Structure of the dihydrolipoamide succinyltransferase (E2) component of the human alpha-ketoglutarate dehydrogenase complex (hKGDHc) revealed by cryo-EM and cross-linking mass spectrometry: Implications for the overall hKGDHc structure.

BACKGROUND: The human mitochondrial alpha-ketoglutarate dehydrogenase complex (hKGDHc) converts KG to succinyl-CoA and NADH. Malfunction of and reactive oxygen species generation by the hKGDHc as well as its E1-E2 subcomplex are implicated in neurodegenerative disorders, ischemia-reperfusion injury, E3-deficiency and cancers. METHODS: We performed cryo-EM, cross-linking mass spectrometry (CL-MS) and molecular modeling analyses to determine the structure of the E2 component of the hKGDHc (hE2k); hE2k transfers a succinyl group to CoA and forms the structural core of hKGDHc. We also assessed the overall structure of the hKGDHc by negative-stain EM and modeling. RESULTS: We report the 2.9 Šresolution cryo-EM structure of the hE2k component. The cryo-EM map comprises density for hE2k residues 151-386 - the entire (inner) core catalytic domain plus a few additional residues -, while residues 1-150 are not observed due to the inherent flexibility of the N-terminal region. The structure of the latter segment was also determined by CL-MS and homology modeling. Negative-stain EM on in vitro assembled hKGDHc and previous data were used to build a putative overall structural model of the hKGDHc. CONCLUSIONS: The E2 core of the hKGDHc is composed of 24 hE2k chains organized in octahedral (8 × 3 type) assembly. Each lipoyl domain is oriented towards the core domain of an adjacent chain in the hE2k homotrimer. hE1k and hE3 are most likely tethered at the edges and faces, respectively, of the cubic hE2k assembly. GENERAL SIGNIFICANCE: The revealed structural information will support the future pharmacologically targeting of the hKGDHc.

Underlying molecular alterations in human dihydrolipoamide dehydrogenase deficiency revealed by structural analyses of disease-causing enzyme variants.

Human dihydrolipoamide dehydrogenase (hLADH, hE3) deficiency (OMIM# 246900) is an often prematurely lethal genetic disease usually caused by inactive or partially inactive hE3 variants. Here we report the crystal structure of wild-type hE3 at an unprecedented high resolution of 1.75 Å and the structures of six disease-causing hE3 variants at resolutions ranging from 1.44 to 2.34 Å. P453L proved to be the most deleterious substitution in structure as aberrations extensively compromised the active site. The most prevalent G194C-hE3 variant primarily exhibited structural alterations close to the substitution site, whereas the nearby cofactor-binding residues were left unperturbed. The G426E substitution mainly interfered with the local charge distribution introducing dynamics to the substitution site in the dimer interface; G194C and G426E both led to minor structural changes. The R460G, R447G and I445M substitutions all perturbed a solvent accessible channel, the so-called H+/H2O channel, leading to the active site. Molecular pathomechanisms of enhanced reactive oxygen species (ROS) generation and impaired binding to multienzyme complexes were also addressed according to the structural data for the relevant mutations. In summary, we present here for the first time a comprehensive study that links three-dimensional structures of disease-causing hE3 variants to residual hLADH activities, altered capacities for ROS generation, compromised affinities for multienzyme complexes and eventually clinical symptoms. Our results may serve as useful starting points for future therapeutic intervention approaches.

DNA binding of sunitinib: Spectroscopic evidence via circular dichroism and nuclear magnetic resonance.

Sunitinib is a non-selective tyrosine kinase inhibitor, but in its chemical structure there can be discovered certain features, which suggest the ability to bind to DNA. These elements are the planar aromatic system and the tertiary amine function, which is protonated at the pH of the organism. In this study, the binding of the drug sunitinib to DNA was investigated using circular dichroism (CD), 1H NMR and UV spectroscopies, along with CD melting. For these studies DNA was isolated from calf thymus (CT), salmon fish sperm (SS), and chicken erythrocyte (CE), however for our purposes an artificially constructed and highly purified plasmid DNA (pUC18) preparation proved to be the most suitable. DNA binding of the drug was confirmed by shifts in the characteristic CD bands of the DNA, the appearance of an induced CD (ICD) signal in the upper absorption region of sunitinib (300 nm-500 nm), and the evidence from CD melting studies and the NMR. Based on the CD and NMR measurements, it can be assumed that sunitinib has a multiple-step binding mechanism.

Crystal structure of the Thermoplasma acidophilum protein Ta1207.

The crystal structure of the Ta1207 protein from Thermoplasma acidophilum is reported. Ta1207 was identified in a screen for high-molecular-weight protein complexes in T. acidophilum. In solution, Ta1207 forms homopentamers of 188 kDa. The crystal structure of recombinant Ta1207 solved by Se-MAD at 2.4 Šresolution revealed a complex with fivefold symmetry. In the crystal lattice, calcium ions induce the formation of a nanocage from two pentamers. The Ta1207 protomers comprise two domains with the same novel α/ß topology. A deep pocket with a binding site for a negatively charged group suggests that Ta1207 functions as an intracellular receptor for an unknown ligand. Homologues of Ta1207 occur only in Thermoplasmatales and its function might be related to the extreme lifestyle of these archaea. The thermostable Ta1207 complex might provide a useful fivefold-symmetric scaffold for future nanotechnological applications.

Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches.

Thermobifidas are thermotolerant, compost inhabiting actinomycetes which have complex polysaccharide hydrolyzing enzyme systems. The best characterized enzymes of these hydrolases are cellulases from T. fusca, while other important enzymes especially hemicellulases are not deeply explored. To fill this gap we cloned and investigated endomannanases from those reference strains of the Thermobifida genus, which have published data on other hydrolases (T. fusca TM51, T. alba CECT3323, T. cellulosilytica TB100T and T. halotolerans YIM90462T). Our phylogenetic analyses of 16S rDNA and endomannanase sequences revealed that T. alba CECT3323 is miss-classified; it belongs to the T. fusca species. The cloned and investigated endomannanases belong to the family of glycosyl hydrolases 5 (GH5), their size is around 50 kDa and they are modular enzymes. Their catalytic domains are extended by a C-terminal carbohydrate binding module (CBM) of type 2 with a 23-25 residues long interdomain linker region consisting of Pro, Thr and Glu/Asp rich repetitive tetrapeptide motifs. Their polypeptide chains exhibit high homology, interdomain sequence, which don't show homology to each other, but all of them are built up from 3-6 times repeated tetrapeptide motifs) (PTDP-Tc, TEEP-Tf, DPGT-Th). All of the heterologously expressed Man5A enzymes exhibited activity only on mannan. The pH optima of Man5A enzymes from T. halotolerans, T. cellulosilytica and T. fusca are slightly different (7.0, 7.5 and 8.0, respectively) while their temperature optima span within the range of 70-75°C. The three endomannanases exhibited very similar kinetic performances on LBG-mannan substrate: 0.9-1.7mM of KM and 80-120 1/sec of turnover number. We detected great variability in heat stability at 70°C, which was influenced by the presence of Ca2+. The investigated endomannanases might be important subjects for studying the structure/function relation behind the heat stability and for industrial applications to hemicellulose degradation.

Protein complex purification from Thermoplasma acidophilum using a phage display library.

We developed a novel protein complex isolation method using a single-chain variable fragment (scFv) based phage display library in a two-step purification procedure. We adapted the antibody-based phage display technology which has been developed for single target proteins to a protein mixture containing about 300 proteins, mostly subunits of Thermoplasma acidophilum complexes. T. acidophilum protein specific phages were selected and corresponding scFvs were expressed in Escherichia coli. E. coli cell lysate containing the expressed His-tagged scFv specific against one antigen protein and T. acidophilum crude cell lysate containing intact target protein complexes were mixed, incubated and subjected to protein purification using affinity and size exclusion chromatography steps. This method was confirmed to isolate intact particles of thermosome and proteasome suitable for electron microscopy analysis and provides a novel protein complex isolation strategy applicable to organisms where no genetic tools are available.

The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together.

Proteasomes execute the degradation of most cellular proteins. Although the 20S core particle (CP) has been studied in great detail, the structure of the 19S regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 26S holocomplex at 9.1 Å resolution. Rpn6 consists of an α-solenoid-like fold and a proteasome COP9/signalosome eIF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP.