Molecular Dynamics Simulations of the Apo-, Holo-, and Acyl-forms of Escherichia coli Acyl Carrier Protein
2008; Elsevier BV; Volume: 283; Issue: 48 Linguagem: Inglês
10.1074/jbc.m805323200
ISSN1083-351X
AutoresDavid I. Chan, Thomas Stockner, D. Peter Tieleman, Hans J. Vogel,
Tópico(s)Chemical Synthesis and Analysis
ResumoAcyl carrier protein (ACP) is an essential co-factor protein in fatty acid biosynthesis that shuttles covalently bound fatty acyl intermediates in its hydrophobic pocket to various enzyme partners. To characterize acyl chain-ACP interactions and their influence on enzyme interactions, we performed 19 molecular dynamics (MD) simulations of Escherichia coli apo-, holo-, and acyl-ACPs. The simulations were started with the acyl chain in either a solvent-exposed or a buried conformation. All four short-chain (≤C10) and one long-chain (C16) unbiased acyl-ACP MD simulation show the transition of the solvent-exposed acyl chain into the hydrophobic pocket of ACP, revealing its pathway of acyl chain binding. Although the acyl chain resides inside the pocket, Thr-39 and Glu-60 at the entrance stabilize the phosphopantetheine linker through hydrogen bonding. Comparisons of the different ACP forms indicate that the loop region between helices II and III and the prosthetic linker may aid in substrate recognition by enzymes of fatty acid synthase systems. The MD simulations consistently show that the hydrophobic binding pocket of ACP is best suited to accommodate an octanoyl group and is capable of adjusting in size to accommodate chain lengths as long as decanoic acid. The simulations also reveal a second, novel binding mode of the acyl chains inside the hydrophobic binding pocket directed toward helix I. This study provides a detailed dynamic picture of acyl-ACPs that is in excellent agreement with available experimental data and, thereby, provides a new understanding of enzyme-ACP interactions. Acyl carrier protein (ACP) is an essential co-factor protein in fatty acid biosynthesis that shuttles covalently bound fatty acyl intermediates in its hydrophobic pocket to various enzyme partners. To characterize acyl chain-ACP interactions and their influence on enzyme interactions, we performed 19 molecular dynamics (MD) simulations of Escherichia coli apo-, holo-, and acyl-ACPs. The simulations were started with the acyl chain in either a solvent-exposed or a buried conformation. All four short-chain (≤C10) and one long-chain (C16) unbiased acyl-ACP MD simulation show the transition of the solvent-exposed acyl chain into the hydrophobic pocket of ACP, revealing its pathway of acyl chain binding. Although the acyl chain resides inside the pocket, Thr-39 and Glu-60 at the entrance stabilize the phosphopantetheine linker through hydrogen bonding. Comparisons of the different ACP forms indicate that the loop region between helices II and III and the prosthetic linker may aid in substrate recognition by enzymes of fatty acid synthase systems. The MD simulations consistently show that the hydrophobic binding pocket of ACP is best suited to accommodate an octanoyl group and is capable of adjusting in size to accommodate chain lengths as long as decanoic acid. The simulations also reveal a second, novel binding mode of the acyl chains inside the hydrophobic binding pocket directed toward helix I. This study provides a detailed dynamic picture of acyl-ACPs that is in excellent agreement with available experimental data and, thereby, provides a new understanding of enzyme-ACP interactions. Fatty acid (FA) 4The abbreviations used are: FA, fatty acid; FAS, FA synthase; ACP, acyl carrier protein; ACPS, ACP synthase; MD, molecular dynamics; RMSF, root mean square fluctuations; SASA, solvent-accessible surface area; NOE, nuclear Overhauser effect. 4The abbreviations used are: FA, fatty acid; FAS, FA synthase; ACP, acyl carrier protein; ACPS, ACP synthase; MD, molecular dynamics; RMSF, root mean square fluctuations; SASA, solvent-accessible surface area; NOE, nuclear Overhauser effect. synthesis occurs in a well structured and characterized cycle of condensation and reduction reactions that elongate acyl chains before they reach maturity and are cleaved off for utilization in various anabolic pathways (1Zhang Y.M. Rock C.O. Nat. Rev. Microbiol. 2008; 6: 222-233Crossref PubMed Scopus (812) Google Scholar). There are two main types of machineries that perform this process, known as the type I fatty acid synthase (FAS) systems found in mammals and fungi and the type II systems existing in bacteria and eukaryotic plastids (2Byers D.M. Gong H. Biochem. Cell Biol. 2007; 85: 649-662Crossref PubMed Scopus (153) Google Scholar). Type I systems are composed of a single long polypeptide chain that contains all the enzymes and other proteins necessary to accomplish fatty acid synthesis. These mega-complexes fold into large machineries, yielding reaction chambers that are readily accessible to the enzymes of FA synthesis for rapid catalysis (3Leibundgut M. Jenni S. Frick C. Ban N. Science. 2007; 316: 288-290Crossref PubMed Scopus (151) Google Scholar, 4Jenni S. Leibundgut M. Boehringer D. Frick C. Mikolasek B. Ban N. Science. 2007; 316: 254-261Crossref PubMed Scopus (172) Google Scholar, 5Maier T. Jenni S. Ban N. Science. 2006; 311: 1258-1262Crossref PubMed Scopus (307) Google Scholar). In sharp contrast, type II or dissociated FAS systems express each of the enzymes necessary for fatty acid synthesis as individual proteins (6White S.W. Zheng J. Zhang Y.M. Rock C.O. Annu. Rev. Biochem. 2005; 74: 791-831Crossref PubMed Scopus (609) Google Scholar). Given how vastly different these two systems are between humans and bacteria, there has been a conscious effort to target FA synthesis in bacteria through novel antibiotics (7Zhang Y.M. White S.W. Rock C.O. J. Biol. Chem. 2006; 281: 17541-17544Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). A number of antibacterial agents acting on FAS components have already been discovered, demonstrating that these systems may be successfully used as a target for new types of antibiotics (8Wright H.T. Reynolds K.A. Curr. Opin. Microbiol. 2007; 10: 447-453Crossref PubMed Scopus (153) Google Scholar, 9Zhang Y.M. Frank M.W. Virga K.G. Lee R.E. Rock C.O. Jackowski S. J. Biol. Chem. 2004; 279: 50969-50975Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 10Wang J. Soisson S.M. Young K. Shoop W. Kodali S. Galgoci A. Painter R. Parthasarathy G. Tang Y.S. Cummings R. Ha S. Dorso K. Motyl M. Jayasuriya H. Ondeyka J. Herath K. Zhang C.W. Hernandez L. Allocco J. Basilio A. Tormo J.R. Genilloud O. Vicente F. Pelaez F. Colwell L. Lee S.H. Michael B. Felcetto T. Gill C. Silver L.L. Hermes J.D. Bartizal K. Barrett J. Schmatz D. Becker J.W. Cully D. Singh S.B. Nature. 2006; 441: 358-361Crossref PubMed Scopus (709) Google Scholar). For example, the pantothenamide class of antibiotics has been shown to incapacitate the FAS system by creating inactive forms of acyl carrier protein (ACP) (9Zhang Y.M. Frank M.W. Virga K.G. Lee R.E. Rock C.O. Jackowski S. J. Biol. Chem. 2004; 279: 50969-50975Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). As a central player in both systems, ACP shuttles the acyl chain intermediates to various enzymes of the FAS systems. In type II systems, ACPs are small, acidic proteins of about 9 kDa. The structure of ACP is well conserved in different organisms, consisting of three major helices (I, II, IV) that run largely parallel to each other, with helix III markedly shorter and almost perpendicular to the bundle (6White S.W. Zheng J. Zhang Y.M. Rock C.O. Annu. Rev. Biochem. 2005; 74: 791-831Crossref PubMed Scopus (609) Google Scholar). The acyl chain intermediates in FA synthesis are bound covalently by means of a prosthetic linker on ACP that is derived from coenzyme A in an enzymatic reaction catalyzed by ACP synthase (ACPS) (Fig. 1). This reaction converts apo-ACP to holo-ACP, which has the phosphopantetheine arm attached to a conserved serine residue (residue 36 in Escherichia coli). The prosthetic linker contains a terminal sulfhydryl group that is utilized to covalently bind the acyl chain intermediates in a thioester linkage (Fig. 1). The hydrophobic acyl chain attached to ACP is positioned in between the four-helix bundle in a pocket that is made up of a core of hydrophobic residues (11Roujeinikova A. Baldock C. Simon W.J. Gilroy J. Baker P.J. Stuitje A.R. Rice D.W. Slabas A.R. Rafferty J.B. Structure. 2002; 10: 825-835Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 12Roujeinikova A. Simon W.J. Gilroy J. Rice D.W. Rafferty J.B. Slabas A.R. J. Mol. Biol. 2007; 365: 135-145Crossref PubMed Scopus (125) Google Scholar, 13Zornetzer G.A. Fox B.G. Markley J.L. Biochemistry. 2006; 45: 5217-5227Crossref PubMed Scopus (77) Google Scholar). During substrate presentation, the acyl chain must somehow overcome stabilizing hydrophobic interactions with the interior of ACP and inject it into the active site of a partner enzyme. The highly dynamic nature of ACP is thought to be a crucial aspect of its ability to interact with a large number of enzymes as well as the plethora of acyl chains and their intermediates (2Byers D.M. Gong H. Biochem. Cell Biol. 2007; 85: 649-662Crossref PubMed Scopus (153) Google Scholar). These adducts include acyl chains ranging from 3 to 18 carbons in length and their β-keto-, β-hydroxy-, as well as enoyl intermediates and unsaturated fatty acyl groups. A number of structures of apo-, holo-, and acyl-ACP are available as well as structures of ACP-enzyme complexes, highlighting important aspects to ACP function such as helix II, which is known as the recognition helix (11Roujeinikova A. Baldock C. Simon W.J. Gilroy J. Baker P.J. Stuitje A.R. Rice D.W. Slabas A.R. Rafferty J.B. Structure. 2002; 10: 825-835Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 12Roujeinikova A. Simon W.J. Gilroy J. Rice D.W. Rafferty J.B. Slabas A.R. J. Mol. Biol. 2007; 365: 135-145Crossref PubMed Scopus (125) Google Scholar, 13Zornetzer G.A. Fox B.G. Markley J.L. Biochemistry. 2006; 45: 5217-5227Crossref PubMed Scopus (77) Google Scholar, 14Parris K.D. Lin L. Tam A. Mathew R. Hixon J. Stahl M. Fritz C.C. Seehra J. Somers W.S. Structure. 2000; 8: 883-895Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 15Rafi S. Novichenok P. Kolappan S. Zhang X.J. Stratton C.F. Rawat R. Kisker C. Simmerling C. Tonge P.J. J. Biol. Chem. 2006; 281: 39285-39293Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In addition, NMR studies have provided insights into the backbone dynamics of apo- and holo-ACP (16Kim Y. Kovrigin E.L. Eletr Z. Biochem. Biophys. Res. Commun. 2006; 341: 776-783Crossref PubMed Scopus (41) Google Scholar). Nevertheless, a dynamic picture of acyl-ACP has been difficult to obtain and a detailed understanding of acyl-chain:ACP interactions remains elusive. It is also unclear how the enzymes of the FAS pathway recognize the appropriate acyl-ACP. Considering that ACP makes up ∼0.25% of all soluble protein in E. coli suggests that there are mechanisms for enzymes to specifically recognize what type of acyl-ACP is bound (17Rock C.O. Jackowski S. J. Biol. Chem. 1982; 257: 10759-10765Abstract Full Text PDF PubMed Google Scholar). To understand this process, it is essential that a dynamic picture of the substrate behavior attached to ACP is obtained. Furthermore, if the pathway of substrate binding by ACP is revealed, potential drug target sites that are not apparent from static structures alone may be identified. To evaluate these questions we performed extensive molecular dynamics (MD) simulations of apo-, holo-, and saturated acyl-ACPs ranging from 4 to 18 carbon groups in length. The holo- and acyl-ACP simulations were conducted both with the prosthetic group in a solvent-exposed state as well as in a solvent-shielded conformation inside the protein. The simulations provide novel information on the manner of substrate protection by ACP as well as differentiating motions between the apo-, holo-, and acylated forms of the protein. Our findings are substantiated by excellent agreement with experimental data in the literature. Simulation Setup—The MD simulations on E. coli ACP were based on the high resolution (1.2 Å) crystal structure of butyryl-ACP (Protein Data Bank code 1L0I), which contains the acyl chain buried inside the hydrophobic pocket of ACP (11Roujeinikova A. Baldock C. Simon W.J. Gilroy J. Baker P.J. Stuitje A.R. Rice D.W. Slabas A.R. Rafferty J.B. Structure. 2002; 10: 825-835Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The Protein Data Bank file was first checked by submitting it to the Biotech Validation Suite for Protein Structures server, and consequently, the side chain of Gln-19 was flipped. Multiple protein conformations for certain atoms were removed based on the best structural agreement in comparison to another crystal structure of Escherichia coli ACP (11Roujeinikova A. Baldock C. Simon W.J. Gilroy J. Baker P.J. Stuitje A.R. Rice D.W. Slabas A.R. Rafferty J.B. Structure. 2002; 10: 825-835Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). 1L0I also contained the mutation I62M, which was used to incorporate a selenomethionine residue into the protein to address the crystallographic phase problem (11Roujeinikova A. Baldock C. Simon W.J. Gilroy J. Baker P.J. Stuitje A.R. Rice D.W. Slabas A.R. Rafferty J.B. Structure. 2002; 10: 825-835Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). For our studies we reversed this mutation through alignments to 1L0H in the program MOLMOL (18Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6487) Google Scholar) and transferring of coordinates. The resulting structure was used as the starting point for all the simulations described here. The simulations conducted can be categorized into two main groups based on the initial orientation of the acyl chain in the starting structures. The first group of simulations contains ACP with its prosthetic linker in a completely solvent-exposed orientation, whereas the second group is composed of ACP with the prosthetic group in a solvent-inaccessible conformation as is seen in 1L0I. Each of these groups is composed of various ACP members, differing in the length of the prosthetic group attachment. Apo- as well as holo-ACP simulations were set up in addition to saturated acyl chain simulations ranging from butyryl-ACP (4 carbon chain) to octadecanoyl-ACP (18 carbon chain) (Table 1). For the solvent-exposed simulations, the prosthetic group was pointed vertically up from the protein, extending along the axis of helix II. This conformation was chosen as an unbiased orientation such that the prosthetic group can move in as many directions as possible. The starting structures for simulations of ACP with solvent-shielded acyl groups were derived from the butyryl-ACP crystal structure 1L0I by extending the butyryl group incrementally by two carbon groups.TABLE 1ACP MD simulations solvent shielded acyl-chain propertiesAcyl chainCarbon groupsSASAAcyl chain volumecCalculated using the program voidoo (28).Average cavity volumeAcyl chainShieldedaBased on the total surface area of the acyl chain.PhosphopantetheinebRefers to Ser-36 side chain only for apo and Ser-36 as well as the phosphopantetheine group for all others.Å2%Å2Å3Å3Apo78.525.9HoloLinker only202.186.7Butyryl42.198.8249.182140.7Hexanoyl63.698.4257.4116170.9Octanoyl83.498.8240.3149200.4Decanoyl1013.495.2258.9182231.9Dodecanoyl1220.494.6275.3214185.8Tetradecanoyl1433.492.2284.0247186.2Hexadecanoyl1663.586.6309.9280163.5Octadecanoyl1867.486.9315.1314181.2a Based on the total surface area of the acyl chain.b Refers to Ser-36 side chain only for apo and Ser-36 as well as the phosphopantetheine group for all others.c Calculated using the program voidoo (28Kleywegt G.J. Jones T.A. Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 178-185Crossref PubMed Scopus (982) Google Scholar). Open table in a new tab The starting structures were initially solvated with explicit simple point charge (SPC) water in a rhombic dodecahedron box. The distance between the protein and edge of the box was ∼1.5 nm, to ensure that the minimum distance between periodic images would not be too low, even with the prosthetic group in an extended conformation directed away from ACP into the solvent. All simulations were performed using the GROMACS 3.2 MD package (19Berendsen H.J.C. van der Spoel D. van Drunen R. Comput. Phys. Commun. 1995; 91: 43-56Crossref Scopus (7194) Google Scholar, 20Lindahl E. Hess B. van der Spoel D. J. Mol. Model. 2001; 7: 306-317Crossref Google Scholar) and the GROMOS96 43a2 force field containing improved alkane dihedrals parameters (21Scott W.R.P. Hunenberger P.H. Tironi I.G. Mark A.E. Billeter S.R. Fennen J. Torda A.E. Huber T. Krüger P. van Gunsteren W.F. J. Phys. Chem. A. 1999; 103: 3596-3607Crossref Scopus (1280) Google Scholar). Virtual sites were used for hydrogens to remove the highest frequency hydrogen vibrations, permitting the use of a 5-fs time-step (22Feenstra K.A. Hess B. Berendsen H.J.C. J. Comput. Chem. 1999; 20: 786-798Crossref Scopus (656) Google Scholar). The neighbor search was conducted according to the grid method, updating the neighbor list every 4 steps and a neighbor list cut-off of 0.9 nm. All the MD simulations were carried out using periodic boundary conditions. Constant temperature (300 K, τT = 0.1ps) and pressure were maintained by coupling to a bath using the Berendsen algorithm (23Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. Dinola A. Haak J.R. J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (23462) Google Scholar). Pressure was kept at 1 bar with isotropic temperature coupling, tau τP of 1.0 ps, and compressibility of 4.5 × 10-5 bar-1. Bond lengths were restrained using the Linear Constraint Solver (LINCS) method (24Hess B. Bekker H. Berendsen H.J.C. Fraaije J.G.E.M. J. Comput. Chem. 1997; 18: 1463-1472Crossref Scopus (11575) Google Scholar). Long range electrostatic interactions were calculated according to Fast Particle-Mesh Ewald (PME) electrostatics (25Darden T. York D. Pedersen L. J. Chem. Phys. 1993; 98: 10089-10092Crossref Scopus (20832) Google Scholar) with a cutoff of 0.9 nm. Van der Waal energies were collected with a twin range cutoff of 0.9/1.4 nm. The bonds and angles of the simple point charge (SPC) water molecules were constrained using the SETTLE algorithm (26Miyamoto S. Kollman P.A. J. Comput. Chem. 1992; 13: 952-962Crossref Scopus (5201) Google Scholar). After equilibration of the water while restraining the protein, the overall charge of the system was neutralized with 15 sodium counterions (14 ions for apo ACP). Subsequently, the protein was gradually released by decreasing the force constant on the protein in 5 distinct steps (1000, 100, 10, 1, and 0 kJ/mol nm2) of 50 ps simulations each. The simulations of ACP with the acyl chain inside the binding pocket as well as the apo- and holo-ACP simulations were conducted for 20 ns each. The simulations starting off with the prosthetic groups in solvent-exposed conformations were conducted for 50 ns each. Analysis of the trajectories was performed using various GROMACS analysis programs as well as SURFNET (27Laskowski R.A. J. Mol. Graph. 1995; 13: 323-330Crossref PubMed Scopus (824) Google Scholar) for the ACP cavity size analysis and VOIDOO (28Kleywegt G.J. Jones T.A. Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 178-185Crossref PubMed Scopus (982) Google Scholar) for acyl volume calculations. Averaged values for the solvent-shielded simulations were calculated for the last 14 ns of the trajectory, which displayed steady r.m.s.d. values. Phosphopantetheine Topology Building—The phosphopantetheine linker that is attached to ACP represents a new topology non existent in the GROMOS96 library of building blocks (Fig. 1). Most of the parameters such as bond lengths, charges, angles, dihedrals, and improper dihedrals were obtained from subcomponents that have been previously characterized in the GROMOS96 43a2 forcefield. Therefore, most parts of the prosthetic group could be modeled based on these previously established groups. The missing partial charges for the thioester group were calculated using quantum mechanics. The quantum mechanics calculations were performed using GAUSSIAN 03 (29Frisch M.J. Trucks G.W. Schlegel H.B. Scuseria G.E. Robb M.A. Cheeseman J.R. Montgomery Jr., J.A. Vreven T. Kudin K.N. Burant J.C. Millam J.M. Iyengar S.S. Tomasi J. Barone V. Mennucci B. Cossi M. Scalmani G. Rega N. Petersson G.A. Nakatsuji H. Hada M. Ehara M. Toyota K. Fukuda R. Hasegawa J. Ishida M. Nakajima T. Honda Y. Kitao O. Nakai H. Klene M. Li X. Knox J.E. Hratchian H.P. Cross J.B. Bakken V. Adamo C. Jaramillo J. Gomperts R. Stratmann R.E. Yazyev O. Austin A.J. Cammi R. Pomelli C. Ochterski J.W. Ayala P.Y. Morokuma K. Voth G.A. Salvador P. Dannenberg J.J. Zakrzewski V.G. Dapprich S. Daniels A.D. Strain M.C. Farkas O. Malick D.K. Rabuck A.D. Raghavachari K. Foresman J.B. Ortiz J.V. Cui Q. Baboul A.G. Clifford S. Cioslowski J. Stefanov B.B. Liu G. Liashenko A. Piskorz P. Komaromi I. Martin R.L. Fox D.J. Keith T. Al-Laham M.A. Peng C.Y. Nanayakkara A. Challacombe M. Gill P.M.W. Johnson B. Chen W. Wong M.W. Gonzalez C. Pople J.A. Gaussian 03, Revision C. 02. Gaussian, Inc., Wallingford, CT2004Google Scholar) and have been previously described in detail (30Kandt C. Xu Z.T. Tieleman D.P. Biochemistry. 2006; 45: 13284-13292Crossref PubMed Scopus (52) Google Scholar). The partial charges used were 0.13 for the sulfur atom, 0.25 for the carbonyl carbon, and -0.38 for the carbonyl oxygen. ACP Protein Dynamics—The r.m.s.d. of the backbone atoms of the trajectories remained low compared with their starting structures, stabilizing between 2 and 2.5 Å. In all the simulations ACP shows no significant degree of unfolding beyond a slight unwinding of helix IV in the extreme C-terminal part of the protein. Throughout the apo-, holo-, and acyl-ACP simulations similar regions of elevated root mean square fluctuations (RMSF) values were identified. Loop I between helices I and II shows highly flexible regions immediately after helix I and just before helix II (Fig. 2). The central region of this loop is remarkably stable, displaying backbone RMSF values around 0.6 Å, which is similar to the least flexible regions of ACP. This is because of hydrogen bonds formed between the backbone atoms of residues Asn-24, Ala-26, and Phe-28 with the backbone atoms of Gln-66, Val-65, and Thr-63, respectively, immediately preceding helix IV. Another noticeably flexible region spans residues 47 to 57, which connects helices II and III. Although the magnitude of the fluctuations in most regions of ACP is consistent between apo-, holo-, and the acyl-ACP simulations, there is a clear difference in this region. ACP displays higher RMSF values from residues 47 to 57 when harboring an acyl chain in its hydrophobic binding pocket in each of the simulations conducted. Averaged over all acyl-ACP simulations with an acyl chain occupying the binding cavity, the RMSF is 0.95 ± 0.12 Å, whereas the RMSF is only 0.79 ± 0.05 Å in apo- and holo-ACP. Side-chain fluctuations display the same pattern, with higher RMSF values in acyl-ACP compared with apo- and holo-ACP in this region. The central portions of the helices in ACP are the most rigid segments of the protein, although the ends of the helices show pronounced flexibility as well (Fig. 2). Buried Acyl-ACP Simulations—Acyl-ACP MD simulations with solvent-shielded acyl groups ranging from 4 to 18 carbons in length were performed based on the butyryl-ACP crystal structure (11Roujeinikova A. Baldock C. Simon W.J. Gilroy J. Baker P.J. Stuitje A.R. Rice D.W. Slabas A.R. Rafferty J.B. Structure. 2002; 10: 825-835Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). All of the simulations retained the acyl chain inside the binding pocket, consistent with what has been described in acyl ACP structures from E. coli and spinach (11Roujeinikova A. Baldock C. Simon W.J. Gilroy J. Baker P.J. Stuitje A.R. Rice D.W. Slabas A.R. Rafferty J.B. Structure. 2002; 10: 825-835Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 12Roujeinikova A. Simon W.J. Gilroy J. Rice D.W. Rafferty J.B. Slabas A.R. J. Mol. Biol. 2007; 365: 135-145Crossref PubMed Scopus (125) Google Scholar, 13Zornetzer G.A. Fox B.G. Markley J.L. Biochemistry. 2006; 45: 5217-5227Crossref PubMed Scopus (77) Google Scholar). The binding pocket is located between the three major helices, with the entrance close to the N-terminal end of helix II and additionally bordered by loop I and helix III. The binding cavity is split up into two different binding pockets that are observed to house the tip of the acyl chains (Fig. 3B). The first pocket is similar to the starting structure, analogous to the conformation found in the crystal and NMR acyl-ACP structures between helices II, III, and IV. The second subpocket of the cavity is located more toward helix I, using the same entrance but now residing between helices I, II, and IV (Fig. 3B, Table 2). Whether the tip of the acyl chain binds the first or the second pocket of the binding cavity is determined through a quasi switch that is mediated by the side-chain orientation of Leu-42 and Leu-46. The χ1 angle of Leu-46 measures ∼68° in the starting conformation but switches to ∼-180° almost always when the acyl chain occupies the second sub-pocket (Fig. 3C). The switch in the dihedral angle of Leu-46 opens up a path into the alternate binding cavity of ACP. As a second requirement for the acyl chain to occupy this side of the binding pocket, the χ1 angle of Leu-42 must switch to the energy minimum around -68°. In the starting structures the χ1 angle of Leu-42 measures ∼180° but during the simulation it commonly switches to about -68°. Although this shift does not necessitate acyl chain entry into sub-pocket II, the Leu-42 χ1 angle always measures -68° when sub-pocket II is occupied (Fig. 3C). Similarly to Leu-46, the movement of Leu-42 is required to open up the pathway into the second binding cavity, such that the tip of the acyl chain may enter. Overall, the majority of the simulation time is spent with the tip of the acyl chain oriented into sub-pocket II. The decanoyl chain is the exception to this pattern, as its group resides in sub-pocket I for most of the simulation. In sub-pocket I, the decanoyl chain wraps around helix III at times rather than stick straight into the cavity. The octadecanoyl group adopts a similar conformation and at times penetrates into the solvent between helices III and IV. This behavior was not seen in any of the other simulations.TABLE 2ACP residues contacted in the acyl-chain binding pocketMD simulationsaResidues given that would yield an NOE to the acyl chain in the solvent-shielded simulations based on r−6 distance dependence.Protein Data Bank structuresCommon residuesSub-pocket 1 onlySub-pocket 2 onlyNOEs C10bResidues converted to the E. coli equivalent according to sequence alignment.,cObtained from Zornetzer et al. (13).NOEs C18bResidues converted to the E. coli equivalent according to sequence alignment.,cObtained from Zornetzer et al. (13).X-ray, C10dObtained from PDB code 2FAE Roujeinikova et al. (12).Val-7, Ile-10, Ile-11Glu-4Phe-28, Val-29Val-29Phe-28, Val-29Thr-39Glu-30, Thr-39Thr-39Leu-42, Val-43, Leu-46Leu-42, Leu-46L46, Glu-47Leu-42, Leu-46, Glu-47Ala-59Ile-54Pro-55, Ala-59Thr-52, Ile-54, Ala-59Glu-60, Ile-62, Ala-68Glu-60, Ile-62, Ala-68Ile-62, Ala-67Glu-60, Ile-62, Thr-63, Ala-68Ile-72Tyr-71Ile-72Tyr-71Tyr-71, Ile-72a Residues given that would yield an NOE to the acyl chain in the solvent-shielded simulations based on r−6 distance dependence.b Residues converted to the E. coli equivalent according to sequence alignment.c Obtained from Zornetzer et al. (13Zornetzer G.A. Fox B.G. Markley J.L. Biochemistry. 2006; 45: 5217-5227Crossref PubMed Scopus (77) Google Scholar).d Obtained from PDB code 2FAE Roujeinikova et al. (12Roujeinikova A. Simon W.J. Gilroy J. Rice D.W. Rafferty J.B. Slabas A.R. J. Mol. Biol. 2007; 365: 135-145Crossref PubMed Scopus (125) Google Scholar). Open table in a new tab Solvent-exposed Acyl-ACP Simulations—Five of the eight 50-ns acyl-ACP MD simulations with the acyl chain oriented into the solvent in the starting structure displayed a transition into the hydrophobic binding pocket. The five forms of ACP are butyryl, hexanoyl, octanoyl, decanoyl, and hexadecanoyl. The structures converged very well with the simulations of solvent-shielded acyl-ACPs, yielding very low r.m.s.d. values (supplemental Fig. 1). In the other three trajectories the acyl chain never traversed into the hydrophobic pocket, and usually the prosthetic linker was engaged in hydrogen bonding with the protein which prevented the arm from sampling a broader region of the conformational space. Notably, the longer acyl-ACP simulations did not immerse into the pocket as frequently, as only one of the four longer (>C10) acyl chains was able to find the binding pocket. In those cases additional simulations were conducted with new sets of randomly generated velocities, yet a transition into the binding pocket was not observed. Typically, the acyl chain starts off in an entirely solvent-exposed state and rapidly fluctuates due to the energetic penalty of having the extended hydrophobic acyl chain in contact with the aqueous solvent (Fig. 4A). Using the solvent-accessible surface areas of the acyl chains, the difference in solvation free energy of the acyl chain going from the entirely solvent-exposed state to the buried conformation was calculated (31Eisenberg D. Mclachlan A.D. Nature. 1986; 319: 199-203Crossref PubMed Scopus (1697) Google Scholar). The differences measure between 7 and 25 kJ/mol, depending on the acyl chain length, with longer chains resulting in larger energy differences. The acyl chain then usua
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