Title: Crystal structure of pyruvate dehydrogenase kinase 3 bound to lipoyl domain 2 of human pyruvate dehydrogenase complex
Abstract: Article28 April 2005free access Crystal structure of pyruvate dehydrogenase kinase 3 bound to lipoyl domain 2 of human pyruvate dehydrogenase complex Masato Kato Masato Kato Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Jacinta L Chuang Jacinta L Chuang Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Shih-Chia Tso Shih-Chia Tso Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author R Max Wynn R Max Wynn Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author David T Chuang Corresponding Author David T Chuang Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Masato Kato Masato Kato Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Jacinta L Chuang Jacinta L Chuang Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Shih-Chia Tso Shih-Chia Tso Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author R Max Wynn R Max Wynn Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author David T Chuang Corresponding Author David T Chuang Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Author Information Masato Kato1, Jacinta L Chuang2, Shih-Chia Tso2, R Max Wynn1,2 and David T Chuang 1,2 1Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA 2Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA *Corresponding author. Departments of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9038, USA. Tel.: +1 214 648 2457; Fax: +1 214 648 8856; E-mail: [email protected] The EMBO Journal (2005)24:1763-1774https://doi.org/10.1038/sj.emboj.7600663 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The human pyruvate dehydrogenase complex (PDC) is regulated by reversible phosphorylation by four isoforms of pyruvate dehydrogenase kinase (PDK). PDKs phosphorylate serine residues in the dehydrogenase (E1p) component of PDC, but their amino-acid sequences are unrelated to eukaryotic Ser/Thr/Tyr protein kinases. PDK3 binds to the inner lipoyl domains (L2) from the 60-meric transacetylase (E2p) core of PDC, with concomitant stimulated kinase activity. Here, we present crystal structures of the PDK3–L2 complex with and without bound ADP or ATP. These structures disclose that the C-terminal tail from one subunit of PDK3 dimer constitutes an integral part of the lipoyl-binding pocket in the N-terminal domain of the opposing subunit. The two swapped C-terminal tails promote conformational changes in active-site clefts of both PDK3 subunits, resulting in largely disordered ATP lids in the ADP-bound form. Our structural and biochemical data suggest that L2 binding stimulates PDK3 activity by disrupting the ATP lid, which otherwise traps ADP, to remove product inhibition exerted by this nucleotide. We hypothesize that this allosteric mechanism accounts, in part, for E2p-augmented PDK3 activity. Introduction The mammalian pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to give rise to acetyl-CoA, linking glycolysis to the Krebs cycle. PDC is a member of the highly conserved mitochondrial α-ketoacid dehydrogenase complexes comprising the PDC, the branched-chain α-ketoacid dehydrogenase complex (BCKDC), and the α-ketoglutarate dehydrogenase complex (Reed et al, 1985; Roche and Patel, 1989; Reed, 2001). PDC is a 9 × 106-Da macromolecular machine organized around a 60-meric transacetylase (E2p) core. To the E2p core, multiple copies of pyruvate dehydrogenase (E1p), dihydrolipoamide dehydrogenase (E3), E3-binding protein (E3BP), pyruvate dehydrogenase kinase (PDK), and pyruvate dehydrogenase phosphatase (PDP) are attached (Reed, 2001). The co-localization of these components greatly enhances the efficiency of multi-step reactions, with the lipoyl domain of E2p participating in most of the reaction steps. Precursors for subunits of these enzyme components are encoded by nuclear genes and imported into the mitochondrial matrix, where they are processed and assembled into PDC (De Marcucci et al, 1988; Lindsay, 1989). PDC in eukaryotes is regulated by reversible phosphorylation (Reed et al, 1985; Harris et al, 2001; Holness and Sugden, 2003). The phosphorylation of serine residues in E1p by PDK results in the inactivation of PDC, whereas the dephosphorylation by PDP restores its activity. To date, four PDK (1–4) isoforms in mitochondria have been identified (Popov et al, 1997). Phosphorylation of E1p occurs at three serine residues (S264: site 1, S271: site 2, and S203: site 3) (Yeaman et al, 1978; Teague et al, 1979; Sale and Randle, 1981). Although phosphorylation of each site alone inactivates PDC, site 1 is most rapidly phosphorylated and the phosphorylation of site 3 is the slowest among the three sites (Sale and Randle, 1981; Korotchkina and Patel, 1995). Interestingly, each PDK isoform exhibits different site specificity. Using E1p mutants with single-functional phosphorylation sites, it was shown that all the four isoforms phosphorylate sites 1 and 2, but only PDK1 modifies site 3 (Kolobova et al, 2001; Korotchkina and Patel, 2001). PDK isoforms exhibit tissue-specific expression; PDK1 is detected in the heart, pancreatic islets, and skeletal muscles; PDK2 is expressed in all tissues; PDK3 is present in the testes, kidney, and brain; PDK4 is abundant in the heart, skeletal muscle, kidney, and pancreatic islets (Bowker-Kinley et al, 1998). The expression of PDK2 and PDK4 is induced in starvation and diabetes, which is reversed by insulin treatment (Wu et al, 1998; Harris et al, 2001). Impaired insulin-induced downregulation of PDK4 (due to the lack of insulin or insensitivity to insulin) leads to the overexpression of PDK4 and shuts off glucose oxidation in diabetic animals (Holness and Sugden, 2003; Roche et al, 2003). Therefore, PDK4 is a potential drug target for the treatment of type II diabetes. PDKs form dimers as the biologically functional unit (Bowker-Kinley et al, 1998; Baker et al, 2000; Korotchkina and Patel, 2001). PDK dimers are recruited to the PDC by preferentially binding to the inner lipoyl (L2) domain of the E2p core (Liu et al, 1995). Binding of L2 to PDKs requires the covalently attached lipoyl group at the lysine 173 of L2 (Radke et al, 1993). Reduction or acetylation of the lipoyl group significantly increases the affinity of L2 for PDKs compared to L2 containing an oxidized lipoyl group (Baker et al, 2000; Roche et al, 2003). The activity of PDKs is stimulated upon binding to E2p, but the response of different isoforms to isolated L2 varies. PDK3 is robustly activated by E2p, and the majority of this activation can be achieved by isolated L2 (Baker et al, 2000; Roche et al, 2003). In contrast, PDK2 activity is augmented by binding to E2p (Baker et al, 2000) or a di-domain (Tuganova and Popov, 2005) consisting of both the L2- and E1p-binding domains, but not binding to L2 alone. Individual isoforms exhibit different binding affinities for L2 with PDK3>PDK1=PDK2>PDK4 (Tuganova et al, 2002). The mammalian PDC contains substoichiometric number of PDK dimers relative to the 20–30 E1p molecules bound to the E2p core (Radke et al, 1993). For efficient phosphorylation of E1p molecules by PDKs, a hand-over-hand model for kinase movements has been proposed (Liu et al, 1995; Roche et al, 2003). According to this model, the PDK dimer moves along the surface of the E2p 60-meric core by repeated dissociation and association, in a relay fashion, with L2 domains from different constitutive E2p monomers. This mechanism presumably enables the limited copies of PDK molecules to phosphorylate the 20–30 copies of E1p molecules bound to the E2p scaffold. Mitochondrial protein kinases comprising PDKs and the related BCKD kinase (BCK) constitute a novel family of protein kinases, in which motifs that are normally present in eukaryotic Ser/Thr/Tyr kinases (Hanks et al, 1988) were not found. Structural studies of rat PDK2 and rat BCK have revealed that these kinases consist of two distinct domains (Machius et al, 2001; Steussy et al, 2001). The C-terminal domain (or K domain in BCK) is an α/β structure with a five-stranded β-sheet, and this fold is shared between the members of the GHKL ATPase/kinase superfamily (http://scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.e.bff.b.html) that includes DNA Gyrase B (Wigley et al, 1991), Hsp90 (Prodromou et al, 1997), histidine kinases including CheA (Bilwes et al, 1999) and EnvZ (Tanaka et al, 1998), as well as MutL (Ban and Yang, 1998). Members of this superfamily share four conserved motifs (N-, G1-, G2-, and G3-boxes), which build a unique ATP-binding fold (Alex and Simon, 1994; Bergerat et al, 1997; Smirnova et al, 1998; Dutta and Inouye, 2000). This fold includes a common structural element known as the 'ATP lid', whose conformational change is coupled to both ATP hydrolysis and protein–protein interactions (Wigley et al, 1991; Ban et al, 1999; Machius et al, 2001). The ATP lid is conserved in the PDK2 and BCK structures. In the PDK2–ADP structure, the ATP lid is partially ordered, whereas in BCK the lid is more ordered in the ADP-bound form than in the ATP-bound structure. The N-terminal domain of PDK2 (or B domain in BCK) consists of a four-helix bundle that resembles the histidine phosphoryl-transfer (HPt) domains of the two-component systems. The HPt domains contain histidine residues for forming phospho-histidine intermediates in a phospho-relay system. However, the phosphoryl-transfer mechanism in mitochondrial protein kinases is distinct and does not involve a histidine residue in the four-helix bundle domain (Davie et al, 1995; Tovar-Mendez et al, 2002). The structural basis for the upregulation of PDKs through binding to E2p is unknown. To address this problem, we produced a stable PDK3–L2 complex, based on the high affinity of PDK3 for the L2 domain, and determined the crystal structures of this complex. Our structural data show a novel mode of L2 binding to PDK3, which induces significant conformational changes in PDK3. We show that these conformational changes markedly reduce the affinity of PDK3 for ADP to mitigate product inhibition by this nucleotide. We suggest that this allosteric mechanism explains, in part, the stimulation of PDK3 activity by E2p binding. Results and discussion L2 binding markedly stimulates the kinase activity of PDK3 There are inconsistencies in the literature regarding the magnitude of L2 domain-stimulated PDK3 activity. The study by the Roche group showed that the L2 domain and E2p stimulate human PDK3 activity by 12.8- and 17-fold, respectively (Baker et al, 2000). These authors concluded that L2 binding is the major determinant for stimulated PDK3 activity and that the small additional activation by E2p over L2 is imparted by the co-localization of the kinase and substrate E1p to E2p. In contrast, the Popov group showed that PDK3 activity is activated by 3- and 38-fold, respectively, by the L2 domain and E2p (Tuganova et al, 2002). To resolve this discrepancy, the kinase activity of human PDK3 was measured by E1p phosphorylation in the absence and presence of a lipoylated L2 construct (residues 126–233, designated as L2), with an intrinsic C-terminal linker (residues 209–233) identical to that used by the Roche group. A maltose-binding protein (MBP) was N-terminally linked to the kinase to produce a soluble MBP-fused PDK3 (MBP–PDK3) fusion protein. The presence of the MBP tag has no effect on the enzymatic properties of the related rat BCK (Davie et al, 1995; Wynn et al, 2000). As shown in Figure 1A and B, at saturating L2 concentrations (20–30 μM), PDK3 activity is stimulated by 11.9-fold, whereas at the 40 nM concentration of E2p used previously (Tuganova et al, 2002), PDK3 activity is maximally stimulated by 16.4-fold. Thus, the fold increases of PDK3 activity stimulated by L2 and E2p in the present study are in good agreement with those reported previously by the Roche group. The significantly lower E2p concentration required for maximally stimulated PDK3 activity presumably results, in part, from the multiple copies of L2 domain present in the 60-meric E2p core. Figure 1.Stimulation of PDK3 kinase activity by the L2 domain and E2p. The kinase activity of MBP–PDK3 (45 nM) was measured by E1p phosphorylation in three independent experiments: (A) in the absence and presence of increasing concentrations (1–30 μM) of the lipoylated L2 domain (residues 126–233, designated as L2) (open bars), (B) in the absence and presence of 40 nM lipoylated E2p/E3BP 60-meric core (shaded bars), and (C) in the absence and presence of L2 (open bar) and a lipoylated L2s construct (residues 126–214) with a shorter C-terminal linker than L2 (solid bars), as described in 'Materials and methods'. The basal (one fold) PDK3 activities in (A), (B), and (C) are similar at 21 nmol 32P incorporated/min/mg PDK3. To calculate the molar concentration of E2p, a molecular mass of 3 458 544 Da for lipoylated E2p/E3BP was used, according to a molecular ratio of E2p:E3BP=48:12 in the 60-meric E2p/E3BP core (Hiromasa et al, 2004). The ordinates in (B) and (C) are the same as in (A). Download figure Download PowerPoint In a separate experiment, PDK3 activity was measured in the absence and presence of an L2 construct (designated as L2s) containing the shorter C-terminal linker (residues 209–214) studied by the Popov group or the above L2 construct used in crystallography. With these two L2 constructs, we obtained three- and 13.3-fold stimulation, respectively, of PDK3 activity (Figure 1C). Our results, therefore, establish that the significantly smaller L2-stimulated PDK3 activity reported by the Popov group is due to the shorter L2 C-terminal linker they used. It follows that the length of the C-terminal linker is critical for the augmentation of PDK3 activity by the L2 domain. In BCK, we have shown that a long C-terminal linker (residues 85–99) of its cognate lipoyl domain is also essential for efficient binding to this kinase (Chuang et al, 2002). However, other than stabilization effects on L2 (Gong et al, 2000), the role of the C-terminal linker in PDK3 activation is not clear, since this region does not appear to interact with PDK3 in the present PDK3–L2 structure (see below). Overall structure of the PDK3–L2 complex We determined three crystal structures of the PDK3–L2 complex (L2 residues 126–233), either without (apo) or with bound nucleotides (ATP or ADP) (Table I). These structures are virtually identical (RMSD: less than 0.55 Å for over 370 Cα atoms), except for the ATP lid (residues 305–327). The refined PDK3 structure is similar to the published PDK2–ADP structure in the absence of L2 (RMSD: 1.07 Å for 321 Cα atoms); however, there are small, yet significant, conformational differences apparently caused by L2 binding. The structure of the cognate BCK is also similar to PDK3 (RMSD: 1.4 Å for 277 Cα atoms), despite low sequence identity (23%) between these two kinases. Table 1. Data collection and refinement statistics of PDK3–L2 structures apo +ADP +ATP Data collectiona Space group P6522 P6522 P6522 Unit cell (Å) a=b 120.81 120.75 120.90 c 238.59 239.15 240.09 Wavelength (Å) 1.54 1.0 1.0 Resolution (Å) 2.60 2.48 2.63 Measurements 316129 422583 231967 Unique reflections 32529 37324 31079 Completeness (%) 100 (100) 99.8 (100) 98.6 (97.2) R-merge (%)b 5.3 (54.7) 3.8 (54.6) 5.9 (54.2) 〈I〉/〈σ(I)〉 28.7 (3.9) 54.1 (3.5) 30.0 (2.1) Multiplicity 9.7 (8.7) 11.3 (9.1) 7.5 (5.1) Refinementa No. of reflections (work/test) 30805/1645 35738/1494 29477/1566 No. of atoms (Mean B value (Å2)) Protein 3803 (62.3) 3803 (64.2) 3849 (66.6) Solvents 53 (46.6) 67 (52.5) 56 (52.3) Nucleotide — 27 (74.4) 31 (45.7) Hetero compound 12 (72.5) 14 (59.0) 14 (66.7) R-work (%)c 21.0 (33.8) 21.0 (38.9) 20.4 (40.7) R-free (%)c 24.8 (38.7) 23.4 (39.9) 23.0 (47.8) RMSD Bond length (Å) 0.016 0.019 0.015 Bond angle (°) 1.700 1.685 1.731 Ramachandran plot Most favored (%) 90.4 91.4 90.3 Allowed (%) 9.6 8.6 9.7 Disallowed (%) 0 0 0 a Values in parentheses refer to data in the highest resolution shell unless otherwise indicated. b R-merge=∑hkl ∑j∣Ij–〈I〉∣/∑hkl ∑j Ij, where 〈I〉 is the mean intensity of j observations from a reflection hkl and its symmetry equivalents c R-work=∑hkl∣∣Fobs∣–k∣Fcalc∣∣/∑hkl∣Fobs∣; R-free=R-work for 4–5% of reflections that were omitted from refinement In the present PDK3 structure, the N-terminal domain comprises residues 12–178 and 366–381, and the C-terminal domain encompasses residues 182–365 (Figure 2A). Structures of these domains are essentially identical to the corresponding domains in PDK2, except that certain disordered regions in PDK2 are ordered in PDK3–L2. One of the ordered regions is the loop between helices α2 and α3 of the N-terminal domain in PDK3. The lipoyl-lysine residue of L2 binds to this region, which is part of the lipoyl-binding pocket, to stabilize the loop conformation. The C-terminal region, which is not visible in PDK2, is ordered in PDK3–L2 (Figure 2A). This region joins the N-terminal domain with the formation of helix α13, and projects out from that domain in an extended conformation. The extended segment (residue 382–401), which is designated as the C-terminal tail, is critical to L2 binding (see below). Figure 2.Monomeric and dimeric structures of the PDK3–L2 complex. (A) The monomeric structure of the PDK3–L2 complex is shown as ribbon models (PDK3 in green, L2 in yellow). The lipoyl-lysine residue of L2 (residues 126–233) bound to the N-terminal domain and ATP bound to the C-terminal domain are shown as space-filling models. The extended C-terminal tail is colored in red. The secondary structures of PDK3 and L2 are labeled as follows: α and A—alpha helix, β and B—beta strand, γ—310 helix. The first 310-helix of PDK3 (γ1), which is not labeled, is located between helices α6 and α7. (B) Stereo view of the dimeric structure of the PDK3–L2 complex with the same view angle as that of the left figure in (A). The C-terminal tails from subunits I (cyan) and II (green) of the PDK3 dimer are highlighted in purple and red, respectively. (C) Each subunit of the PDK3 dimer is shown in molecular-surface representation with the same color configuration as in (B). The two bound L2 (yellow) are shown as ribbon models with L2 bound to subunit I and L2′ to subunit II. The lipoyl-lysine residue of L2 is shown as a space-filling model. In the left panel, the extended C-terminal tail (red) from subunit II crosses over with the equivalent (purple) from subunit I to interact with both its N-terminal domain and L2 bound to subunit I. In the right panel, the arrows point to the active-site clefts between the N- and C-terminal domains of each subunit. The bound ATP is colored in blue. Download figure Download PowerPoint The L2 domain is a member of the biotinyl/lipoyl-carrier protein superfamily (http://scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.c.baf.b.b.html). In the complex, L2 adopts a β-barrel structure comprising two antiparallel β-sheets (Figure 2A). This fold is similar, but not identical, to the NMR structure previously reported (RMSD: 2.1 Å for 65 Cα atoms) (Supplementary Figure S1B) (Howard et al, 1998). The crystal structure of L2 shows a helix (A1) in the C-terminal region, whereas in the NMR structure this region is in an extended conformation. The lipoyl-lysine residue at position 173 is located in a short loop connecting strands β4 and β5. The long side chain of the lipoyl-lysine protrudes into its binding pocket in the N-terminal domain of PDK3 (Figure 2). Each asymmetric unit contains one PDK3 monomer bound to one L2 (Figure 2A), with two symmetry-related monomers forming a functional dimer (Figure 2B and C) (Bowker-Kinley et al, 1998; Baker et al, 2000; Korotchkina and Patel, 2001). This result is consistent with the presence of PDK3 dimers in solution, as indicated by analytical ultracentrifugation (Hiromasa and Roche, 2003) and gel filtration (JL Chuang, unpublished data, 2004). The dimer formation of PDK3 is achieved by extensive interactions in a head-to-tail fashion between two β-sheets each from the C-terminal domains of the two subunits. This dimerization mode is similar to that described for PDK2 (Steussy et al, 2001) and BCK dimers (Machius et al, 2001). The PDK3 dimer binds two L2 with no interactions observed between these L2 domains (Figure 2C, left). Remarkably, the extended C-terminal tail from PDK3 subunit II crosses over to subunit I and interacts with L2 bound to subunit I, becoming an integral part of the lipoyl-binding pocket in the latter subunit. The present structure is consistent with earlier biochemical studies that indicated the subunit stoichiometry of PDK3:L2=1:1 (Tuganova et al, 2002; Hiromasa and Roche, 2003). Isothermal titration calorimetry (ITC) was performed to dissect interactions between MBP–PDK3 and lipoylated L2 (Figure 3). The binding isotherm gives rise to a dissociation constant (Kd) of 1.17±0.23 μM for L2 binding to PDK3. This constant is 10-fold lower than those for L2 binding to PDK1 and PDK2 previously determined by gel filtration (Tuganova et al, 2002). The close to unity (0.74) subunit stoichiometry (PDK3 monomer: L2 monomer) and the iterative-best-fit monophasic binding isotherm indicate that both L2-binding sites within the PDK3 dimer are equivalent. Figure 3.Binding of L2 domain to wild-type and C-terminally truncated PDK3. The binding of lipoylated L2 to wild-type and C-terminally truncated (Δ392–406) MBP–PDK3 was measured by ITC as described in 'Materials and methods'. The upper panel shows the raw isothermal data for the titration of L2 against the truncated and wild-type MBP–PDK3 (upper and lower data, respectively). The lower panel represents the binding isotherms versus the molar ratio of L2 monomer: MBP–PDK3 monomer (•, wild-type MBP–PDK3; ○, truncated MBP-PDK3). The solid line in the lower panel depicts the fitting of the binding isotherm, based on a single type of the L2-binding site using ORIGIN software package. The wild-type MBP–PDK3 shows a dissociation constant (Kd) of 1.17±0.23 μM (n=3) for L2; for the truncated MBP–PDK3 (Δ392–406), there is no detectable L2 binding. Download figure Download PowerPoint The C-terminal domain of PDK3 binds ATP or ADP through conserved residues in the common motifs of the GHKL superfamily (Dutta and Inouye, 2000). The ATP-binding site is located on the sidewall of the C-terminal domain facing the cleft between the N- and C-terminal domains (Figure 2A). The γ-phosphate of bound ATP is accessible to the E1p substrate from within the cleft, which is likely to be the active site for the phosphoryl-transfer reaction. Based on previous studies with PDK2 (Tuganova et al, 2001), the invariant D247 in PDK3 located in the active-site cleft presumably serves as the general base for the activation of phosphorylatable serine residues in E1p. The bound L2 in the N-terminal domain of subunit I is located more than 30 Å away from the active-site cleft of this subunit, and is at more than 23 Å distance from the opposing active-site cleft in subunit II (Figure 2B and C). Thus, there appears no direct interaction between the bound L2 and either of the active-site clefts, suggesting that L2 exerts its effects on PDK3 through an allosteric mechanism. Extensive interactions exist between PDK3 and L2 Figure 4A shows the 619 Å2 surface area in the N-terminal domain of PDK3 subunit I that constitutes the binding interface with L2. Superimposition of electrostatic potential surfaces on PDK3 subunit I and L2 (Figure 4C) with interacting surfaces on the corresponding subunit/domain (Figure 4A) reveals two types of interactions: (1) hydrophobic interactions in the lipoyl-binding pocket and its surrounding regions, and (2) electrostatic interactions in the lower part of the N-terminal domain including helix α13. In the PDK2 structure without L2, the C-terminal region comprising α13 is largely disordered (Steussy et al, 2001); therefore, the formation of helix α13 in the complex structure is apparently induced by L2 binding. Figure 4.Interaction surfaces between PDK3 and L2. Interfaces between PDK3 and L2 are shown in molecular surface representations. L2 is separated from PDK3 and rotated by 180° to show the binding interfaces on both proteins. (A) A single L2 binds to the N-terminal domain of PDK3 subunit I. The corresponding interfaces are in green. (B) The C-terminal tail of PDK3 subunit I binds to L2′ in complex with subunit II (cf. Figure 2A). Binding surfaces between the tail and L2′ are in navy blue. (C) The electrostatic potential surfaces of PDK3 and L2 were calculated with APBS based on the Poisson–Boltzmann equation (Baker et al, 2001). Positive-charged regions are shown in blue, and negative-charged regions in red. The interfaces between the N-terminal domain of PDK3 and L2 (cf. A) consist of hydrophobic interactions in the lipoyl-binding pocket and its surrounding regions as well as electrostatic interactions on the lower portion of the N-terminal domain. Conserved residues in human PDKs and the lipoyl domains of human PDC are indicated. Download figure Download PowerPoint The hydrophobic interactions occur mainly in the lipoyl-binding pocket in PDK3. The pocket is formed by the loop between helices α1 and α2 as well as helices α2 and α3, with helix α8 serving as the backwall (Figure 5). These structural components confer a 9-Å deep cylindrical pocket. The long aliphatic side chain of the lipoyl-lysine residue in L2 bends approximately 60° at the C4 atom, allowing the lipoamide group to protrude into the lipoyl-binding pocket. Hydrophobic residues L27, F32, F35, F48, L49, L164, L171, and F172, which are highly conserved among human PDKs (Supplementary Figure S1), constitute the inner lining of the pocket and appear to be essential for hydrophobic interactions with the aliphatic side chain of lipoamide. In particular, the reduced dithiolane moiety of lipoamide (Figure 5, inset) is sandwiched between the invariant F35 and F48. Crystallization of the PDK3–L2 complex was carried out in the presence of 10 mM DTT; at this DTT concentration the oxidized form of lipoamide is readily reduced (Bao et al, 2004a; Hiromasa et al, 2004); this apparently facilitated the crystallization of the PDK3–L2 complex. The reduced form of lipoamide in L2 has higher affinity for PDKs than its oxidized counterpart, as a feedback inhibitory mechanism in the PDC (Baker et al, 2000; Roche et al, 2003; Bao et al, 2004b). Hydrophobic residues in the lipoyl-binding pocket are also conserved in the related BCK (Supplementary Figure S1). The conservation of these residues strongly suggests that the recognition of the lipoyl-lysine residue by PDKs and BCK is similar. Surrounding the lipoyl-binding pocket in PDK3, highly conserved residues F22, P26, and V55 as well as methylene groups of the K51 side chain contribute to interactions with highly conserved hydrophobic residues in L2 (L140, P142, A174, and I176) (Supplementary Figures S1 and S2). The structural data explain the preliminary findings that substitutions of L140 and A174 of L2 both remove 80% of L2-stimulated PDK3 activity (Roche et al, 2003). Figure 5.Structure of the hydrophobic lipoyl-binding pocket in PDK3. The lipoyl-binding pocket is formed by a loop region between helices α1 (not shown) and α2, as well as helices α2, α3, and α8. These regions form a cylindrical pocket containing conserved hydrophobic residues (brown). The lipoyl-lysine residue (yellow) projects into the hydrophobic pocket. The final 2Fo−Fc electron density (blue, contoured at 1σ) is superimposed on the lipoyl-lysine residue. The inset shows a different view of the bound reduced dihydrolipoamide. The C-terminal tail (red) from the other PDK3 subunit is integrated into the lipoyl-binding pocket. The first and last residue numbers of the C-terminal tail are indicated. Download figure Download PowerPoint The second interface between the N-terminal domain of PDK3 and L2 consists of electrostatic interactions. Basic residues R21 (α1), K374, and R378 (α13), which are highly conserved in human PDKs (Supplementary Figure S1), are located underneath the lipoyl-binding pocket (Figure 4C). This basic region interacts with an a