Structural basis for recognition and methylation of p97 by METTL21D, a valosin-containing protein lysine methyltransferase.

p97 is a human AAA+ (ATPase associated with diverse cellular activities, also known as valosin-containing protein [VCP]) ATPase, which is involved in diverse cellular processes such as membrane fusion and proteolysis. Lysine-specific methyltransferase of p97 (METTL21D) was identified as a class I methyltransferase that catalyzes the trimethylation of Lys315 of p97, a so-called VCP lysine methyltransferase (VCPKMT). Interestingly, VCPKMT disassembles a single hexamer ring consisting of p97-D1 domain and methylates Lys315 residue. Herein, the structures of S-adenosyl-L-methionine-bound VCPKMT and S-adenosyl-L-homocysteine-bound VCPKMT in complex with p97 N/D1 (N21-Q458) were reported at a resolution of 1.8 Å and 2.8 Å, respectively. The structures revealed the molecular details for the recognition and methylation of monomeric p97 by VCPKMT. Using biochemical analysis, we also investigated whether the methylation of full-length p97 could be sufficiently enhanced through cooperation between VCPKMT and the C terminus of alveolar soft part sarcoma locus (ASPL). Our study provides the groundwork for future structural and mechanistic studies of p97 and inhibitors.

Structural basis for the interaction between human Npl4 and Npl4-binding motif of human Ufd1.

The heterodimer of human ubiquitin fusion degradation 1 (hUfd1) and human nuclear protein localization 4 (hNpl4) is a major cofactor of human p97 adenosine triphosphatase (ATPase). The p97-Ufd1-Npl4 complex translocates the ubiquitin-conjugated proteins from the endoplasmic reticulum membrane to the cytoplasm. Ubiquitinated proteins are then degraded by the proteasome. The structures of Npl4 and Ufd1-Npl4 (UN) complex in Saccharomyces cerevisiae have been recently reported; however, the structures of hNpl4 and the human UN complex remain unknown. Here, we report the crystal structures of the human UN complex at a resolution of 2.7 Å and hNpl4 at a resolution of 3.0 Å. We also present atomic details and characterization of the human UN complex. Crystallographic studies and site-directed mutagenesis of the hUfd1 residues involved in the interaction with hNpl4 revealed the atomic details of the two proteins.

The Crystal Structure of L-Leucine Dehydrogenase from Pseudomonas aeruginosa.

Leucine dehydrogenase (LDH, EC 1.4.1.9) catalyzes the reversible deamination of branched-chain L-amino acids to their corresponding keto acids using NAD+ as a cofactor. LDH generally adopts an octameric structure with D4 symmetry, generating a molecular mass of approximately 400 kDa. Here, the crystal structure of the LDH from Pseudomonas aeruginosa (Pa-LDH) was determined at 2.5 Å resolution. Interestingly, the crystal structure shows that the enzyme exists as a dimer with C2 symmetry in a crystal lattice. The dimeric structure was also observed in solution using multiangle light scattering coupled with size-exclusion chromatography. The enzyme assay revealed that the specific activity was maximal at 60°C and pH 8.5. The kinetic parameters for three different amino acid and the cofactor (NAD+) were determined. The crystal structure represents that the subunit has more compact structure than homologs' structure. In addition, the crystal structure along with sequence alignments indicates a set of non-conserved arginine residues which are important in stability. Subsequent mutation analysis for those residues revealed that the enzyme activity reduced to one third of the wild type. These results provide structural and biochemical insights for its future studies on its application for industrial purposes.

Molecular and Enzymatic Features of Homoserine Dehydrogenase from Bacillus subtilis.

Homoserine dehydrogenase (HSD) catalyzes the reversible conversion of L-aspartate-4- semialdehyde to L-homoserine in the aspartate pathway for the biosynthesis of lysine, methionine, threonine, and isoleucine. HSD has attracted great attention for medical and industrial purposes due to its recognized application in the development of pesticides and is being utilized in the large scale production of L-lysine. In this study, HSD from Bacillus subtilis (BsHSD) was overexpressed in Escherichia coli and purified to homogeneity for biochemical characterization. We examined the enzymatic activity of BsHSD for L-homoserine oxidation and found that BsHSD exclusively prefers NADP+ to NAD+ and that its activity was maximal at pH 9.0 and in the presence of 0.4 M NaCl. By kinetic analysis, Km values for L-homoserine and NADP+ were found to be 35.08 ± 2.91 mM and 0.39 ± 0.05 mM, respectively, and the Vmax values were 2.72 ± 0.06 µmol/min-1 mg-1 and 2.79 ± 0.11 µmol/min-1 mg-1, respectively. The apparent molecular mass determined with size-exclusion chromatography indicated that BsHSD forms a tetramer, in contrast to the previously reported dimeric HSDs from other organisms. This novel oligomeric assembly can be attributed to the additional C-terminal ACT domain of BsHSD. Thermal denaturation monitoring by circular dichroism spectroscopy was used to determine its melting temperature, which was 54.8°C. The molecular and biochemical features of BsHSD revealed in this study may lay the foundation for future studies on amino acid metabolism and its application for industrial and medical purposes.

Biochemical and Molecular Characterization of Glycerol Dehydrogenas from Klebsiella pneumoniae.

Glycerol dehydrogenase (GlyDH) catalyzes the oxidation of glycerol to dihydroxyacetone (DHA), which is the first step in the glycerol metabolism pathway. GlyDH has attracted great interest for its potential industrial applications, since DHA is a precursor for the synthesis of many commercially valuable chemicals and various drugs. In this study, GlyDH from Klebsiella pneumoniae (KpGlyDH) was overexpressed in E. coli and purified to homogeneity for biochemical and molecular characterization. KpGlyDH exhibits an exclusive preference for NAD+ over NADP+. The enzymatic activity of KpGlyDH is maximal at pH 8.6 and pH 10.0. Of the three common polyol substrates, KpGlyDH showed the highest kcat/Km value for glycerol, which is three times higher than for racemic 2,3-butanediol and 32 times higher than for ethylene glycol. The kcat value for glycerol oxidation is notably high at 87.1 ± 11.3 sec-1. KpGlyDH was shown to exist in an equilibrium between two different oligomeric states, octamer and hexadecamer, by size-exclusion chromatography analysis. KpGlyDH is structurally thermostable, with a Tm of 83.4°C, in thermal denaturation experiment using circular dichroism spectroscopy. The biochemical and biophysical characteristics of KpGlyDH revealed in this study should provide the basis for future research on its glycerol metabolism and possible use in industrial applications.

Ufd1 phosphorylation at serine 229 negatively regulates endoplasmic reticulum-associated degradation by inhibiting the interaction of Ufd1 with VCP.

Misfolded proteins in the endoplasmic reticulum (ER) are removed through multistep processes termed ER-associated degradation (ERAD). Valosin-containing protein (VCP) plays a crucial role in ERAD as the interaction of ubiquitin fusion degradation protein 1 (Ufd1) with VCP via its SHP box motif (228F-S-G-S-G-N-R-L235) is required for ERAD. However, the mechanisms by which the VCP-Ufd1 interaction is regulated are not well understood. Here, we found that the serine 229 residue located in the Ufd1 SHP box is phosphorylated in vitro and in vivo by cyclic adenosine monophosphate-dependent protein kinase A (PKA), with this process being enhanced by either forskolin (an adenylyl cyclase activator) or calyculin A (a protein phosphatase inhibitor). Moreover, a phosphomimetic mutant (S229D) of Ufd1 as well as treatment by forskolin, calyculin A, or activated PKA strongly reduced Ufd1 binding affinity for VCP. Consistent with this, the Ufd1 S229D mutant significantly inhibited ERAD leading to the accumulation of ERAD substrates such as a tyrosinase mutant (C89R) and 3-hydroxy-3-methylglutaryl coenzyme A reductase. However, a non-phosphorylatable Ufd1 mutant (S229A) retained VCP-binding ability and was less effective in blocking ERAD. Collectively, our results support that Ufd1 S229 phosphorylation status mediated by PKA serves as a key regulatory point for the VCP-Ufd1 interaction and functional ERAD.

Structural insights into the interaction of human p97 N-terminal domain and SHP motif in Derlin-1 rhomboid pseudoprotease.

The interaction of the rhomboid pseudoprotease Derlin-1 and p97 is crucial for the retrotranslocation of polyubiquitinated substrates in the endoplasmic reticulum-associated degradation pathway. We report a 2.25 Å resolution structure of the p97 N-terminal domain (p97N) in complex with the Derlin-1 SHP motif. Remarkably, the SHP motif adopts a short, antiparallel ß-strand that interacts with the ß-sheet of p97N-a site distinct from that to which most p97 adaptor proteins bind. Mutational and biochemical analyses contributed to defining the specific interaction, demonstrating the importance of a highly conserved binding pocket on p97N and a signature motif on SHP. Our findings may also provide insights into the interactions between other SHP-containing proteins and p97N.

Structural Details of Ufd1 Binding to p97 and Their Functional Implications in ER-Associated Degradation.

The hexameric ATPase p97 has been implicated in diverse cellular processes through interactions with many different adaptor proteins at its N-terminal domain. Among these, the Ufd1-Npl4 heterodimer is a major adaptor, and the p97-Ufd1-Npl4 complex plays an essential role in endoplasmic reticulum-associated degradation (ERAD), acting as a segregase that translocates the ubiquitinated client protein from the ER membrane into the cytosol for proteasomal degradation. We determined the crystal structure of the complex of the N-terminal domain of p97 and the SHP box of Ufd1 at a resolution of 1.55 Å. The 11-residue-long SHP box of Ufd1 binds at the far-most side of the Nc lobe of the p97 N domain primarily through hydrophobic interactions, such that F225, F228, N233 and L235 of the SHP box contact hydrophobic residues on the surface of the p97 Nc lobe. Mutating these key interface residues abolished the interactions in two different binding experiments, isothermal titration calorimetry and co-immunoprecipitation. Furthermore, cycloheximide chase assays showed that these same mutations caused accumulation of tyrosinase-C89R, a well-known ERAD substrate, thus implying decreased rate of protein degradation due to their defects in ERAD function. Together, these results provide structural and biochemical insights into the interaction between p97 N domain and Ufd1 SHP box.

Structural insights into the interaction of p97 N-terminus domain and VBM in rhomboid protease, RHBDL4.

RHBDL4 is an active rhomboid that specifically recognizes and cleaves atypical, positively charged transmembrane endoplasmic reticulum-associated degradation (ERAD) substrates. Interaction of valosin-containing protein (p97/VCP) and RHBDL4 is crucial to retrotranslocate polyubiquitinated substrates for ERAD pathway. Here, we report the first complex structure of VCP-binding motif (VBM) with p97 N-terminal domain (p97N) at 1.88 Šresolution. Consistent with p97 adaptor proteins including p47-ubiquitin regulatory X (UBX), gp78-VCP-interacting motif (VIM), OTU1-UBX-like element, and FAF1-UBX, RHBDL4 VBM also binds at the interface between the two lobes of p97N. Notably, the RF residues in VBM are involved in the interaction with p97N, showing a similar interaction pattern with that of FPR signature motif in the UBX domain, although the directionality is opposite. Comparison of VBM interaction with VIM of gp78, another α-helical motif that interacts with p97N, revealed that the helix direction is inversed. Nevertheless, the conserved arginine residues in both motifs participate in the majority of the interface via extensive hydrogen bonds and ionic interactions with p97N. We identified novel VBM-binding mode to p97N that involves a combination of two types of p97-cofactor specificities observed in the UBX and VIM interactions. This highlights the induced fit model of p97N interdomain cleft upon cofactor binding to form stable p97-cofactor complexes. Our mutational and biochemical analyses in defining the specific interaction between VBM and p97N have elucidated the importance of the highly conserved VBM, applicable to other VBM-containing proteins. We also showed that RHBDL4, ubiquitins, and p97 co-operate for efficient substrate dislocation.

APIP, an ERBB3-binding partner, stimulates erbB2-3 heterodimer formation to promote tumorigenesis.

Despite the fact that the epidermal growth factor (EGF) family member ERBB3 (HER3) is deregulated in many cancers, the list of ERBB3-interacting partners remains limited. Here, we report that the Apaf-1-interacting protein (APIP) stimulates heregulin-ß1 (HRG-ß1)/ERBB3-driven cell proliferation and tumorigenesis. APIP levels are frequently increased in human gastric cancers and gastric cancer-derived cells. Cell proliferation and tumor formation are repressed by APIP downregulation and stimulated by its overexpression. APIP's role in the ERBB3 pathway is not associated with its functions within the methionine salvage pathway. In response to HRG-ß1, APIP binds to the ERBB3 receptor, leading to an enhanced binding of ERBB3 and ERBB2 that results in sustained activations of ERK1/2 and AKT protein kinases. Furthermore, HRG-ß1/ERBB3-dependent signaling is gained in APIP transgenic mouse embryonic fibroblasts (MEFs), but not lost in Apip-/- MEFs. Our findings offer compelling evidence that APIP plays an essential role in ERBB3 signaling as a positive regulator for tumorigenesis, warranting future development of therapeutic strategies for ERBB3-driven gastric cancer.

Crystal structure of Rv2258c from Mycobacterium tuberculosis H37Rv, an S-adenosyl-l-methionine-dependent methyltransferase.

The Mycobacterium tuberculosis Rv2258c protein is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MTase). Here, we have determined its crystal structure in three forms: a ligand-unbound form, a binary complex with sinefungin (SFG), and a binary complex with S-adenosyl-L-homocysteine (SAH). The monomer structure of Rv2258c consists of two domains which are linked by a long α-helix. The N-terminal domain is essential for dimerization and the C-terminal domain has the Class I MTase fold. Rv2258c forms a homodimer in the crystal, with the N-terminal domains facing each other. It also exists as a homodimer in solution. A DALI structural similarity search with Rv2258c reveals that the overall structure of Rv2258c is very similar to small-molecule SAM-dependent MTases. Rv2258c interacts with the bound SFG (or SAH) in an extended conformation maintained by a network of hydrogen bonds and stacking interactions. Rv2258c has a relatively large hydrophobic cavity for binding of the methyl-accepting substrate, suggesting that bulky nonpolar molecules with aromatic rings might be targeted for methylation by Rv2258c in M. tuberculosis. However, the ligand-binding specificity and the biological role of Rv2258c remain to be elucidated due to high variability of the amino acid residues defining the substrate-binding site.

Death effecter domain for the assembly of death-inducing signaling complex.

Death-inducing signaling complex (DISC) is a platform for the activation of initiator caspase in extrinsic apoptosis. Assembly of DISC is accomplished by two different types of homotypic interaction: one is between death domains (DDs) of a death receptor and FADD, and the other is between death effecter domains (DEDs) of FADD, procaspase-8/-10 and cFLIP. Recent biochemical investigations on the stoichiometry of DISC have revealed that single-DED-containing FADD exists in DISC in a substantially lower abundance than the sum of tandem-DEDs-containing components that are procaspase-8 and cFLIP. In addition, the homology models of the tandem DEDs in procaspase-8 and cFLIP show that two different interaction faces, H1-H4 face and H2-H5 face, are exposed for possible inter-molecular DED-DED interactions. These recent findings led to a proposal of the DED chain model for the interactions between FADD, procaspase-8 and cFLIP in DISC. This emerging view provides new insights on the topology of DED-DED network in DISC and furthermore on how procaspase-8 and cFLIP cluster for dimerization and proteolytic activation.

A significant enhancement of water vapour uptake at low pressure by amine-functionalization of UiO-67.

The functionalization of UiO-67 with -NH2 groups enhances CO2 and CH4 adsorption at 1 bar and 298 K and positively influences the framework's interaction with water as evidenced by the significant enhancement of water vapour adsorption at 0.1 < P/P0 < 0.3 and 298 K.

Structure of Mycobacterium smegmatis Eis in complex with paromomycin.

The Rv2416c gene of Mycobacterium tuberculosis (Mtb) encodes the enhanced intracellular survival (Eis) protein that enhances intracellular survival of the pathogen in host macrophages during infection. The Mtb Eis protein is released into the cytoplasm of the phagocyte during intracellular infection and modulates the host immune response. It also contributes to drug resistance by acetylating multiple amine groups of aminoglycosides. Interestingly, the nonpathogenic M. smegmatis (Msm) contains a homologous eis gene (MSMEG_3513). The overall structures of Mtb Eis and Msm Eis are highly similar to each other, reflecting the high level (58%) of amino-acid sequence identity between them. Both Mtb Eis and Msm Eis are active as aminoglycoside acetyltransferases, while only Mtb Eis functions as an N(ℇ)-acetyltransferase to acetylate Lys55 of dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase 7 (MKP-7), leading to the suppression of host immune responses. Here, the crystal structure of Msm Eis in the paromomycin-bound form is reported, revealing detailed interactions between an aminoglycoside antibiotic and Msm Eis. The crystal structure of Msm Eis in the paromomycin-bound form has been determined at 3.3 Šresolution. This work provides potentially useful information for structure-guided discovery of Eis inhibitors as a novel antituberculosis drug against drug-resistant Mtb.

Crystal structure of JHP933 from Helicobacter pylori J99 shows two-domain architecture with a DUF1814 family nucleotidyltransferase domain and a helical bundle domain.

The jhp0933 gene in the plasticity region of Helicobacter pylori J99 encodes a hypothetical protein (JHP933), which may play some roles in pathogenesis. Here, we have determined the crystal structure of JHP933 at 2.17 Å. It represents the first crystal structure of the DUF1814 protein family. JHP933 consists of two domains: an N-terminal domain of the nucleotidyltransferase (NTase) fold and a C-terminal helix bundle domain. A highly positively charged surface patch exists adjacent to the putative NTP binding site. Structural similarity of JHP933 to known NTases is very remote, suggesting that it may function as a novel NTase.

Poly[bis-(µ4-2,3,5,6-tetra-fluoro-benzene-1,4-di-carboxyl-ato-κ(4) O (1):O (1'):O (4):O (4'))bis-(tetra-hydro-furan-κO)dizinc].

The title compound, [Zn2(C8F4O4)2(C4H8O)2] n , has a three-dimensional metal-organic framework structure. The asymmetric unit consists of two Zn(II) atoms, two tetrahydrofuran ligands, one 2,3,5,6-tetra-fluoro-benzene-1,4-di-carboxyl-ate ligand and two half 2,3,5,6-tetra-fluoro-benzene-1,4-di-carboxyl-ate ligands, which are completed by inversion symmetry. One Zn(II) atom has a distorted trigonal-bipyramidal coordination geometry, while the other has a distorted octa-hedral geometry. Two independent tetra-hydro-furan ligands are each disordered over two sets of sites with occupancy ratios of 0.48 (4):0.52 (4) and 0.469 (17):0.531 (17).

Structural and biochemical basis for the inhibition of cell death by APIP, a methionine salvage enzyme.

APIP, Apaf-1 interacting protein, has been known to inhibit two main types of programmed cell death, apoptosis and pyroptosis, and was recently found to be associated with cancers and inflammatory diseases. Distinct from its inhibitory role in cell death, APIP was also shown to act as a 5-methylthioribulose-1-phosphate dehydratase, or MtnB, in the methionine salvage pathway. Here we report the structural and enzymatic characterization of human APIP as an MtnB enzyme with a Km of 9.32 µM and a Vmax of 1.39 µmol min(-1) mg(-1). The crystal structure was determined at 2.0-Å resolution, revealing an overall fold similar to members of the zinc-dependent class II aldolase family. APIP/MtnB exists as a tetramer in solution and exhibits an assembly with C4 symmetry in the crystal lattice. The pocket-shaped active site is located at the end of a long cleft between two adjacent subunits. We propose an enzymatic reaction mechanism involving Glu139* as a catalytic acid/base, as supported by enzymatic assay, substrate-docking study, and sequence conservation analysis. We explored the relationship between two distinct functions of APIP/MtnB, cell death inhibition, and methionine salvage, by measuring the ability of enzymatic mutants to inhibit cell death, and determined that APIP/MtnB functions as a cell death inhibitor independently of its MtnB enzyme activity for apoptosis induced by either hypoxia or etoposide, but dependently for caspase-1-induced pyroptosis. Our results establish the structural and biochemical groundwork for future mechanistic studies of the role of APIP/MtnB in modulating cell death and inflammation and in the development of related diseases.

A docking study of enhanced intracellular survival protein from Mycobacterium tuberculosis with human DUSP16/MKP-7.

The intracellular pathogen Mycobacterium tuberculosis (Mtb) causes tuberculosis, and one of its secreted effector proteins, called enhanced intracellular survival (Eis) protein, enhances its survival in macrophages. Mtb Eis activates JNK-specific dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7) through the acetylation on Lys55, thus inactivating JNK by dephosphorylation. Based on the recently reported crystal structure of Mtb Eis, a docking model for the binding of Mtb Eis to DUSP16/MKP-7 was generated. In the docking model, the substrate helix containing Lys55 of DUSP16/MKP-7 fits nicely into the active-site cleft of Mtb Eis; the twisted ß-sheet of Eis domain II embraces the substrate helix from one side. Most importantly, the side-chain of Lys55 is inserted toward acetyl-CoA and the resulting distance is 4.6 Å between the NZ atom of Lys55 and the carbonyl carbon of the acetyl group in acetyl-CoA. The binding of Mtb Eis and DUSP16/MKP-7 is maintained by strong electrostatic interactions. The active-site cleft of Mtb Eis has a negatively charged surface formed by Asp25, Glu138, Asp286, Glu395 and the terminal carboxylic group of Phe396. In contrast, DUSP16/MKP-7 contains five basic residues, Lys52, Lys55, Arg56, Arg57 and Lys62, which point toward the negatively charged surface of the active-site pocket of Mtb Eis. Thus, the current docking model suggests that the binding of DUSP16/MKP-7 to Mtb Eis should be established by charge complementarity in addition to a very favorable geometric arrangement. The suggested mode of binding requires the dissociation of the hexameric Mtb Eis into dimers or monomers. This study may be useful for future studies aiming to develop inhibitors of Mtb Eis as a new anti-tuberculosis drug candidate.

Poly[(µ6-benzene-1,3,5-tri-carboxyl-ato-κ(6) O (1):O (1'):O (3):O (3'):O (5):O (5'))tris-(N,N-di-methyl-formamide-κO)tris-(µ3-formato-κ(3) O:O:O')trizinc(II)].

The asymmetric unit of the title compound, [Zn3(HCO2)3(C9H3O6)(C3H7NO)3] n , contains one Zn ion, one formate ligand, one N,N-di-methyl-formamide (DMF) ligand and one-third of a benzene-1,3,5-tri-carboxyl-ate (btc) ligand situated on a crystallographic 3 axis. Each Zn(II) atom is coordinated by one O atom from a DMF ligand, two O atoms from two btc ligands and three O atoms from three formate ligands in a distorted octa-hedral geometry. The Zn(II) atoms are connected by the formate and btc ligands, forming hexanuclear cores, which are linked by btc ligands, constructing a two-dimensional layered network along the ab plane.

Crystallization and preliminary X-ray crystallographic analysis of human Apaf-1-interacting protein.

Apaf-1-interacting protein (APIP) is known to inhibit two different types of cell death: caspase-1-dependent pyroptosis and caspase-9-dependent apoptosis. APIP is also involved in the methionine-salvage pathway, where it is called 5-methylthioribulose-1-phosphate dehydratase (MtnB). The enzyme activity seems to be essential for inhibition of pyroptosis by APIP, but not for inhibition of apoptosis. In this study, human APIP was overproduced in Escherichia coli, purified and crystallized. An X-ray diffraction data set was collected to 2.40 Šresolution and the crystals belonged to space group C222(1), with unit-cell parameters a=106.61, b=107.50, c=189.76 Å. Given that four APIP molecules exist in the asymmetric unit, the Matthews coefficient is 2.70 Å3 Da(-1) and the corresponding solvent content is 54.4%.