Cross-talk between thiamin diphosphate binding and phosphorylation loop conformation in human branched-chain alpha-keto acid decarboxylase/dehydrogenase

The decarboxylase/dehydrogenase (E1b) component of the 4-megadalton human branched-chain alpha-keto acid dehydrogenase (BCKD) metabolic machine is a thiamin diphosphate (ThDP)-dependent enzyme with a heterotetrameric cofactor-binding fold. The E1b component catalyzes the decarboxylation of alpha-keto acids and the subsequent reductive acylation of the lipoic acid-bearing domain (LBD) from the 24-meric transacylase (E2b) core. In the present study, we show that the binding of cofactor ThDP to the E1b active site induces a disorder-to-order transition of the conserved phosphorylation loop carrying the two phosphorylation sites Ser(292)-alpha and Ser(302)-alpha, as deduced from the 1.80-1.85 A apoE1b and holoE1b structures. The induced loop conformation is essential for the recognition of lipoylated LBD to initiate E1b-catalyzed reductive acylation. Alterations of invariant Arg(287)-alpha, Asp(295)-alpha, Tyr(300)-alpha, and Arg(301)-alpha that form a hydrogen-bonding network in the phosphorylation loop result in the disordering of the loop conformation as elucidated by limited proteolysis, accompanied by the impaired binding and diminished reductive acylation of lipoylated LBD. In contrast, k(cat) values for E1b-catalyzed decarboxylation of the alpha-keto acid are higher in these E1b mutants than in wild-type E1b, with higher K(m) values for the substrate in the mutants. ThDP binding that orders the loop prevents phosphorylation of E1b by the BCKD kinase and averts the inactivation of wild-type E1b, but not the above mutants, by this covalent modification. Our results establish that the cross-talk between the bound ThDP and the phosphorylation loop conformation serves as a feed-forward switch for multiple reaction steps in the BCKD metabolic machine.     

Tetrameric assembly and conservation in the ATP-binding domain of rat branched-chain alpha-ketoacid dehydrogenase kinase

We showed previously that the rat branched-chain alpha-ketoacid dehydrogenase (BCKD) kinase is capable of autophosphorylation. However, despite its sequence similarity to bacterial histidine protein kinases, BCKD kinase does not function as a histidine protein kinase. In the present study, we report that the rat BCKD kinase exists as a homotetramer of M(r) = 185,000, based on results of gel filtration and dynamic light scattering. This is in contrast to the related mammalian pyruvate dehydrogenase kinase isozymes that occur as homodimers. The tetrameric assembly of BCKD kinase was confirmed by the presence of four 5'-adenylyl-imidodiphosphate-binding sites (K(D) = 4.1 x 10(-6)m) per molecule of the kinase. Incubation of the BCKD kinase with increasing concentrations of urea resulted in dissociation of the tetramer to dimers and eventually to monomers as separated on a sucrose density gradient. Both tetramers and dimers, but not the monomer, maintained the conformation capable of binding ATP and undergoing autophosphorylation. BCKD kinase depends on a fully lipoylated transacylase for maximal activity, but the interaction between the kinase and the transacylase is impeded in the presence of high salt concentrations. Alterations of conserved residues in the ATP-binding domain led to a marked reduction or complete loss in the catalytic efficiency of the BCKD kinase. The results indicate that BCKD kinase, similar to pyruvate dehydrogenase kinase isozymes, belongs to the superfamily of ATPase/kinase.     

Subcloning,expression, purification,and characterization of recombinant human leptin-binding domain

A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.     

Sequence-specific 1H-NMR assignments and secondary structure of the lipoyl domain of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex

The lipoyl domain (residues 1-85) of the lipoate-acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been subjected to detailed structural analysis by means of two-dimensional (2D) 1H-NMR spectroscopy at 400 MHz. Sequence-specific proton resonance assignments were made, but at this field strength not all of the side-chain protons could be assigned, especially from complex spin systems like those of leucine, proline and lysine residues. Measurement of short-range interproton distances identified two extensive regions of beta-sheet, each containing four anti-parallel peptide strands. The lipoyl-lysine residue (Lys42) is located in a tight turn at a corner of one sheet, the N-terminal and C-terminal residues of the domain are close together in two adjacent beta-strands in the other. The lipoylated and unlipoylated forms of the domain have almost identical spectra, indicating that there is little, if any, conformational change in the protein as a result of the post-translational modification.     

Androgen receptor ligand-binding domain interaction and nuclear receptor specificity of FXXLF and LXXLL motifs as determined by L/F swapping

The androgen receptor (AR) ligand-binding domain (LBD) binds FXXLF motifs, present in the AR N-terminal domain and AR-specific cofactors, and some LXXLL motifs of nuclear receptor coactivators. We demonstrated that in the context of the AR FXXLF motif many different amino acid residues at positions +2 and +3 are compatible with strong AR LBD interaction, although a preference for E at +2 and K or R at +3 was found. Pairwise systematic analysis of F/L swaps at +1 and +5 in FXXLF and LXXLL motifs showed: 1) F to L substitutions in natural FXXLF motifs abolished AR LBD interaction; 2) binding of interacting LXXLL motifs was unchanged or increased upon L to F substitutions; 3) certain noninteracting LXXLL motifs became strongly AR-interacting FXXLF motifs; whereas 4) other nonbinders remained unaffected by L to F substitutions. All FXXLF motifs, but not the corresponding LXXLL motifs, displayed a strong preference for AR LBD. Progesterone receptor LBD interacted with some FXXLF motifs, albeit always less efficiently than corresponding LXXLL motifs. AR LBD interaction of most FXXLF and LXXLL peptides depended on classical charge clamp residue K720, whereas E897 was less important. Other charged residues lining the AR coactivator-binding groove, K717 and R726, modulated optimal peptide binding. Interestingly, these four charged residues affected binding of individual peptides independent of an F or L at +1 and +5 in swap experiments. In conclusion, F residues determine strong and selective peptide interactions with AR. Sequences flanking the core motif determine the specific mode of FXXLF and LXXLL interactions.     

Structure of the membrane domain of subunit b of the Escherichia coli F0F1 ATP synthase.

The structure of the N-terminal transmembrane domain (residues 1-34) of subunit b of the Escherichia coli F0F1-ATP synthase has been solved by two-dimensional 1H NMR in a membrane mimetic solvent mixture of chloroform/methanol/H2O (4:4:1). Residues 4-22 form an alpha-helix, which is likely to span the hydrophobic domain of the lipid bilayer to anchor the largely hydrophilic subunit b in the membrane. The helical structure is interrupted by a rigid bend in the region of residues 23-26 with alpha-helical structure resuming at Pro-27 at an angle offset by 20 degrees from the transmembrane helix. In native subunit b, the hinge region and C-terminal alpha-helical segment would connect the transmembrane helix to the cytoplasmic domain. The transmembrane domains of the two subunit b in F0 were shown to be close to each other by cross-linking experiments in which single Cys were substituted for residues 2-21 of the native subunit and b-b dimer formation tested after oxidation with Cu(II)(phenanthroline)2. Cys residues that formed disulfide cross-links were found with a periodicity indicative of one face of an alpha-helix, over the span of residues 2-18, where Cys at positions 2, 6, and 10 formed dimers in highest yield. A model for the dimer is presented based upon the NMR structure and distance constraints from the cross-linking data. The transmembrane alpha-helices are positioned at a 23 degrees angle to each other with the side chains of Thr-6, Gln-10, Phe-14, and Phe-17 at the interface between subunits. The change in direction of helical packing at the hinge region may be important in the functional interaction of the cytoplasmic domains.     

Single phosphorylation of Tyr304 in the cytoplasmic tail of ephrin B2 confers high-affinity and bifunctional binding to both the SH2 domain of Grb4 and the PDZ domain of the PDZ-RGS3 protein.

The B class cell-attached ephrins mediate contact-dependent cell-cell communications and transduce the contact signals to the host cells through the binding interactions of their cytoplasmic domains. Two classes of intracellular effectors of B ephrins have been identified: one contains the PSD-95/Dlg/ZO-1 (PDZ) domain (for example PDZ-RGS3), and the second the Src homology 2 (SH2) domain (e.g. the Grb4 adaptor protein). The interaction with Grb4 requires phosphorylation of tyrosine residues on the conserved cytoplasmic C-terminal region of B ephrins, while binding to the PDZ domain is independent of tyrosine phosphorylation. However, the exact phosphorylation site(s) required for signaling remained obscure and it is also unknown whether the two classes of effectors can bind to B ephrins simultaneously or if the binding of one affects the binding of the other. We report here that phosphorylation of Tyr304 in the functional C-terminal region (residues 301-333) of ephrin B2 confers high-affinity binding to the SH2 domain of the Grb4 protein. Tyrosine phosphorylation at other candidate sites resulted in only minor change of the binding of Tyr304-phosphorylated ephrin B peptide (i.e. ephrinB2(301-333)-pY304) with the SH2 domain. (1)H-(15)N NMR HSQC experiments show that only the ephrinB2(301-333)-pY304 peptide forms a stable and specific binding complex with the SH2 domain of Grb4. The SH2 and PDZ domains were found to bind to the Tyr304 phosphopeptide both independently and at the same time, forming a three-component molecular complex. Taken together, our studies identify a novel SH2 domain binding motif, PHpY304EKV, on the cytoplasmic domains of B ephrins that may be essential for reverse signaling via the Grb4 adaptor protein alone or in concert with proteins containing PDZ domains.     

Modulation of retinoic acid receptor alpha activity by lysine methylation in the DNA binding domain

Metabolic labeling and detection with a methylated lysine-specific antibody confirm lysine methylation of RAR alpha in mammalian cells. We previously reported Lys (347) trimethylation of mouse retinoic acid receptor alpha (RAR alpha) in the ligand binding domain (LBD) that affected ligand sensitivity of the dissected LBD. Here we report two monomethylated residues, Lys (109) and Lys (171) identified by LC-ESI-MS/MS in the DNA binding domain (DBD) and the hinge region, which affect retinoic acid (RA) sensitivity, coregulator interaction and heterodimerization with retinoid X receptor (RXR) in the context of the full-length protein. Constitutive negative mutation at Lys (109), but not Lys (171), reduces RA-dependent activation. Methylation at Lys (109) plays a more dominant role than trimethylation at Lys (347) in terms of RA activation of the full-length receptor. Lys (109) is located in a homologous sequence (CEGC K GFFRRS) of the DBD in RARs and is conserved in the nuclear receptor superfamily even across the species boundary. This study uncovers a potential role for monomethylation at Lys (109) in coordinating the synergy between DBD and LBD for ligand-dependent activation of RAR alpha.     

Solution structures of the C-terminal domain of cardiac troponin C free and bound to the N-terminal domain of cardiac troponin I.

The N-terminal domain of cardiac troponin I (cTnI) comprising residues 33-80 and lacking the cardiac-specific amino terminus forms a stable binary complex with the C-terminal domain of cardiac troponin C (cTnC) comprising residues 81-161. We have utilized heteronuclear multidimensional NMR to assign the backbone and side-chain resonances of Ca2+-saturated cTnC(81-161) both free and bound to cTnI(33-80). No significant differences were observed between secondary structural elements determined for free and cTnI(33-80)-bound cTnC(81-161). We have determined solution structures of Ca2+-saturated cTnC(81-161) free and bound to cTnI(33-80). While the tertiary structure of cTnC(81-161) is qualitatively similar to that observed free in solution, the binding of cTnI(33-80) results mainly in an opening of the structure and movement of the loop region between helices F and G. Together, these movements provide the binding site for the N-terminal domain of cTnI. The putative binding site for cTnI(33-80) was determined by mapping amide proton and nitrogen chemical shift changes, induced by the binding of cTnI(33-80), onto the C-terminal cTnC structure. The binding interface for cTnI(33-80), as suggested from chemical shift changes, involves predominantly hydrophobic interactions located in the expanded hydrophobic pocket. The largest chemical shift changes were observed in the loop region connecting helices F and G. Inspection of available TnC sequences reveals that these residues are highly conserved, suggesting a common binding motif for the Ca2+/Mg2+-dependent interaction site in the TnC/TnI complex.     

ESI-MS and FTIR studies of the interaction between the second PDZ domain of hPTP1E and target peptides.

The specificity of interaction between the second PDZ domain of human protein tyrosine phosphatase1E (PDZ2) and a C-terminal peptide, ENEQVSAV, from the guanine nucleotide exchange factor RA-GEF-2 was investigated using Fourier transform infrared (FTIR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Specificity of the binding interaction and the importance of Ser in the -2 position of the target peptide were demonstrated using alternate peptides ENEQVCAV and KDDEVYYV. FTIR-monitored thermal denaturation in the amide I region showed a 10 degrees C increase in melting temperature (Tm) for the PDZ2-ENEQVSAV complex compared with that of free PDZ2, and the spectra revealed increased absorption in the beta-sheet region (1628 cm(-1)) of PDZ2 on peptide binding. Neither of these results were observed with peptides containing either Cys or Tyr in the -2 position. Complex formation with the Ser-containing peptide was further demonstrated by direct measurement of a 1:1 PDZ-peptide complex by ESI-MS in 100% aqueous solutions without the need for organic co-solvents. Our results demonstrate that even a single atom (O --> S) substitution from Ser to Cys in the -2 position disrupts C-terminal peptide binding to PDZ2.     

Ligand- and coactivator-mediated transactivation function (AF2) of the androgen receptor ligand-binding domain is inhibited by the cognate hinge region

Transactivation functions (AF2) in the ligand-binding domains (LBD) of many steroid receptors are well characterized, but there is little evidence to support such a function for the LBD of the androgen receptor (AR). We report a mutant AR, with residues 628-646 in the hinge region deleted, which exhibited transactivation activity that was more than double that of the wild type (WT) AR. Although no androgen-dependent AF2 activity could be observed for the WT ARLBD fused to a heterologous DNA-binding domain, the mutant ARLBD(Delta628-646) was 30-40 times more active than the WT ARLBD. In the presence of the p160 coactivator TIF2, AR(Delta628-646) was significantly more active than similarly treated WT AR. Deletion of residues 628-646 also enhanced TIF2-ARLBD activity 8-fold, an effect not present when the LBD-interacting LXXLL motifs of TIF2 were mutated, suggesting that the negative modulatory activity of residues 628-646 were exerted via coactivator pathways. Although the AP-1 (c-Jun/c-Fos) system and NcoR have been reported to interact with and repress the activity of some steroid receptors, c-Jun, c-Fos, c-Jun/c-Fos, nor NcoR function was consistently affected by the absence or presence of residues 628-646, implying that the AR hinge region exerts its silencing effects in a manner independent of these corepressors. Our data provide evidence for the novel finding that strong androgen-dependent AF2 exists in the ARLBD and is the first report of a negative regulatory domain in the AR. Because mutations in this region are commonly associated with prostate cancer, it is important to characterize the mechanisms by which the hinge region exerts its repressor effect on ligand-activated and coactivator-mediated AF2 activity of the ARLBD.     

Crystal structure of a coiled-coil domain from human ROCK I

The small GTPase Rho and one of its targets, Rho-associated kinase (ROCK), participate in a variety of actin-based cellular processes including smooth muscle contraction, cell migration, and stress fiber formation. The ROCK protein consists of an N-terminal kinase domain, a central coiled-coil domain containing a Rho binding site, and a C-terminal pleckstrin homology domain. Here we present the crystal structure of a large section of the central coiled-coil domain of human ROCK I (amino acids 535-700). The structure forms a parallel α-helical coiled-coil dimer that is structurally similar to tropomyosin, an actin filament binding protein. There is an unusual discontinuity in the coiled-coil; three charged residues (E613, R617 and D620) are positioned at what is normally the hydrophobic core of coiled-coil packing. We speculate that this conserved irregularity could function as a hinge that allows ROCK to adopt its autoinhibited conformation.     

High precision NMR structure and function of the RING-H2 finger domain of EL5, a rice protein whose expression is increased upon exposure to pathogen-derived oligosaccharides

EL5, a RING-H2 finger protein, is rapidly induced by N-acetylchitooligosaccharides in rice cell. We expressed the EL5 RING-H2 finger domain in Escherichia coli and determined its structure in solution by NMR spectroscopy. The EL5 RING-H2 finger domain consists of two-stranded beta-sheets (beta1, Ala(147)-Phe(149); beta2, Gly(156)-His(158)), one alpha-helix (Cys(161)-Leu(166)), and two large N- and C-terminal loops. It is stabilized by two tetrahedrally coordinated zinc ions. This structure is similar to that of other RING finger domains of proteins of known function. From structural analogies, we inferred that the EL5 RING-H2 finger is a binding domain for ubiquitin-conjugating enzyme (E2). The binding site is probably formed by solvent-exposed hydrophobic residues of the N- and C-terminal loops and the alpha-helix. We demonstrated that the fusion protein with EL5-(96-181) and maltose-binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and a rice E2 protein, OsUBC5b. This supported the idea that the EL5 RING finger domain is essential for ubiquitin-ligase activity of EL5. By NMR titration experiments, we identified residues that are critical for the interaction between the EL5 RING-H2 finger and OsUBC5b. We conclude that the RING-H2 finger domain of EL5 is the E2 binding site of EL5.     

Genetic analysis of a potential zinc-binding domain of the adenovirus E4 34k protein

E4 34k, the product of adenovirus early region 4 (E4) open reading frame 6, modulates viral late gene expression, viral DNA replication, apoptosis, double strand break repair, and transformation through multiple interactions with components in infected and transformed cells. Conservation of several cysteine and histidine residues among E4 34k sequences from a variety of adenovirus serotypes suggests the presence of a zinc binding domain important for function. Consistent with the hypothesis that E4 34k is a zinc metalloprotein, zinc binding by baculovirus-expressed E4 34k protein was demonstrated in a zinc blotting assay. To investigate the relationship between the potential zinc-binding region and E4 34k function, a series of mutant genes containing single amino acid substitutions at each of the conserved cysteine and histidine residues in E4 34k were constructed. The mutant proteins were examined for the ability to complement the late protein synthetic defect of an E4 deletion mutant, to physically interact with the viral E1b 55-kDa protein (E1b 55k) and cellular p53 protein, to relocalize E1b 55k, and to destabilize the p53 protein. These analyses identified a subset of cysteine and histidine residues required for stimulation of late gene expression, physical interaction with E1b 55k, and p53 destabilization. These data suggest that a zinc-binding domain participates in the formation of the E4 34k-E1b 55k physical complex and that the complex is required in late gene expression and for p53 destabilization.     

Molecular basis for autoregulatory interaction between GAE domain and hinge region of GGA1

Golgi-localizing, gamma-adaptin ear domain homology, ADP ribosylation factor-binding (GGA) proteins and the adaptor protein (AP) complex, AP-1, are involved in membrane traffic between the trans Golgi network and the endosomes. The gamma-adaptin ear (GAE) domain of GGAs and the gamma1 ear domain of AP-1 interact with an acidic phenylalanine motif found in accessory proteins. The GAE domain of GGA1 (GGA1-GAE) interacts with a WNSF-containing peptide derived from its own hinge region, although the peptide sequence deviates from the standard acidic phenylalanine motif. We report here the structure of GGA1-GAE in complex with the GGA1 hinge peptide, which revealed that the two aromatic side chains of the WNSF sequence fit into a hydrophobic groove formed by aliphatic portions of the side chains of conserved arginine and lysine residues of GGA1-GAE, in a similar manner to the interaction between GGA-GAEs and acidic phenylalanine sequences from the accessory proteins. Fluorescence quenching experiments indicate that the GGA1 hinge region binds to GGA1-GAE and competes with accessory proteins for binding. Taken together with the previous observation that gamma1 ear binds to the GGA1 hinge region, the interaction between the hinge region and the GAE domain underlies the autoregulation of GGA function in clathrin-mediated trafficking through competing with the accessory proteins and the AP-1 complex.     

Repeating functional domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Each polypeptide chain in the lipoate acetyltransferase (E2) core of the pyruvate dehydrogenase complex from Escherichia coli contains three repeating sequences in the N-terminal half of the molecule. The repeats are highly homologous in primary structure and each includes a lysine residue that is a potential site for lipoylation. We have shown that all three sites are lipoylated, at least in part, and that the three lipoylated segments of the E2 chain can be isolated as distinct functional domains after limited proteolysis. Each domain becomes partly acetylated in the intact complex in the presence of substrate. In the primary structure, the domains are separated by regions of polypeptide chain oddly rich in alanine and proline residues. These regions are probably the conformationally mobile segments observed in the 1H-n.m.r. spectrum of the complex and which are removed by tryptic cleavage at Lys-316. The C-terminal half of the molecule contains the acetyltransferase active site and the binding sites for E1, E3 and other E2 subunits. The pyruvate dehydrogenase complex of E. coli, which has a heterogeneous quaternary structure, is thus far unique among the 2-oxo acid dehydrogenase complexes in possessing more than one lipoyl domain per E2 chain, but this may be a general feature of the enzyme from Gram-negative organisms.