FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2099202202> ?p ?o ?g. }

• W2099202202 endingPage "5994" @default.
• W2099202202 startingPage "5983" @default.
• W2099202202 abstract "Article23 November 2006free access A synchronized substrate-gating mechanism revealed by cubic-core structure of the bovine branched-chain α-ketoacid 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 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 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 Chad A Brautigam Chad A Brautigam Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Myra Custorio Myra Custorio 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 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 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 Chad A Brautigam Chad A Brautigam Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Myra Custorio Myra Custorio 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, R Max Wynn1,2, Jacinta L Chuang2, Chad A Brautigam2, Myra Custorio2 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. Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA. Tel.: +1 214 648 2457; Fax: +1 214 648 8856; E-mail: [email protected] The EMBO Journal (2006)25:5983-5994https://doi.org/10.1038/sj.emboj.7601444 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The dihydrolipoamide acyltransferase (E2b) component of the branched-chain α-ketoacid dehydrogenase complex forms a cubic scaffold that catalyzes acyltransfer from S-acyldihydrolipoamide to CoA to produce acyl-CoA. We have determined the first crystal structures of a mammalian (bovine) E2b core domain with and without a bound CoA or acyl-CoA. These structures reveal both hydrophobic and the previously unreported ionic interactions between two-fold-related trimers that build up the cubic core. The entrance of the dihydrolipoamide-binding site in a 30-Å long active-site channel is closed in the apo and acyl-CoA-bound structures. CoA binding to one entrance of the channel promotes a conformational change in the channel, resulting in the opening of the opposite dihydrolipoamide gate. Binding experiments show that the affinity of the E2b core for dihydrolipoamide is markedly increased in the presence of CoA. The result buttresses the model that CoA binding is responsible for the opening of the dihydrolipoamide gate. We suggest that this gating mechanism synchronizes the binding of the two substrates to the active-site channel, which serves as a feed-forward switch to coordinate the E2b-catalyzed acyltransfer reaction. Introduction The mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC), pyruvate dehydrogenase complex (PDC) and 2-oxoglutarate dehydrogenase complex (OGDC) are a family of highly conserved macromolecular catalytic machines (Reed and Hackert, 1990; Reed, 2001). These complexes catalyze multistep reactions (Reaction 1) that lead to the oxidative decarboxylation of α-ketoacids giving rise to acetyl-CoA, acyl-CoA or succinyl-CoA. These products are subsequently utilized in the Krebs cycle. Inherited defects in these complexes produce severe metabolic diseases in humans; for example, maple syrup urine disease (MSUD) involves defective BCKDC (Chuang and Shih, 2001) and lactic acidemia results from mutated PDC (Robinson and Sherwood, 1984). Both metabolic disorders are manifested by often-fatal α-ketoacidosis, neurological derangement and mental retardation. In addition, the association of OGDC with Alzheimer's disease and Parkinson's disease has been reported (Mizuno et al, 1994; Kish, 1997). The core components of α-ketoacid dehydrogenase complexes are the major autoantigens in primary biliary cirrhosis (Yeaman et al, 2000). The BCKDC is a 4-MDa catalytic machine organized around a core scaffold consisting of dihydrolipoamide acyltransferase (E2b, with ‘b’ for branched-chain) subunits, to which multiple copies of branched-chain α-ketoacid dehydrogenase complex (E1b) and dihydrolipoamide dehydrogenase (E3) are noncovalently tethered (Pettit et al, 1978). This architectural design is conserved in PDC and OGDC with specific pyruvate dehydrogenase (E1p) and acetyltransferase (E2p) for the former complex and 2-oxoglutarate dehydrogenase (E1o) and succinyltransferase (E2o) for the latter (Reed, 2001). When examined by electron microscopy, the E2b of BCKDC and the E2o of OGDC from all species, and the E2p of PDC from Gram-negative bacteria exhibit a 24-meric cubic core, whereas the E2p's of PDC from Gram-positive bacteria, fungi and mammals show a 60-meric dodecahedral scaffold (Oliver and Reed, 1982). In the eukaryotic BCKDC, two regulatory enzymes, that is, the BCKD kinase (Chuang et al, 2002) and presumably the BCKD phosphatase also bind to the E2b core, which control BCKDC activity by reversible phosphorylation (Harris et al, 1997). The E2b subunit of BCKDC consists of an N-terminal lipoyl-bearing domain (LBD), an internal subunit-binding domain (SBD) and a C-terminal core (catalytic) domain (E2bCD) (Griffin et al, 1988; Lau et al, 1991). The E2bCD possesses a catalytic site for the acyltransfer reaction and is responsible for the formation of the cubic core. The LBD and SBD are tethered to the E2bCD through flexible linker regions with E1b and E3 binding to the same SBD in a mutually exclusive manner (Lessard et al, 1996). In BCKDC, as in PDC and OGDC, LBD plays an important role in connecting distinct reaction steps catalyzed by the E1b, E2b and E3 components. A lipoylamide moiety covalently attached to LBD (Lip-LBD) of E2b serves as a ‘swinging arm’ to extract an acyl group from the E1b active site. The resultant S-acyldihydrolipoamide moiety on LBD is transferred to the E2b active site (the ‘swinging arm’ active-site coupling mechanism) (Koike et al, 1963; Perham, 2000), where the acyl group is conjugated with CoA, producing acyl-CoA by the reversible E2bCD-catalyzed acyltransfer reaction (in the forward direction of Reaction 2). Finally, the reduced Lip-LBD visits the E3 active site, where the dihydrolipoamide [Lip(SH)2] moiety on LBD is reoxidized, with NAD+ as the ultimate electron acceptor. High-resolution crystal structures of full-length E2's have not been deciphered presumably owing to the flexibility of the linker segments. However, a significant amount of structural information has been obtained for individual domains of E2. The NMR structures of LBD and SBD have been reported (Perham, 2000; Reed, 2001). The crystal structures of cubic E2CD cores from Azotobacter vinelandii PDC (E2pCD) and Escherichia coli OGDC (E2oCD) as well as dodecahedral E2pCD cores from Bacillus stearothermophilus and Enterococcous faecalis PDCs have been determined (Mattevi et al, 1992; Knapp et al, 1998; Izard et al, 1999). These E2CD structures corroborated the proposition, based on sequence similarities, that the topology of E2CD is analogous to that of E. coli chloramphenicol acetyltransferase (CAT) (Guest, 1987; Leslie et al, 1988). E2CD forms a disk-shaped homotrimer with a pseudo three-fold axis in the center. Each of the eight or 20 trimers of E2CDs occupies one vertex of a hollow cube (24-mer) with octahedral 432 symmetry, or a dodecahedron (60-mer) with icosahedral 532 symmetry. In the forward acyltransfer reaction catalyzed by E2bCD (Reaction 2), the two substrates CoA and the S-acyldihydrolipoamide moiety on LBD bind to the active site of E2bCD. The A. vinelandii E2pCD structure has shown that the CoA substrate binds to one entrance of the 30 Å long active-site channel formed by two three-fold-related subunits within a trimer. The second substrate, LBD-free dihydrolipoamide, binds to the opposing entrance of the channel facing the outside of the cubic core (Mattevi et al, 1993). The invariant histidine residue (His391′ in bovine E2bCD) located in the center of the active-site channel functions as a general-base catalyst to abstract a proton from CoA for activation of the substrate (Russell and Guest, 1990; Meng and Chuang, 1994; Hendle et al, 1995). However, the regulation of this bimolecular reaction in the active-site channel of the E2CDs has not been elucidated. The mammalian BCKDC degrades branched-chain α-ketoacids derived from the branched-chain amino acids leucine, isoleucine and valine with the concomitant conversion of CoA to isovaleryl-CoA, α-methylbutyryl-CoA and isobutyryl-CoA (IB-CoA), respectively (Pettit et al, 1978). MSUD mutations have been identified in the E1b-α, E1b-β, E2b and E3 subunits of the human BCKDC (Chuang and Shih, 2001). The lack of a mammalian E2bCD structure has hampered the understanding of how MSUD mutations impede the function and assembly of the E2b core of human BCKDC. The available structures of cognate E2pCD and E2oCD are from bacteria, which share less than 30% sequence identity with their human counterpart. In the present study, we determined the crystal structure of the cubic core of bovine E2bCD, which shares 93% sequence identity with the human homolog. Our structural and biochemical data describe, for the first time, adverse effects of certain MSUD mutations on the assembly and catalysis of E2bCD. Significantly, the structures of bovine E2bCD reveal a novel gating mechanism in which the binding of CoA to one entrance of the active-site channel synchronizes the opening of the otherwise closed gate on the opposite end for S-acyldihydrolipoamide binding. The CoA-induced gate opening results in markedly increased affinity of E2bCD for dihydrolipoamide as measured by isothermal titration calorimetry (ITC). To our knowledge, this synchronized substrate-gating mechanism is the first example of regulating a bimolecular reaction through distant communication between the two-substrate entrances of the same active-site channel. We propose that this synchronized gating mechanism serves as a feed-forward switch to coordinate and maintain the catalytic efficiency of the E2b-catalyzed acyltransfer reaction. This molecular switch is likely a common feature in the catalytic cores of mammalian α-ketoacid dehydrogenase complexes. Results and discussion Monomer and oligomer structures of bovine E2bCD We determined four structures of bovine E2bCD (residues 162–421): an apo form at 2.7 Å resolution, two CoA-bound forms (produced with both a short 15-min and a long overnight soak) at 2.7 and 2.17 Å, respectively, and a product isobutyryl-CoA (IB-CoA)-bound form at 2.5 Å (Supplementary Table SI). In all structures, the linker region between SBD and E2bCD (residues 162–187) is disordered. Eight monomers of E2bCD were found in the asymmetric unit of the crystal: two trimers and two monomers, each of the latter from one of the two additional trimers that are related by crystallographic symmetry. The structures of these eight monomers in the same crystal are essentially identical (the r.m.s. deviations are less than 0.4 Å for all Cα atoms), except the monomers in the E2bCD-CoA (long soak) structure. The overall structure of bovine E2bCD monomer is similar to those of E. coli E2oCD (r.m.s.d=1.3 Å for the corresponding 217 Cα atoms) and A. vinelandii E2pCD (r.m.s.d=1.2 Å for the corresponding 223 Cα atoms) (Figure 1A and B) (Mattevi et al, 1992; Knapp et al, 1998), despite the less than 30% sequence identities between these core domains (Figure 2). Chloramphenicol acetyltransferase (CAT) has a similar topology to those of E2CDs (Leslie et al, 1988), and the r.m.s.d between E2bCD and CAT is 1.7 Å for the corresponding 153 Cα atoms (Figure 1B). Figure 1.Monomer and oligomer structures of bovine E2bCD. (A) Monomer structure of bovine E2bCD. The bound CoA is shown as a stick model. (B) Stereo diagram of the monomer structures of E. coli E2oCD (blue, PDB code 1E2O) (Knapp et al, 1998), A. vinelandii E2pCD (green, 1EAA) (Mattevi et al, 1992) and E. coli CAT (gray, 3CLA) (Leslie et al, 1988) superimposed on bovine E2bCD (red). The bound CoA of E2bCD is shown as a stick model in red. (C) Trimer structure of bovine E2bCD. Each subunit is differently colored. A dashed line (left panel) and a small filled triangle (right panel) indicate a pseudo three-fold axis of the trimer. The bound CoA molecules are depicted as space-filling models. (D) The 24-mer cubic structures of bovine E2bCD (left), E. coli E2oCD (center) and A. vinelandii E2pCD (right) shown as space-filling models. For clarity, one of the eight trimers in the cube is colored the same as (C). White ovals highlight the trimer–trimer interactions that dictate the cubic core assembly. Download figure Download PowerPoint Figure 2.Sequence alignments and secondary structure assignments of the three E2CD's. The sequence alignments of bovine E2bCD, E. coli E2oCD and A. vinelandii E2pCD were performed with Clustal W (Thompson et al, 1994) and modified with ESPript (Gouet et al, 1999); red box: identical residues, yellow box: conserved, open box: semi-conserved. Secondary structures and residue numbers for each protein are indicated above and below the alignments, respectively, with the same color coordination as in the protein nomenclatures. Triangles immediately above aligned residues depict the following functions or effects in bovine E2bCD: black, CoA binding; red, MSUD mutations; blue, active-site residues; orange, hydrophobic-socket residues for the trimer–trimer interaction; magenta, gatekeepers for the dihydrolipoamide-binding site. Download figure Download PowerPoint Like bacterial E2CDs, three monomers of bovine E2bCD assemble into a trimer of 61 Å in height and 63 Å in diameter, whose monomers are related by a single three-fold rotational axis (Figure 1C). The subunit interfaces between one E2bCD monomer and the other two are 2900 Å2, including 47 hydrogen bonds (H-bonds). These values are comparable to those of E. coli E2oCD (subunit interface: 2740 Å2, 52 H-bonds and six salt bridges) (Knapp et al, 1998). A. vinelandii E2pCD has a more extended N-terminal region that crosses over a neighboring subunit from the edge to the center of the trimer, thereby providing the largest subunit interface for trimer formation (3900 Å2, 48 H-bonds involved) among the three E2 core structures (Mattevi et al, 1992). The cubic core of bovine E2bCD generated with crystallographic symmetry-related molecules is similar to those of bacterial E2CDs in both the shape and dimensions (∼130 Å edge) (Figure 1D). Eight trimers of E2bCD, which are building blocks of the cube, are located at corners of the cube with octahedral 432 symmetry. The trimer–trimer interactions are accomplished solely through C-terminal contacts between the 2-fold-related subunits, each from a separate neighboring trimer. Each octahedral face of these cubes has a large rectangular hole, which provides the passage for CoA to approach the CoA-binding site inside the cubic core (Mattevi et al, 1992). Distinct trimer–trimer interactions in bovine E2bCD In the two-fold trimer–trimer interface of bovine E2bCD, the two C-terminal helices (H7 and H7′) from respective monomers of the two interacting trimers line up with each other in the center of the interface (Figure 3A and B). The side chain of Leu418 (or Leu418′) from each helix projects, like a ‘knob’, into a hydrophobic ‘socket’ of the opposing subunit, as observed in E. coli E2oCD and A. vinelandii E2pCD (Figure 3 and Supplementary Figure S1) (Mattevi et al, 1992; Knapp et al, 1998). This ‘socket’ in bovine E2bCD is formed by hydrophobic residues Leu227′, Leu233′, Ile236′, Leu244′, Phe249′ and Phe307′. Residues Leu417 or Leu417′ confer additional hydrophobic interactions between the two C-terminal helices. Removal of the two C-terminal leucine residues in porcine E2o corresponding to Leu417 and Leu418 of bovine E2b completely prevents cubic-core formation (Koike et al, 2000). Many of the residues in the hydrophobic ‘knob-and-socket’ interactions are conserved in bacterial E2oCD and E2pCD (Figure 2). However, among the three E2CDs, bovine E2bCD possesses the largest interface area between trimers (1161 Å2). Figure 3.Trimer–trimer interactions in the bovine E2bCD cubic core. (A) Hydrophobic contacts in the ‘knob-and-socket’ interactions between two-fold-related subunits (blue and green) from each neighboring trimer in the bovine E2bCD core. The residues in the hydrophobic sockets are colored in orange. (B) The same trimer–trimer interface shown in (A) with the residues forming H-bonds and ion pairs as stick models. (C) Pattern diagrams showing trimer–trimer interactions in the cubic cores of the bovine E2bCD (left), E. coli E2oCD (middle) and A. vinelandii E2pCD (right). The protruding C-terminal helix is labeled with a white letter C. The hydrophobic ‘knob-and-socket’ interface is colored in orange. White lines represent H-bonds and ion pairs. (D) Sedimentation coefficients of wild-type and mutant bovine E2bCDs. The sedimentation coefficients (s20,w) of the wild-type (red) and R240C mutant (blue) are 17.1S and 17.3S, respectively, which are close to the calculated coefficient (17.5S) from the theoretical molecular mass of the 24-mer E2bCD cubic core. The sedimentation coefficients of the K252N (green) and ΔC2 (orange) mutants are both 4.4S. When modeled as discrete species, the calculated masses of the mutants agree well with the theoretical mass of a E2bCD trimer. Download figure Download PowerPoint A distinct feature in bovine E2bCD is that many H-bonds and salt bridges contribute to additional trimer–trimer interactions (Figure 3B). Notably, the C-terminal residue Lys421 makes H-bonds with Lys421′ as well as Lys252′ and Asn302′ from the other subunit, with the latter residues forming a ‘clamp’ holding the backbone of Lys421. As a result of the symmetrical interactions, the two clamps provided by Lys421 and Lys421′ practically embrace each other. Thus, in addition to the hydrophobic ‘knob-and-socket’ interactions, bovine E2bCD employs the second ionic clamping mechanism to augment the trimer–trimer contacts, which we term ‘clamp-and-socket’ interactions (Figure 3C). The significance of the clamping mechanism is corroborated by the K252N MSUD mutation (Chuang and Shih, 2001) that eliminates one H-bond for Lys421 in bovine E2bCD (Figure 3B). This mutation prevents formation of the cubic E2bCD core, resulting in an exclusively trimeric species, as indicated by a sedimentation coefficient of 4.4S (Figure 3D). Similarly, when the last two C-terminal residues (Leu420 and Lys421) are truncated, E2bCD (ΔC2) is no longer able to form the cubic core and shows the same sedimentation coefficient. In contrast, the R240C MSUD mutation (Chuang and Shih, 2001) that abolishes a salt bridge with Asp419′ in the trimer–trimer interface (Figure 3B) does not affect the cubic assembly of E2bCD, judging from its sedimentation coefficient of 17.3S, which is similar to the wild type (17.1S) (Figure 3D). The presence of a His-tag at the C-terminus of bovine E2bCD also does not impede the formation of the cubic core (unpublished data). In contrast, the introduction of a C-terminal His-tag in E. coli E2oCD produces exclusively trimers (Knapp et al, 2000). Through an apparent steric hindrance, the cubic core of the A. vinelandii E2p dissociates upon binding of either E1p or E3 (Bosma et al, 1984). These results suggest that the clamp interactions between the two C-terminal lysine residues are critical to the increased stability of the bovine E2bCD core, compared to its bacterial counterparts. Two different conformations between the bound CoA and the bound acyl-CoA In the bovine E2bCD-CoA (short soak) structure, all eight monomers in the asymmetric unit bind an intact CoA with its pantetheine tail extended in the active-site channel (Figure 4A). The bound CoA is held in place by as many as 12 direct and five water-mediated (indirect) H-bonds. Arg230 is highly conserved in the E2 family (Figure 2); this residue forms an H-bond with the 3′-phosphate group of the bound CoA. The R230G MSUD mutation (Chuang et al, 1997) increases the Kd of E2bCD for CoA by 10-fold, and reduces the kcat/Km by six-fold over the wild type (Table I). Asn339 makes three direct H-bonds to the adenine ring and pyrophosphate of the bound CoA. In the majority of E2bCDs and E2pCDs from Gram-negative bacteria, this residue is replaced by a serine, whose side chain is not able to form an H-bond with CoA, as is the case with Ser559 in A. vinelandii E2pCD (Figure 2) (Mattevi et al, 1993). Furthermore, Gln317, which is present only in the mammalian E2bCD (Figure 2), forms direct and indirect H-bonds with the pyrophosphate moiety of the bound CoA. This position in all other E2CDs is occupied by a glycine, alanine or serine residue, whose side chains are too short to form H-bonds with CoA. These structural properties suggest that the bovine E2bCD may have evolved the increased specificity and affinity for CoA over the prokaryotic E2 cores. Figure 4.A CoA-mediated gating mechanism for synchronized dihydrolipoamide binding in the active-site channel of bovine E2bCD. (A) Stereo view of the CoA-binding site of E2bCD-CoA (short soak). The bound CoA in the ‘in’ conformation and its surrounding residues are shown in stick models. Residues from the three-fold-related subunit are shown in cyan. The residues in magenta are the gatekeepers at the dihydrolipoamide entrance of the active-site channel. Water molecules are shown as small spheres. The 2Fo-Fc density of the bound CoA is superimposed at a 1-σ contour level. (B) The bound IB-CoA in the ‘out’ conformation in E2bCD-IB-CoA. The 2Fo-Fc densities are at the 1-σ level. (C) The oxidized CoA in the ‘in’ conformation in E2bCD-CoA (long soak). Three of the eight E2bCD monomers in the asymmetric unit of this crystal show this structure. The omit density of the oxidized CoA was at the 1-σ level. (D) The disordered pantetheine tail of the CoA (the ‘out’ conformation) bound to the remaining five monomers in E2bCD-CoA (long soak). The 2Fo-Fc density is contoured at the 1-σ level. (E) Superimposed gating turns and bound cofactors; white: apo, green: E2bCD-CoA (short soak), orange: E2bCD-IB-CoA. The bound CoA (carbon atoms in green) in E2bCD-CoA (short soak) is in the ‘in’ conformation, whereas the pantetheine tail of the bound IB-CoA (carbons in orange) in E2bCD-IB-CoA is disordered (the ‘out’ conformation). The gray arrow indicates the direction of the dihydrolipoamide-binding site. The gatekeeper residues are in magenta. (F) Surface model of the closed dihydrolipoamide gate in the apo E2bCD structure. Three gatekeeper residues (Leu293, Val295 and Leu353) are colored in magenta and the gating turn in red. (G) The open gate for the dihydrolipoamide-binding site in E2bCD-CoA (short soak). The thiol group of the bound CoA in the ‘in’ conformation is shown in yellow. (H) The closed dihydrolipoamide gate in E2bCD-IB-CoA. Download figure Download PowerPoint Table 1. CoA-binding and kinetic constants of wild-type and mutant E2bCDs CoA bindinga IV-CoA bindinga Kineticsb Oligomeric statec Mutant category Protein Kd (μM) ΔG (kcal/mol) Kd (μM) ΔG (kcal/mol) kcat (s−1) Km (μM) kcat/Km (s−1 μM−1) Wild type 3.0±0.35 −7.5 6.8±0.35 −7.1 1874±122 219±14 8.6 24 — R230Gd 29±2.5 −6.1 — — 1294±56 1349±58 0.95 24 CoA binding R240Cd 9.1±1.6 −6.7 — — 1713±89 222±11.5 7.7 24 Trimer–trimer K252Nd 11±1.2 −6.6 — — 818±58 340±24 2.4 3 Trimer–trimer ΔC2 5.0±0.71 −7.3 — — 2019±161 211±16 9.5 3 Trimer–trimer L293A 25±1.4 −6.6 — — 347±23 1005±61 0.35 24 Gating D288A 13±1.1 −6.5 — — 828±68 386±18 2.1 24 Gating All measurements for CoA or IV-CoA binding and kinetics were in triplicate. a Kd and ΔG for CoA or IV-CoA were measured by ITC. b Kinetics was measured in the reverse direction of the acyltransfer reaction (Reaction 2) with dihydrolipoamide and IV-CoA as substrates. Km is for IV-CoA. c Oligomeric states were judged by gel filtration and designated as follows: 24: 24-mer, 3: trimer. d MSUD mutations. The extended conformation of the bound CoA in bovine E2bCD-CoA (short soak) (Figure 4A) is similar to the ‘in’ conformation previously reported in the A. vinelandii E2pCD structure (Mattevi et al, 1993). In this conformation, the thiol group of the bound CoA is poised for activation by the catalytic residue His391′. A conserved serine residue Ser338 forms an H-bond with a peptide nitrogen atom in the pantetheine tail. Ser338 was shown to be involved in the acyltransfer reaction presumably through stabilization of the tetrahedral intermediate (Meng and Chuang, 1994). On the other hand, in the structure of bovine E2bCD-IB-CoA, the pantetheine tail of the bound IB-CoA, which is a substrate for the reverse acyltransfer reaction (Reaction 2), is largely disordered, with several acetate molecules from the crystallization buffer occupying the active-site channel (Figure 4B). Acetate at the concentration present in the crystallization buffer (250 mM) has minimal effects on the binding of IV-CoA to bovine E2bCD as determined by ITC (data not shown). This suggests that the disordering of the pantetheine tail is not caused by the presence of acetate ions in the crystal. We refer to the partially disordered conformation of the bound IB-CoA as the ‘out’ conformation, which is reminiscent of the compact conformation of the bound CoA with the folded pantetheine tail observed outside the active-site channel of A. vinelandii E2pCD (Mattevi et al, 1993). A novel CoA-mediated gating mechanism for the dihydrolipoamide-binding site The entrance of the dihydrolipoamide-binding site in the active-site channel opposing the CoA-binding site is open in all reported structures of A. vinelandii E2pCD (apo, CoA-bound and dihydrolipoamide-bound forms) as well as in the apo structure of E. coli E2oCD (Supplementary Figure S2) (Mattevi et al, 1993; Knapp et al, 1998). Surprisingly, in the structures of the apo and IB-CoA-bound bovine E2bCD, the entrance for dihydrolipoamide is completely closed (Figure 4F and H), whereas the same entrance is open in the E2bCD-CoA (short soak) structure (Figure 4G). The gate at the dihydrolipoamide entrance is formed by Leu293, Val295 and Leu353 as well as part of the helix H1. In the closed-gate state, there are hydrophobic interactions between the side chains of these gatekeeper residues clustered at the channel entrance (Figure 4F and H). Gate opening is brought about by the conformational change on a β-hairpin turn that runs from strands E2 to F1 (designated as the gating turn). This gating turn is part of the active-site channel separating the two opposite entrances for CoA and dihydrolipoamide. When the dihydrolipoamide gate is open, the gating turn moves toward the CoA-binding site by maximally ∼2.5 Å with a concomitant shift and flip for the Leu293 and Asp288 side chains, respectively, which are located on this turn (Figure 4E). The open- and closed-gate conformations at the dihydrolipoamide-binding site are correlated with the conformation of the bound CoA. All monomers in bovine E2bCD-CoA (short soak) with the open gate bind CoA in the ‘in’ conformation (Figure 4A and E). The gating turn in the open-gate conformation is stabilized by several direct and indirect H-bonds between the pantetheine tail of the bound CoA and Asp288 on the gating turn, as well as by a salt bridge between the side chains of Asp288 and Arg401′. In contrast, E2bCD monomers with a closed-gate conformation are either in the apo form or with the bound IB-CoA in the ‘out’ conformation (Figure 4E). The absence of interactions between the pantetheine tail and the gating turn apparently locks the gate in the closed conformation. Two conformati" @default.
• W2099202202 created "2016-06-24" @default.
• W2099202202 creator A5003311962 @default.
• W2099202202 creator A5012092032 @default.
• W2099202202 creator A5038727241 @default.
• W2099202202 creator A5046753083 @default.
• W2099202202 creator A5070862060 @default.
• W2099202202 creator A5077177876 @default.
• W2099202202 date "2006-11-23" @default.
• W2099202202 modified "2023-10-01" @default.
• W2099202202 title "A synchronized substrate-gating mechanism revealed by cubic-core structure of the bovine branched-chain α-ketoacid dehydrogenase complex" @default.
• W2099202202 cites W1518644765 @default.
• W2099202202 cites W1527238249 @default.
• W2099202202 cites W1539796472 @default.
• W2099202202 cites W1576270438 @default.
• W2099202202 cites W1582602870 @default.
• W2099202202 cites W1965098923 @default.
• W2099202202 cites W1966553114 @default.
• W2099202202 cites W1975317507 @default.
• W2099202202 cites W1982323560 @default.
• W2099202202 cites W1985629904 @default.
• W2099202202 cites W1986851354 @default.
• W2099202202 cites W1993671723 @default.
• W2099202202 cites W1997955496 @default.
• W2099202202 cites W2001057655 @default.
• W2099202202 cites W2007829111 @default.
• W2099202202 cites W2013004671 @default.
• W2099202202 cites W2016765195 @default.
• W2099202202 cites W2022436829 @default.
• W2099202202 cites W2024852530 @default.
• W2099202202 cites W2038840577 @default.
• W2099202202 cites W2044718961 @default.
• W2099202202 cites W2045731233 @default.
• W2099202202 cites W2045830082 @default.
• W2099202202 cites W2049257750 @default.
• W2099202202 cites W2058089145 @default.
• W2099202202 cites W2060000581 @default.
• W2099202202 cites W2060809301 @default.
• W2099202202 cites W2061868451 @default.
• W2099202202 cites W2079901091 @default.
• W2099202202 cites W2082584140 @default.
• W2099202202 cites W2084661994 @default.
• W2099202202 cites W2087402489 @default.
• W2099202202 cites W2090787083 @default.
• W2099202202 cites W2091679238 @default.
• W2099202202 cites W2095753590 @default.
• W2099202202 cites W2095974497 @default.
• W2099202202 cites W2106882534 @default.
• W2099202202 cites W2120842521 @default.
• W2099202202 cites W2131256962 @default.
• W2099202202 cites W2131392379 @default.
• W2099202202 cites W2144081223 @default.
• W2099202202 cites W2151977734 @default.
• W2099202202 cites W2151997912 @default.
• W2099202202 cites W21982396 @default.
• W2099202202 cites W3192061832 @default.
• W2099202202 cites W4247106131 @default.
• W2099202202 doi "https://doi.org/10.1038/sj.emboj.7601444" @default.
• W2099202202 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1698891" @default.
• W2099202202 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17124494" @default.
• W2099202202 hasPublicationYear "2006" @default.
• W2099202202 type Work @default.
• W2099202202 sameAs 2099202202 @default.
• W2099202202 citedByCount "48" @default.
• W2099202202 countsByYear W20992022022012 @default.
• W2099202202 countsByYear W20992022022013 @default.
• W2099202202 countsByYear W20992022022014 @default.
• W2099202202 countsByYear W20992022022015 @default.
• W2099202202 countsByYear W20992022022016 @default.
• W2099202202 countsByYear W20992022022018 @default.
• W2099202202 countsByYear W20992022022019 @default.
• W2099202202 countsByYear W20992022022020 @default.
• W2099202202 countsByYear W20992022022021 @default.
• W2099202202 countsByYear W20992022022022 @default.
• W2099202202 countsByYear W20992022022023 @default.
• W2099202202 crossrefType "journal-article" @default.
• W2099202202 hasAuthorship W2099202202A5003311962 @default.
• W2099202202 hasAuthorship W2099202202A5012092032 @default.
• W2099202202 hasAuthorship W2099202202A5038727241 @default.
• W2099202202 hasAuthorship W2099202202A5046753083 @default.
• W2099202202 hasAuthorship W2099202202A5070862060 @default.
• W2099202202 hasAuthorship W2099202202A5077177876 @default.
• W2099202202 hasBestOaLocation W20992022021 @default.
• W2099202202 hasConcept C121332964 @default.
• W2099202202 hasConcept C12554922 @default.
• W2099202202 hasConcept C1276947 @default.
• W2099202202 hasConcept C153911025 @default.
• W2099202202 hasConcept C159985019 @default.
• W2099202202 hasConcept C18903297 @default.
• W2099202202 hasConcept C192562407 @default.
• W2099202202 hasConcept C194544171 @default.
• W2099202202 hasConcept C199185054 @default.
• W2099202202 hasConcept C2164484 @default.
• W2099202202 hasConcept C2777289219 @default.
• W2099202202 hasConcept C55493867 @default.
• W2099202202 hasConcept C62520636 @default.
• W2099202202 hasConcept C86803240 @default.
• W2099202202 hasConcept C89611455 @default.

• next