Mammalian Fatty Acid Synthase: X-ray Structure of a Molecular Assembly Line Francisco J. Asturias* Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
F
atty acids are ubiquitous cellular components with a variety of structural and metabolic functions, from energy storage to modulation of gene expression. In plants and bacteria, de novo synthesis of fatty acids is carried out by individual enzymes. In contrast, in animal cells, the enzymes involved in fatty acid synthesis are organized in a single fatty acid synthase polypeptide (Figure 1). Encompassing all of the activities required to retain and extend an acyl chain from acetyl-CoA and malonyl-CoA precursors and then release a full-length, long-chain fatty acid, mammalian fatty acid synthase (FAS) constitutes a true molecular assembly line (1 ). Human FAS is a target for drug development against obesity and related diseases, and FAS inhibitors have been shown to have antitumor activity. Although the biochemistry of fatty acid synthesis was established over 30 years ago, structural understanding of FAS had, until recently, been limited (high-resolution structures of individual prokaryotic enzymes and a couple of FAS domains were available). The situation has changed dramatically with the recent publication of the first X-ray structure of FAS by Nenad Ban and colleagues (2 ). In mammalian cells, the functional form of FAS is a homodimer (MW ~540 000 Da) with two centers for fatty acid synthesis. The model for FAS organization that prevailed for over 20 years was based on a logical but naïve interpretation of the possible structure of an FAS monomer (3 ). It was observed that the bifunctional compound dibromopropanone could cross‑link www.acschemicalbiolog y.o rg
the thiol in the active site cysteine of the N-terminal b‑ketoacyl synthase (KS) domain of one monomer with the thiol in the phosphopantetheine group of the acyl carrier protein (ACP) located near the C‑terminus of the other monomer. Therefore, a model with two extended FAS monomers in an antiparallel arrangement was proposed, thereby providing a simple explanation for the complementary interaction of the N-terminal condensing domains of one monomer with the C‑terminal reducing and chain terminating domains of the other. However, functional complementation studies of baculovirusexpressed active site FAS mutants revealed a number of intramonomer interactions that could not be explained by the extended, antiparallel model of FAS (4 ). Careful re-examination of the dibromopropanone experiment (5 ) also revealed both intra‑ and intermonomer cross-links. These investigations eventually resulted in the formulation of an alternative model for FAS organization in which the two monomers were intertwined (6 ) (Box 1). The paucity of structural information on FAS that prevailed until very recently illustrates some of the challenges in structural analysis of large macromolecular complexes. A relatively new technique that can often be used to determine low (10–30 Å)‑resolution structures of large macromolecules is molecular electron microscopy (EM). Analysis of FAS by EM was complicated by the flexibility of the molecule, and a first low-resolution 3‑D EM reconstruction of FAS was interpreted
A B S T R A C T Mammalian fatty acid synthase (FAS) is a homodimeric, multifunctional polypeptide which comprises two full sets of catalytic subunits that carry out fatty acid synthesis. A recently published X-ray structure of FAS reveals, for the first time, the organization of all active sites involved in acyl chain elongation and provides a structural framework for interpretation of extensive functional studies. Further analysis with techniques capable of providing information about single molecule conformations will eventually provide a more complete understanding of FAS.
* To whom correspondence should be addressed. E-mail:
[email protected].
Published online April 21, 2006 10.1021/cb6001448 CCC: $33.50 © 2006 by American Chemical Society
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1
KS
MAT
DH
C161
S581
H878
G1672
S2151
ER
A C P
KR G1888
2505
TE S2302
consistent with the biochemical and functional information (8 ). b This represented a significant advance, but the organization of the different active sites in FAS remained obscure. Our understanding of FAS has been taken to the next level with the publication of its X-ray structure (2 ). This was a long-awaited result, as needle-like crystals of FAS were reported over 30 years ago, in one of the first papers to describe a protocol for FAS purification. Because the resolution of the X-ray electron density map extended to only 4.5 Å, it was not possible to identify individual residues or trace the polypetide backbone of the FAS monomers. However, secondary structure elements were clearly identifiable, and the electron density map was interpreted by fitting high-resolution structures Figure 1. Active sites in the primary sequence of an FAS monomer and the fatty acid catalytic cycle. of individual domain homoa) Arrangement of domains in the primary sequence of a fatty acid synthase monomer. Critical active logues (Figure 2). The overall site residues are indicated. The N‑terminal b‑ketoacyl synthase (KS, orange) domain is followed by X-shape of the FAS structure is malonyl/acetyl transferase (MAT, red), dehydratase (DH, light green), a,b‑enoyl reductase (ER, dark green), very similar to the most recent b‑ketoacyl reductase (KR, yellow), acyl carrier protein (ACP, maroon) with its prosthetic phosphopanteteine EM reconstruction (8 ) and, group, and finally the C-terminal thioesterase (TE, light blue) domain. The core region between the DH and ER domains has no catalytic activity. b) Cycle of reactions catalyzed by FAS, which culminate in like it, appears asymmetric. synthesis of a long-chain fatty acid. The cycle is initiated when MAT catalyzes transfer of the acyl moiety As predicted by biochemical of acetyl-CoA (initiation substrate) to the ACP. The acyl moiety is momentarily transfered to the KS domain, and EM analysis (8, 9 ), the KS and MAT catalyzes transacylation of the malonyl group of malonyl-CoA (elongation substrate) to the domains are dimeric and, along ACP. KS catalyzes decarboxylative condensation to an acetoacetyl‑ACP. KR catalyzes NADPH‑dependent with the dehydratase (DH) and reduction of the b‑carbon, and DH dehydrates the resulting b‑hydroxyacyl‑ACP to an a,b‑enoyl intermediate. ER catalyzes the NADPH‑dependent reduction of the enoyl to produce a four‑carbon acyl b-enoyl reductase (ER) domains, chain to which two‑carbon units derived from malonyl‑CoA are attached in subsequent elongation form the central portion of the cycles. When the acyl chain reaches a length of 16–18 carbons, it is released from the ACP by TE. FAS structure. The ER domains are also dimeric (contributing as providing evidence for the extended was asymmetric and clearly inconsistent significantly to the monomer–monomer antiparallel model, disregarding the with the predictions of the extended anti interface), and the DH domains adopt a inconsistency with the results from FAS parallel model. Analysis of the 3‑D structure pseudo-dimeric fold within each monofunctional complementation assays (7 ). In of an FAS monomer and localization of the mer. Protruding from this central portion a more recent EM study, variability in FAS N-termini (by tagging an FAS mutant with of the structure are pseudo-symmetrically conformation was reduced by the use of a a metal cluster directly visible in images placed malonyl/acetyl transferase (MAT) KS point mutant imaged in the presence of of single FAS particles) to the center of the and b-ketoacyl reductase (KR) domains, substrates. This approach yielded a more FAS structure were combined to propose a both of which are monomeric. A large, faithful 3‑D reconstruction of FAS, which revised model for FAS organization that was catalytically inactive region that follows 1 36
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Figure 2. Organization of FAS active sites. Overlaid on an outline of the X-ray structure are the different active sites. The KS dimer forms the bottom part of the central portion of the structure. Protruding from the bottom are the (monomeric) MAT domains. The DH domains sit atop the KS dimer, and the ER domains (also dimeric) form the top central portion of the structure. Finally, extending from the top are the monomeric KR domains. The noncatalytic “core” in the FAS primary sequence appears to be distributed among the other active sites. The active sites on either side of the pseudo-symmetric structure are arranged around a reaction chamber. Extra density after the KR domain on the right likely corresponds to part of the ACP and TE domains in that monomer. The active sites are in all cases located near the center of the domains. The length of the prosthetic group in the ACP is long enough to reach each active site from the periphery of the corresponding domain, but the ACP itself must be highly mobile to reach the different domains around each reaction chamber. The scale bar corresponds to 100 Å.
the DH domain appears to be distributed throughout the central portion of the FAS structure, contributing to the interfaces between the KS, MAT, and DH domains. The arrangement of domains around two reaction chambers formed by the coiled FAS monomers makes intuitive sense, in that it accommodates necessary functional interactions without requiring major domain rearrangements. Perhaps the most significant issue that remains unresolved is the localization and range of motion of the C-terminal ACP and thioesterase (TE) domains not included in the X-ray structure of FAS. The prosthetic phosphopantetheine group of the ACP plays a critical role by translocating substrates from one FAS active site to another, and must therefore be able to reach those sites. From the available ACP structures (10, 11 ), it is clear that the phosphopantetheine “arm” could only extend far enough to reach from the edge of a given domain to the domain’s internal active site. This implies that the ACP must have a significant degree of mobility. Harder to understand is the reason for the high mobility of the C-terminal TE domain, which results from more than its attachment to the mobile ACP, as the ACP and TE domains are connected by what appears www.acschemicalbiolog y.o rg
to be a highly flexible, disordered linker whose length affects TE functionality (12 ). The X-ray structure (2 ) does not address the issue of monomer organization in FAS, but consideration of the EM results (8 ) suggests that the two monomers likely cross over each other in the central portion of the structure. Regardless, the static X‑ray structure cannot explain both the intraand intermonomer functional interactions revealed by functional complementation studies (4 ) (ACP domains can work with the KS and MAT in the same or the other monomer, see Box 1). Those interactions must be facilitated by the combined effect of large ACP mobility and conformational changes in the portions of the FAS structure included in the current X-ray map. The existence of a significant degree of flexibility in FAS has been established by alignment and multivariate statistical analysis of single particle images and by normal-mode elastic deformation analysis of the low-resolution structure of FAS (8, 13 ). FAS has long been considered a paradigm for understanding the modular structure of the giant polyketide synthases (PKS) responsible for biosynthesis of some anti biotics and other important biologically active compounds. The X-ray structure of FAS
now reveals that the dimerization interface comprises domains that are often absent in PKS modules. Interestingly, along with the structure of mammalian FAS, Ban and colleagues also reported on the structure of a vastly different fungal FAS (14 ). Previous studies had established fundamental differences in structure between mammalian and yeast FAS (15 ), but the X-ray structures now illustrate how two synthases with completely different organization carry out the same biosynthetic task. Perhaps some of the strategies that made possible calculation of an FAS X-ray structure could be adapted to enable similar analysis of PKS modules. Differences between the FAS structures calculated by X-ray crystallography and EM (particularly apparent when top or bottom views of the structures compared) suggest that the role that dimerization plays in FAS function and the reasons behind the asymmetric nature of the FAS structure are yet to be understood. The X-ray structure of FAS determined by Ban and colleagues (2 ) will prove very valuable in designing experiments and interpreting results from techniques such as optical, fluorescence, and electron microscopy, that can record “snapshots” of individual FAS molecules under functionally relevant conditions. VOL.1 NO. 3 • 135—138 • 2 0 0 6
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Box 1. Functional characterization of FAS using baculovirusexpressed active site point mutants. Extensive functional A KS MAT DH ER KR CP TE studies have been carried out to characterize the interactions G1888A S2302A C161Q S581A H878A between FAS active sites, taking advantage of point mutations that knock-out the activity of individual domains. a) Point b C161A mutations that inactivate each of the FAS active sites have A His KS MAT DH ER KR CP TE been identified, and FAS mutants with a given mutation can be expressed in Sf9 insect cells and purified by affinity chroma A FLAG KS MAT DH ER KR CP TE tography. b) Reversible dissociation of the FAS monomers S2151A induced by low temperature (4 °C) incubation makes possible assembly of heterodimers with specific mutations, which can c be used to characterize inter‑ and intramonomer functional interactions. c) Functional complementation studies using a variety of mutants have resulted in a map of active site interactions. The acyl carrier protein (ACP) groups can interact with the b-ketoacyl synthase (KS) and malonyl/acetyl transferase (MAT) sites of either FAS monomer. In general, 65–80% of elongation cycles involve interactions between one ACP and the KS group of the other monomer. However, the other 20–35% of elongation cycles involve intramonomer ACP/KS interactions, a finding that was in clear conflict with the initial model for FAS organization depicting two extended monomers in an antiparallel orientation. In fact, the ultimate demonstration of the significance of intramonomer functional complementation came from an experiment in which a wild-type FAS monomer was dimerized with a fully inactive one. The resulting dimer showed fatty acid synthesis activity corresponding to about 30% that of a wild-type FAS dimer. These functional complementation studies were carried out in the last several years by Stuart Smith and his group at Children’s Hospital Oakland Research Institute.
a
G1672V
S2151A
6
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