, and thus themodel for the acylated phosphopantetheine moietyhas been built and refined in one prevalent conforma-tion in each of the two molecules.

Two different binding modes of acylatedphosphopantetheine group

In the crystal structures of hexanoyl-, heptanoyl-and decanoyl-ACPs, the aliphatic chain of thesubstrate and the β-mercaptoethylamine moiety ofthe phosphopantetheine group are sequestered intothe hydrophobic tunnel-like cavity in the core of thefour-helix bundle (Figure 2(a)–(e)) with the thioester

Figure 2. (a)–(e) Cross-sections across the molecules of (a)(e) decanoyl-ACP showing the hydrophobic cavities of ACP inorientation is the same for all structures. The acyl chain, the pshown in CPK representation and coloured according to atomin red and sulphur in orange. The protein moiety is shown asacylated phosphopantetheine group in the crystal structurepeptides of the protein residues that form hydrogen bondsuperimposed structures of hexanoyl-ACP and subunit A of hand those of butyryl-ACP, subunit B of heptanoyl-ACP and dethree structures, a thin line is used to show amodelled positionof the 4′-phosphopantetheine group for which no interpretablthioester bonds are depicted as yellow spheres. (g) Changes inACP induced by the binding of fatty acyl chains (for calculatiohave been excluded from the model). For hexanoyl- and heptaasymmetric unit is shown. For decanoyl-ACP, the volume habound in its hydrophobic pocket.

bond being shielded from the solvent. In all acylderivatives, the acyl chain adopts a linear orienta-tion in the tunnel with the terminal methyl grouppointing towards the end of the cavity situatedbetween the C-terminal ends of helices α2 and α4.Comparative analysis of the crystal structures of allacyl-ACPs from E. coli including the previouslyreported structure of butyryl(C4)-ACP22 reveals thattwo distinctly different binding modes for theacylated phosphopantetheine group are present. Inthe crystal structures of the butyryl-ACP, subunit Bof heptanoyl-ACP and subunit A of decanoyl-ACPthe acylated prosthetic group is stabilised mainlythrough hydrophobic interactions between thealiphatic chain of the substrate and apolar side-chains of the residues lining the hydrophobic cavity(Phe28, Val29, Leu42, Val43, Leu46, Ile54, Ala59,Ile62/Met62 and Ala68). Additional contributions

unliganded, (b) butyryl-, (c) hexanoyl-, (d) heptanoyl- andthe unliganded state and with the acyl chains bound. Thehosphopantetheine group and the side-chain of Ser36 aretype, with carbon atoms in green, nitrogen in blue, oxygenribbon structure. (f) Two observed binding modes for thes of acyl-ACPs. Only the side-chains or the main-chains with the prosthetic group are shown for clarity. Theeptanoyl-ACP are shown in cyan and green, respectively,canoyl-ACP coloured magenta, red and pink. In the latterof the solvent-exposed β-alanine and pantoic acidmoietiese electron density was observed. The sulphur atoms in thethe probe-accessible volume of the hydrophobic cavity inns, atoms of the acyl chain and phosphopantetheine groupnoyl-ACP, the average value for the two monomers in thes been calculated for the monomer that has an acyl chain

Figu

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139Crystallographic Analysis of Acyl-ACPs

140 Crystallographic Analysis of Acyl-ACPs

are made by a hydrogen bond from the amidenitrogen atom of the β-mercaptoethylamine moietyto the main-chain carbonyl group of Glu60 and byvan der Waals contacts formed by the latter moietywith residues Thr39, Ala59, Ile62/Met62, and Thr63(Figure 2(f)). In this binding mode, the phospho-pantetheine moiety is tethered to the protein at twoends i.e. by the covalent link to Ser36 and throughthe acyl chain that is attached to its other end, and isembedded in the protein hydrophobic core. Thepantoic acid and β-alanine moieties of the prostheticgroup are exposed to the solvent and display asignificant degree of thermal or static disorder as noelectron density has been observed for this part ofthe molecule. The contacts observed in the butyrylderivative between the carbon at position 4 in theacyl chain andMet62 in the I62M variant used in ourearlier structure determination22 suggest that burialof longer acyl chain derivatives in the manner ob-served in our structures of hexanoyl- and heptanoyl-derivatives of wild-type ACP (see below) would beunfavourable in the I62M variant. In our originalstructure determination, althoughwe had data on thebutyryl derivative of wild-type ACP, we focusedupon the I62M variant because it showed betteroccupancy for the acyl chain, presumably arisingfrom favourable interactions with Met62.22

A different binding mode for the acylated 4′-phosphopantetheine group has been found in thetwo monomers in the asymmetric unit of thehexanoyl-ACP crystals and in subunit A of thecrystal of heptanoyl-ACP (Figure 2(f)). In thesestructures, the thioester-linked acyl chain is insertedapproximately 5 Å deeper into the hydrophobiccavity with the thioester bond being sequestered inthe very core of the protein. The protein moietyforms multiple contacts with the phosphopan-tetheine arm along its full length. These contactsinclude a number of hydrogen bonds with residueslocated on the two opposite sides of the bindingpocket and help to anchor the arm to the proteinsurface in a clamping arrangement. The side-chainhydroxyl of Thr39 of helix α2 accepts a hydrogenbond from the hydroxyl group of the pantoic acidmoiety and donates a hydrogen bond to thecarbonyl group of the β-alanine moiety, whereasthe amide nitrogen atom of the β-mercaptoethyla-mine moiety forms a bifurcated hydrogen bondwiththe main-chain carbonyl oxygen atoms of Ala59 andIle62 (Figure 2(f)).Superposition of the protein moieties of the

representatives for the two different bindingmodes reveals that the side-chain of Thr39 appearsto play a key role in stabilization of the polarmoieties of the phosphopantetheine group in thestructures where the thioester bond between thelatter and the acyl chain is sequestered deep in theprotein core. In the remaining set of structureswhere a large part of the phosphopantetheine groupis disordered, the Thr39 side-chain has a differentorientation, forming an intra-helix hydrogen bondwith the main-chain carbonyl group of Ser36. Thereare no other significant structural differences

between the protein moieties with the two differentconfigurations of the prosthetic group. Analysis ofthe complete set of the acyl-ACP structures revealsno obvious correlation between the binding mode ofthe prosthetic group and the length of the acyl chain.This suggests that the two observed configurationsof the prosthetic group are likely to represent twodifferent conformational states of E. coli acyl-ACPthat exist in solution.

Hydrophobic cavity expansion

The hydrophobic tunnel-like cavity accommoda-ting the acyl chain in hexanoyl-, heptanoyl- anddecanoyl-ACP, has a wide opening near the Nterminus of helix α2, runs through the core of thefour-helix bundle almost parallel to helix α2 andnarrows down staying short of opening into asurface pocket located on the other side of theprotein between the C-terminal ends of helices 2 and4 (Figure 2(a)–(e)). The protein scaffold around thecavity has a fissure that starts at the mouth of thecavity and runs between helices α2 and α3. This sidegap is filled with ordered water molecules. Thehydrophobic surface of the cavity is formed by theburied aliphatic side-chains of the residues from thefour amphipathic α-helices and the loops connectingthem: Val7, Ile11 (of helix α1), Phe28, Val29 (of loopL1-2), Leu42, Val43, Leu46, Phe50 (of helix α2), Ile54(of loop L2-3), Ala59 (of helixα3), Ile62 (of loop L3-4),Ala68, Tyr71 and Ile72 (of helix α4). The entranceto the cavity is lined by the polar residues: Thr39(of helix α2), Glu60 (of helix α3), and Thr63 (ofloop L3-4).The hydrophobic cavities of unliganded protein

(PDB code 1LOH),22 butyryl-, hexanoyl-, heptanoyl-and decanoyl-ACP are illustrated and compared inFigure 2. The solvent-accessible volume of theinternal cavity of ACP (calculated using a proberadius of 1.4 Å30) increases from a value close to zerofor the unliganded protein to 164 Å3 for decanoyl-ACP. As illustrated in Figure 2(g), the cavity volumefollows the order of the length of the acyl chain,demonstrating a high structural plasticity of theacyl-chain binding site.Pairwise superpositions of the structure of the

protein moiety of decanoyl-ACP (subunit A) withthose of unliganded protein, i.e. where the attachedacyl chain is not observed in the hydrophobic cavityin the crystal structure, butyryl-, hexanoyl- (subunitA) and heptanoyl-ACP (subunit A) reveal thevariation in structure and give overall rmsd valuesfor Cα atoms of 0.94, 0.53, 0.20 and 0.20 Å,respectively. In the residue-by-residue rmsd plot(Figure 3(a)), the highest rmsd values, i.e. thoseshowing the greatest variation in structure, arelocated in the region comprising the short helix α3the loops connecting α3 to helices α2 and α4 and inthe C-terminal region. When the segments 51–64and 73–76 are excluded from comparison, theoverall rmsd values drop to 0.55, 0.39, 0.19 and0.18 Å, respectively. An immediate observation thatcan be made from the analysis of the rmsd plot and

Figure 3. (a) rmsd of Cα atomsfor pairwise superpositions of theprotein moiety of hexanoyl-ACP(subunit A) with those of unligan-ded protein (red), butyryl- (dark-purple), hexanoyl- (subunit A, dark-blue) and heptanoyl-ACP (subunitA, blue) as a function of residuesnumber (colour coding is consistentwith (c)). (b) Stereo ribbon diagramof the superimposed structures ofunliganded ACP (red) and decan-oyl-ACP (green) showing angulardisplacements of the four helicesinduced by the binding of theacylated prosthetic group. The lat-ter is drawn in ball-and stick repre-sentation and the axes of the helicesare shown as thin rods. (c) Stereo-diagram highlighting differencesbetween the superimposed crystalstructures of unliganded (red),butyryl- (dark-purple), hexanoyl-(dark-blue), heptanoyl- (blue) anddecanoyl-ACP (green) in the regionthat forms the hydrophobic pocket.To mark the position of the acylchain-binding site, the decanoylmoiety and the phosphopante-theine group are shown for de-canoyl-ACP in a ball-and-stickrepresentation. The protein moi-eties in butyryl-, hexanoyl- andheptanoyl-ACP adopt conforma-tions that are intermediate betweenthe structure of the unligandedprotein and that of decanoyl-ACP.Binding of the acyl chains of anincreasing length results in gradualoverall swelling of the global pro-tein fold.

141Crystallographic Analysis of Acyl-ACPs

the overall superposition of the whole set ofstructures (Figure 3) is that the structures of differentacyl-ACPs form a cluster of structures that are muchmore similar to each other than to the structure ofthe unliganded protein. As illustrated in Figure 3(b)and (c), variation of the volume of the hydrophobiccavity in ACP is achieved without major changes inthe protein fold. Upon insertion of the acyl chaininto the hydrophobic core of the protein, severalhydrophobic residues that line the internal cavityincluding Phe28, Leu42, Val43, Leu46, Ile54, Ala59,Ile62, Tyr71 and Ile72 are shifted. However, only the

side-chains of Phe28, Ile54 and Ile72 undergo aconformational re-organisation to avoid unfavour-able interactions with the fatty acyl chain. Re-orientation of these residues upon introduction ofthe acyl chain is accompanied by small angulardisplacements of the helices, which move as rigidfragments. The superposition of the two “ultimate”structures in the set, those of the unliganded protein(solvent-accessible cavity volume ∼0 Å3) anddecanoyl-ACP (cavity volume 164 Å3) (Figure 3(b))shows that in order to accommodate the substrate,helices α1 and α3 and the N-terminal end of helix α2

142 Crystallographic Analysis of Acyl-ACPs

move outward, with the maximum shift of 3.0 Åbeing observed for the Cα atom of Glu57 (helix α3).The protein moieties in butyryl-, hexanoyl- andheptanoyl-ACP adopt conformations that are inter-mediate between the structure of the unligandedprotein and that of decanoyl-ACP and, as illustratedin Figure 3(c), binding of the acyl chains of anincreasing length results in gradual overall “swelling”of the global protein fold.

Discussion

The function of ACP in fatty acid biosynthesis is toshuttle the substrates attached to its phosphopan-tetheine arm as thioesters between the FAS enzymes.Analysis of the crystal structures of hexanoyl-,heptanoyl- and decanoyl-ACP reveals the detailsof how the protein stabilizes the growing fatty acylchain by sequestering the aliphatic chain of thesubstrate and the β-mercaptoethylamine moiety ofthe phosphopantetheine group into the hydropho-bic cavity in the core of the four-helix bundle. Ourfindings are supported by previous NMR27,28 anddifference spectroscopy31 studies that provided theevidence of interactions between the covalentlyattached fatty acyl chain and protein residuesPhe28, Phe50, Ile54, Ala59 and Tyr71. In allstructures analysed, the thioester bond is shieldedfrom the solvent, in line with previous observationsof stability of ACP thioesters to base hydrolysis.25

Conserved residues Phe28, Ile54 and Tyr71 impli-cated in interactions with the acyl chain also play animportant role in maintaining the protein fold. Theside-chain of Phe28 occupies a key position in thehydrophobic core, anchoring the middle of the loopL12 to the protein. The side-chains of Ile54 and Tyr71stack against each other, providing an importantcontact between the loop L23 and the C terminus ofhelix α4. Previous biochemical and mutagenesisstudies have shown that Ile54 is indispensable forACP activity and stability. Ile54 has been implicatedin interactions of ACP with β-ketoacyl-(ACP)reductase,32 acyl carrier protein synthase20 andprotein acyltransferase HlyC.33 The I54A variant ofthe E. coli ACP homologue from Vibrio harveyi doesnot fold34 and an E. coliACP I54C variant is partiallydefective in the reconstitution of type II FAS in crudeextracts.33 The remaining apolar residues constitu-ting the acyl chain binding site are conservativelysubstituted throughout the sequences of ACPs fromdifferent bacterial sources,23 which highlights theimportance of non-specific hydrophobic interactionsin the binding of the aliphatic chains of the fatty acidbiosynthesis intermediates by ACP.Analysis of the crystal structures of acyl-ACPs

reveals that binding of the fatty acyl chain inducesexpansion of the hydrophobic tunnel-like cavity,and the cavity volume gradually increases as thelength of the bound acyl chain increases from four toten carbon atoms. Ligand-length-dependent varia-tion of the cavity volume implies that structuralplasticity of the hydrophobic core is essential for

ACP to carry out its physiological role. Our analysishas shown that expansion of the hydrophobic cavityupon binding of the acyl chain of up to ten carbonatoms in length is achieved without major changesin the overall protein fold. The size of the cavity indecanoyl-ACP is in good agreement with thatreported for the solution structure of spinach C10-ACP24. The studies on the spinach ACP alsodemonstrated that its fold only undergoes subtlefurther changes on binding of a longer (C18) acylchain. Although there is little change in the fold ofthe spinach ACP, there is a large change in the size ofthe cavity (from 128 Å3 to 305 Å3) and a remarkableincrease in loop dynamics upon going from a C10 toa C18 acylated form. Given the small size of theE. coli ACP (8.5 kDa), these observations support theview that for the interactions with the FAS enzymeswhen intermediates of varying lengths are cova-lently linked to ACP, the protein moiety of the lattermay form an important and invariant part of therecognition surface.Our observation that the thiol-activated moiety of

the substrate can be sequestered within the carrierprotein raises an important question as to howindividual FAS enzymes discriminate between thedifferent pathway intermediates (e.g. β-keto, β-hydroxyl, acetoacetyl-, malonyl-ACP) when theacyl chain is transported in this mode. As revealedby our analysis, the entrance into the acyl-chainbinding cavity is lined with polar residues whereasthe surface of the expandable tunnel is mainlyformed by apolar side-chains. The charged thioester-linked group in malonyl-ACP is likely to remainsolvent-exposed and possibly stabilized against thesurface of the protein. When the acyl chain issequestered into the protein cavity, the polar sidegroups in β-keto and β-hydroxyl-acyl-ACP aremore likely to be bound in the polar environmentat the opening of the tunnel with possible hydrogenbonding to the side-chain of Thr39 or the main-chainpeptides of Ala59, Glu60 or Ile62 with the aliphatictail of the substrate buried in the hydrophobicpocket. As a result β-keto and β-hydroxyl inter-mediates of equivalent chain length may be buriedto different extents and perhaps not as deeply asobserved for the hexanoyl and heptanoyl chains inour structures. This might provide a means ofspecific recognition of a particular bound intermedi-ate. The prediction of the location of the thioesterlink in trans Δ2 enoyl intermediates and hence theirrecognition and discrimination from saturatedforms by partner enzymes, poses a slightly moredifficult problem. A possibility is favourable stac-king interactions between the double bond in theacyl chain and the side-chain of Phe28, which isknown to have a critical function and has been seento adopt different rotamers upon acyl chainloading.22,27 In turn this might give rise to a uniqueconformation for the phosphopantetheine moietyand/or the protein surface and hence create arecognition feature. The precise positioning ofindividual intermediates is likely to be defined bythe hydrophilic/hydrophobic nature of the thioe-

143Crystallographic Analysis of Acyl-ACPs

ster-linked reactive moiety (groups at positions 1 to3) and the possible hydrogen-bonding network withthe protein but to be independent of the length of theacyl chain. Our observation that at least twodifferent binding modes exist for the acylatedprosthetic group in the crystal forms of ACPsupports the view that the 4′-phosphopantetheinearm tethering the substrate to Ser36 is fairly flexibleand may adopt a set of different conformations toallow optimal binding of individual intermediatesto partner enzymes during the biosynthetic cycle.This suggests a mechanism whereby the nature ofthe buried intermediates of the fatty acid biosynth-esis cycle might be “reported” to the FAS enzymesthrough differences associated with the accessiblesurface features of ACP, perhaps in large part arisingfrom alternative conformations of the phosphopan-tetheine linker itself. Upon binding of a partnerenzyme to ACP, we would anticipate furtherstructural rearrangements of ACP so as to enablefull access to the thioester linkage or reactive sidegroups for catalysis. However, our results suggestthat maybe during a round of fatty acid elongation,the intermediates could simply adjust their relativepositions in the binding pocket on ACP so as toexpose particular functional groups and keep theacyl chain buried. Structures of complexes ofdifferent ACP-bound fatty acid biosynthesis inter-mediates with partner enzymes may help to resolvethis question. Our structures illustrate that themechanism for recognition of a particular acylintermediate is not straightforward and if it doesinvolve the conformation adopted by the phospho-pantetheine group then it is also complicated by anyvariability in the extent of burial of this moietydependent upon the length of attached acyl chain.We therefore hypothesize that the 4′-phosphopan-tetheine prosthetic group forms a variable part of therecognition surface on ACP that helps to define itsspecificity towards different partners within FAS.

Materials and Methods

Preparation and crystallization of acyl-ACPs

E. coli hexanoyl-, heptanoyl- and decanoyl-ACP wereprepared following the previously described procedure.29

Crystals of hexanoyl-ACP were obtained by using 3 μlhanging drops containing 12 mg/ml protein mixed withthe same volume of the reservoir solution containing 16–20% (w/v) polyethyleneglycol (PEG) 1500, 20 mM zincacetate, 50 mM sodium cacodylate (pH 6.0), andequilibrated against the reservoir solution at 17 °C. Thecrystals belong to space group P21212 with unit-celldimensions a=48.4 Å, b=105.0 Å, c=27.9 Å and with twomolecules in the asymmetric unit. Crystals of heptanoyl-and decanoyl-ACP were obtained using 8–12% (w/v)PEG 4000, 30 mM zinc acetate, 50 mM sodium cacodylate(pH 6.0). These crystals belonged to the same spacegroup as those of hexanoyl-ACP and had very similarunit cell dimensions of a=49.3 Å, b=106.2 Å, c=28.2 Å(heptanoyl-ACP) and a=47.2 Å, b=107.5 Å, c=28.0 Å(decanoyl-ACP).

X-ray data collection and structure determination

Diffraction data were collected at cryogenic tempera-tures on station ID14.2 of the ESRF. All the data wereprocessed and scaled using the DENZO/SCALEPACKpackage35 and subsequently handled using CCP4software.36 Data collection statistics are summarized inTable 1.The structure of hexanoyl-ACP was solved by mole-

cular replacement using the coordinates of the 1.2 Åresolution structure of E. coli butyryl-ACP I62M (PDB1L0I)22 with the prosthetic group, ions and watermolecules excluded. Molecular replacement was per-formed using CNS,37 which revealed that the asymmetricunit contained two independent molecules. Modelbuilding and simulated annealing refinement werecarried out using programs TURBO-FRODO38 andCNS, respectively, with no non-crystallographic symmet-ry restraints and no cutoff on structure factor amplitudesimposed, and the stereochemistry was analysed withPROCHECK.39 Water molecules were introduced duringthe course of the refinement at geometrically reasonablepositions, but they were only retained after refinement iftheir B-factors remained below 60 Å2. Metal (Zn and Na)ions could be detected and distinguished from watermolecules as strong density peaks in the (Fo–Fc)difference maps with the characteristic coordinationspheres of oxygen atoms. At the final stages of refine-ment the hexanoyl-phosphopantetheine groups werefitted into the corresponding regions of the electrondensity maps in the two molecules in the asymmetricunit and their coordinates have been refined to conver-gence. Restrained individual temperature factors wererefined isotropically for all non-hydrogen atoms. Similarprocedures were employed in determining the structuresof heptanoyl- and decanoyl-ACP. In one of the monomersof decanoyl-ACP, the quality of the electron density mapdid not permit modelling of the acylated phosphopan-tetheine group and thus only the phosphate moiety wasincorporated into the model for this subunit. Refinementstatistics for the three crystal structures are summarisedin Table 1. Solvent-accessible volumes were calculated forthe internal cavity of ACP using the server CASTp with aprobe radius of 1.4 Å.30

Protein Data Bank accession codes

The coordinates and structure factors ofE. coli hexanoyl-,heptanoyl- and decanoyl-ACP have been deposited in theRCSB Protein Data Bank under ID codes 2FAC, 2FAD and2FAE, respectively.

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Edited by M. Guss

(Received 29 June 2006; received in revised form 13 September 2006; accepted 19 September 2006)Available online 23 September 2006