Firefly Luciferase (FLuc)
Created by Richard Vargo
Firefly luciferase (FLuc) (PDB ID: 3IES) is an ATP-hydrolyzing, decarboxylating 4-oxidoreductase that needs luciferin, ATP, oxygen, and Mg2+ for the bioluminescence reaction (Marques & Esteves da Silva, 2008). FLuc has one subunit. The 551-residue firefly luciferase enzyme has a molecular weight of 60.844 kDa (Gasteiger et al, 2005). Its isoelectric point is 6.42 (Gasteiger et al., 2005).
The main function of FLuc is bioluminescence. Because of this bioluminescence, FLuc is a reporter enzyme that requires the presence of ATP and is broadly used in chemical biology and drug discovery assays (Auld et al., 2010). Specifically, luciferase is often used as a reporter enzyme in cell-based gene expression assays (Fan & Wood, 2007). The bioluminescence of the reaction catalyzed by the enzyme makes it ideal for such assays, as it can be measured and quantified. Intracellularly, this is a bimolecular reaction between ATP and D-luciferin with the FLuc serving as the catalyst (Auld et al., 2008). It is this reaction that ultimately gives off the distinctive flash of light that makes Photinus pyralis, the source of the enzyme, a bioluminescent organism.
FLuc was the first enzyme in the superfamily of adenylate-forming enzymes for which the X-ray structure was determined (Conti, Franks, & Brick, 1996). The enzyme has two domains that are linked by a flexible hinge region, and the N-terminal and C-terminal domains both contain alpha + beta structure (Conti et al., 1996). The large N-terminal domain consists of residents 4-436, and the secondary structure present in this domain include a distorted antiparallel beta-barrel and two beta-sheets, which are flanked on either side by alpha-helices (Conti et al., 1996). The C-terminal domain of the protein consists of residues 440-544 (Conti et al., 1996). This domain is not shown in the 3D representation of FLuc. The entire protein contains fifteen alpha-helices-- twelve are in the N-terminal domain and only three are in the smaller C-terminal domain--and five beta-sheets named A-E, of which A,B, and C are in the N-terminal domain and D and E are in the C-terminal domain (Conti et al., 1996).
In the large N-terminal domain, the two beta-sheet subdomains are assembled to form a five-layered alpha-beta-alpha-beta-alpha tertiary structure (Conti et al., 1996). Thus, the two alpha-helices are located between the sheets, and the other helices are packed against the outer faces (Conti et al., 1996). In the N-terminal region, the two beta-sheets are tilted which results in the C-termini of the parallel strands being closer together. Each of the two beta-sheet subdomains is composed of eight strands, and each displays a right-handed twist (Conti et al., 1996). Conti et al. elucidate the structure of the beta-sheets A and B. Beta-sheet A includes five parallel and three antiparallel beta-strands with six associated helices, and it is constructed from a single portion of the polypeptide chain (77-222), excluding A8 and helix 12 (399-405) (Conti et al., 1996). Beta-sheet B consists of six parallel and two antiparallel beta-strands with six associated alpha-helices, and this portion of the protein arises from two non-contiguous portions of polypeptide chain, specifically segments 22-70 and 236-351 (Conti et al., 1996). The core of each beta-sheet consists of parallel strands joined to alpha-helices with standard right-handed cross-over connections, resulting in the arrangement of helices, which are antiparallel to the strands, on either side of the sheet (Conti et al., 1996).
The packing of the two beta-sheets creates a long surface groove, shaped by the C termini of the strands and by the N termini of the helices between the sheets (Conti et al., 1996). The antiparallel beta-barrel closes one end of the groove, and it is distorted in that it is formed by three distinct faces (Conti et al., 1996). Two sides each consist of three-stranded antiparallel beta-sheets with one of the beta-strands, C2, hydrogen bonding to the last strand of beta-sheet B (Conti et al., 1996). The third and final side of the beta-barrel of FLuc is formed by strands 7 and 8 of beta-sheet B and by the loop that connects these two strands (Conti et al., 1996). This packing of the barrel against the side of the two beta-sheets forms two shallow depressions on the molecule's concave surface (Conti et al., 1996). These two depressions plus the groove that arises from the packing of the three N-terminal subdomains, form a Y-shaped "valley" system on the surface of the large N-terminal domain (Conti et al., 1996). Finally, the small C-terminal domain is separated from the larger N-terminal domain by a wide cleft, and this cleft forms a "lid" over the beta-barrel and strands 7 and 8 of the beta-sheets (Conti et al., 1996). There is a distorted, flexible hinge that connects the two domains, and it is comprised of residues 436-440 (Conti et al., 1996).
The active site for the enzyme is located in the N-terminal domain near the hinge region (Conti et al., 1996). When viewing the molecule with beta-sheet A on the left, beta-sheet B on the right, and the beta-barrel at the top while looking down onto the Y-shaped "valley" system, the active site is visible at the N-terminal domain surface (Conti et al., 1996). Additionally, Conti et al. report that there are three invariant, conserved residues in the active site of FLuc and other luciferases, and these include Lys-206, Glu-344, Asp-422 (1996). Other well-conserved residues in the adenylate-forming enzyme superfamily which they note include Tyr 401, Ser-198, Tyr-340, Ser-420, Gly-421, and Glu-389 (Conti et al., 1996). The side chain of Ser-198 hydrogen bonds to the carboxylate oxygen of Glu-344(Conti et al., 1996). The Asp422 carboxylate groups hydrogen bond to the side chain of Tyr-340, a well-conserved residue (Conti et al., 1996). The hydroxyl group of Ser-420 can hydrogen bond to the backbone nitrogens of both Asp-422 and Gly-421, and Gly-421 also interacts with the carboxylate of Glu389(Conti et al., 1996). Additionally, researchers have found that Lys-529--which is not shown because it is located in the C-terminal region--is a critical residue that functions to effectively orient the substrate and to provide favorable interactions that are necessary for transition state stabilization that ultimately leads to efficient adenylate production (Branchini, Murtiashaw, Magyar, & Anderson, 2000). Moreover, when ATP binds to the enzyme, the two domains of the protein come together (Franks, Jenkins, Conti, Lieb, & Brick, 1998). However, this cannot be shown because only the N-terminal domain is featured in this 3D representation of 3IES.
The ligand for FLuc is PTC124-AMP (5'-O-[(R)-[({3-[5-(2-fluorophenyl)-1,2,4- oxadiazol-3-yl]phenyl}carbonyl)oxy] (hydroxy)phosphoryl]adenosine) (Auld et al., 2010). The planar nature of the 3,5-diaryl oxadiazole allows for an orientation in the active site of FLuc that is similar to that of its natural substrate, D-luciferin (Auld et al., 2010). Due to the heterocyclic structure of D-luciferin, other small, heterocyclic compounds of low molecular weight can inhibit the enzyme (Auld et al., 2008). Auld et al. also report that the PTC124-AMP substrate molecule represents a structural mimicry of the luciferyl-AMP intermediate, often denoted LH2-AMP (2010). While conducting an assay, researchers discovered that the chemical PTC124 is a strong inhibitor of FLuc activity (Auld et al., 2010). However, this chemical also serves another unique but contradictory function. PTC124 and its analogs actually increase cellular FLuc activity levels by posttranslational stabilization (Auld et al., 2010). In particular, this increase is accomplished by ligand-induced stabilization which offsets the short, three-to-four hour half-life of FLuc (Thompson et al., 1997).
There are many residues that are important for the enzyme firefly luciferase in the PTC124-inhibited structure. Auld et al. report that the PTC124-AMP molecule fully occupies the active site of FLuc (2010). The AMP forms six hydrogen bonds with luciferase active site residues (Auld et al., 2010). The PTC124-AMP molecule makes critical hydrogen bonds with several invariant FLuc active site residues, including the conserved Asp-422 (Auld et al, 2010). Other residues which hydrogen bond with the PTC124-AMP adduct include His-245, Gly-316, Gly-339, Leu-319, and Thr-343 (Auld et al., 2010). The diaryl oxadiazole group of the PTC124 portion is positioned in a hydrophobic pocket near Ala-348 and Ile-351, and the PTC124 portion also shows aromatic stacking with Phe-247 (Auld et al., 2010). When the ligand binds, there is movement of the loop between residues Ser-314 and Leu-319 which allows for ligand binding, and this is the main structural difference between the apo form of FLuc (3IEP) and the FLuc-ligand complex (Auld et al., 2010).
Finally, FLuc functions to bring the "near attack conformers" (NACs) of the two reactants, PTC124 and ATP, within van der Waals distance allowing bond formation (Auld et al., 2010). The m-isomer of PTC124 provides the optimal NAC, and this explains the reactivity of the meta-carboxylate of PTC124 (Auld et al., 2010). The high-affinity binding of PTC124 to FLuc arises from the enzyme-catalyzed formation of a multisubstrate adduct inhibitor (MAI) through an important meta-carboxylate group on PTC124 (Auld et al., 2010). The PTC124-AMP adduct binds to the FLuc active site with a KD of 120 pM (Auld et al., 2010). Ultimately, FLuc catalyzes the reaction between ATP, its natural substrate, and PTC124 to form the inhibitor PTC124-AMP (Auld et al., 2010). This is similar to the fact that the oxidation of the intermediate LH2-AMP forms L-AMP, a strong FLuc inhibitor, and this is a rare example of an enzyme catalyzing the formation of its own inhibitor (Auld et al., 2010).