separate protein [14]. Central to FAS, an acyl carrier protein (ACP) acts as a metabolic scaffold, tethering the growing fatty acid as it is shuttled iteratively between catalytic domains of the synthase. Fatty acid biosynthesis begins by post-translational modification of the ACP catalyzed by a phosphopantetheinyl transferase (PPTase), which transfers 49-phosphopantetheine from coenzyme A to a conserved serine residue on ACP. This converts inactive apo-ACP to its active holo form bearing a flexible prosthetic arm for attachment of fatty acids via thioester linkage. Once holo- ACP is loaded with an acyl starter unit, fatty acid synthesis occurs on the ACP by sequential action of ketosynthase (KS), ketor- eductase (KR), dehydratase (DH), and enoyl reductase (ER) enzymes, each cycle resulting in a net addition of two carbons to the growing chain. During chain elongation, ACP buries the growing fatty acid in its hydrophobic core to protect it from hydrolysis [15]. Functional interaction with each FAS domain induces a conformational change in ACP that draws the acyl chain out of the pocket (‘switchblade’ mechanism) for further processing by downstream enzymes [16]. When a mature fatty acid has been assembled on the ACP, a thioesterase (TE) catalyzes acyl transfer from ACP to its active site cysteine residue, priming the thioester for attack by water. Based on our previous work in bacteria [17] as well as plant studies to manipulate fatty acid chain length [7], we propose that in algal fatty acid biosynthesis, the TE must functionally interact with ACP to receive the acyl chain, and thus significant protein-protein interactions between the ACP and TE are required to mediate the hydrolysis of fatty acids. We chose to investigate Chlamydomonas reinhardtii, as it is one of a small number of algal species for which all three genomes have been sequenced [18]. Most importantly, it is one of only ten algae that can be transformed with current genetic technologies [19], and techniques have been developed that facilitate robust expression of transgenes in both the nuclear and plastid genome of C. reinhardtii [20,21]. Together, these make C. reinhardtii an ideal model organism to explore algal fatty acid biosynthesis and decipher ACP-TE interactions. Such dynamic protein-protein interactions within the cell are well-known to govern many biological processes [22,23]. Typical methods to decipher protein- protein interactions include yeast 2-hybrid and TAP-tag systems [24], although these techniques can produce false positive readings. Chemical crosslinking can also be used to identify partner proteins, but this procedure suffers from a lack of specificity [24]. To investigate ACP-TE interactions in vitro, we employ a novel approach that circumvents issues associated with traditional methods. Activity-based chemical crosslinking is a powerful means to capture the transient interactions occurring between FAS domains and ACP [17]. We have used this methodology extensively in prokaryotic systems to study protein- protein interactions, successfully implementing activity-based probes designed for a wide array of activities [25,26]. Here, we illustrate the use of activity-based probes to investigate ACP-TE interactions towards engineering fatty acid chain length in C. reinhardtii. By employing chemical probes specifically designed for the enzymatic activity of TEs, we report the first demonstration of metabolic product control through protein-protein interactions. This work emphasizes the importance of protein-protein recogni- tion between the ACP and catalytic enzymes in mediating fatty acid biosynthesis in algal plastids. We address this hypothesis in silico, in vitro and in vivo using C. reinhardtii as a model system. Characterizing algal FAS domains and their interactions will further enable us to optimize heterologous expression of fatty acid biosynthetic enzymes in microalgae and significantly alter the fatty acid profile of transgenic production strains. Fatty Acid Biosynthetic Manipulation in Microalgae Results Sequence alignment of plant TEs and comparison to CrTE To critically compare the C. reinhardtii TE sequence to activity and its structure to function, we first conducted a multiple sequence alignment between plant FatA and FatB TEs and CrTE (Figs. 1, S1). Structure-based analysis of known TEs and comparison to CrTE places it in thioesterase family TE14 along with FatA and FatB TEs [27]. Sequence alignment with plant TEs shows that CrTE contains a catalytic cysteine residue as part of a Cys-Asn-His catalytic triad (Fig. 1). FatB TEs have a conserved hydrophobic 18-amino acid domain [28], while FatA TEs contain distinctive sub-motifs in an N-terminal 60 amino acid transit peptide sequence [29]. Although ChloroP1.1 identifies a 60 amino acid plastid targeting sequence at its N-terminus (Fig. S1), CrTE is distinct from FatA and FatB TEs in this region, which has been proposed as the source for substrate specificity in TEs [9]. BLAST analysis indicates that CrTE shares the closest homology to predicted TEs from Volvox carteri and Chlorella variabilis, a multicellular chlorophyte alga and a unicellular green alga, respectively, both closely related to C. reinhardtii. An exhaustive search of medium chain-specific plant TEs for homology to the CrTE resulted in a panel of fourteen plant TEs (Text S1). Because there are currently no crystal structures available for plant or algal TEs, homology models were constructed, and TEs were screened computationally for func- tional binding to CrACP. In silico modeling of ACP-TE protein-protein interactions and TE-substrate interactions While there are no plant or algal TE structures deposited in the PDB, two crystal structures (PDB: 2OWN and 2ESS) display homology and high sequence identity (37.9% and 41.4%, respectively) to CrTE around the active site residues Cys-His- Asn (8 A˚). The modeled CrTE obtained from two different homology algorithms are nearly superimposable and in line with previous modeled TE structures including those from Arabidopsis [30] and Jatropha [29]. To investigate the binding mode of a fatty acid by CrTE and its substrate specificity, the modeled CrTE was subjected to non-biased docking of a stearate-pantetheine substrate (Fig. S2). Consistently, the calculated top 10 models display the model with pantetheine substrates bound to CrTE in its binding tunnel (Fig. 2C) with the thioester exposed to the tentative active site. Experimentally observed fatty acyl substrates (14:0, 16:0, 16:4, 18:0, 18:1 and 18:3) were docked onto the model structure of CrTE and all fatty acids dock to the binding tunnel of CrTE (Fig. S2). However, the 16:4 and 18:3 substrates do not bind in a favorable orientation, as expected since desaturations are typically derived from post-synthase desaturase enzymes. C. reinhardtii chloroplastic ACP (Cr-cACP) was modeled and docked onto TEs using the Cluspro server (Fig. 2). We generated the homology model of Cr-cACP based on the template of the ACP from Aquifex aeolicus (PDB: 2EHTA), which shows a sequence identity of 52.6%, a q-meanscore of 0.7, and a residual RMSD of 0.08 between template and model. Models were compared with those obtained from the i-Tasser server [31] and no significant deviations between the two homology modeling algorithms were observed. The models generated from protein-protein docking were examined for binding of apo-ACP close to the TE active site and for juxtaposition of the ACP serine in relation to the TE active site cysteine. Docking of Cr-cACP to CrTE displayed large surface interactions between helix II of Cr-cACP and the CrTE as well as a productive orientation of the ACP serine to TE cysteine (Fig. 2A), consistent with previous studies of protein-protein interactions PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e42949