The three-dimensional structure of VIM-2, a Zn-beta-lactamase from Pseudomonas aeruginosa in its reduced and oxidised form

The crystal structures of the universally widespread metallo-β-lactamase (MBL) Verona integron-encoded MBL (VIM)-2 from Pseudomonas aeruginosa have been solved in their native form as well as in an unexpected oxidised form. This carbapenem-hydrolysing enzyme belongs to the so-called B1 subfamily of MBLs and shares the folding of αβ/βα sandwich, consisting of a core of β-sheet surrounded by α-helices. Surprisingly, it showed a high tendency to be strongly oxidised at the catalytic cysteine located in the Cys site, Cys221, which, in the oxidised structure, becomes a cysteinesulfonic residue. Its native structure was obtained only in the presence of Tris(2-carboxyethyl)phosphine. This oxidation might be a consequence of a lower affinity for the second Zn located in the Cys site that would also explain the observed susceptibility of VIM-2 to chelating agents. This modification, if present in nature, might play a role in catalytic down-regulation. Comparison between native and oxidised VIM-2 and a predicted model of VIM-1 (which shows one residue different in the Cys site compared with VIM-2) is performed to explain the different activities and antibiotic specificities.     

Spectroscopy and a high-resolution crystal structure of Tyr263 mutants of cyanobacterial phytochrome Cph1

Phytochromes are biliprotein photoreceptors that can be photoswitched between red-light-absorbing state (Pr) and far-red-light-absorbing state (Pfr). Although three-dimensional structures of both states have been reported, the photoconversion and intramolecular signaling mechanisms are still unclear. Here, we report UV-Vis absorbance, fluorescence and CD spectroscopy along with various photochemical parameters of the wild type and Y263F, Y263H and Y263S mutants of the Cph1 photosensory module, as well as a 2.0-Å-resolution crystal structure of the Y263F mutant in its Pr ground state. Although Y263 is conserved, we show that the aromatic character but not the hydroxyl group of Y263 is important for Pfr formation. The crystal structure of the Y263F mutant (Protein Data Bank ID: 3ZQ5) reaffirms the ZZZssa chromophore configuration and provides a detailed picture of its binding pocket, particularly conformational heterogeneity around the chromophore. Comparison with other phytochrome structures reveals differences in the relative position of the PHY (phytochrome specific) domain and the interaction of the tongue with the extreme N-terminus. Our data support the notion that native phytochromes in their Pr state are structurally heterogeneous.     

A novel lipid binding site formed by the MAP kinase insert in p38 alpha

The p38 mitogen-activated protein (MAP) kinases function as signaling molecules essential for many cellular processes, particularly mediating stress response. The activity of p38 MAP kinases is meticulously regulated to reach the desired cellular phenotype. Several alternative activation and attenuation mechanisms have been characterized recently which include new phosphorylation sites. Here we present the crystal structure of p38α MAP kinase in complex with n-octyl-β-glucopyranoside detergent. The complex unveils a novel lipid-binding site formed by a local conformational change of the MAP kinase insert. This binding is the first attribution for a possible role of the MAP kinase insert in p38. The binding site can accommodate a large selection of lipidic molecules. In addition, we also show via biophysical methods that arachidonic acid and its derivatives bind p38α in vitro. Based on our analysis we propose that the binding of lipids could fine-tune p38α catalytic activity towards a preferred phenotype.     

The crystal structure of Cupriavidus necator nitrate reductase in oxidized and partially reduced states

The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein that belongs to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes and catalyzes the reduction of nitrate to nitrite. The protein comprises a large catalytic subunit (NapA, 91 kDa) containing the molybdenum active site plus one [4Fe-4S] cluster, as well as a small subunit (NapB, 17 kDa), which is a diheme c-type cytochrome involved in electron transfer. Crystals of the oxidized form of the enzyme diffracted beyond 1.5 Å at the European Synchrotron Radiation Facility. This is the highest resolution reported to date for a nitrate reductase, providing true atomic details of the protein active center, and this showed further evidence on the molybdenum coordination sphere, corroborating previous data on the related Desulfovibrio desulfuricans NapA. The molybdenum atom is bound to a total of six sulfur atoms, with no oxygen ligands or water molecules in the vicinity. In the present work, we were also able to prepare partially reduced crystals that revealed two alternate conformations of the Mo-coordinating cysteine. This crystal form was obtained by soaking dithionite into crystals grown in the presence of the ionic liquid [C₄mim]Cl⁻. In addition, UV-Vis and EPR spectroscopy studies showed that the periplasmic nitrate reductase from C. necator might work at unexpectedly high redox potentials when compared to all periplasmic nitrate reductases studied to date.     

The crystal structure of an ADP complex of Bacillus subtilis pyridoxal kinase provides evidence for the parallel emergence of enzyme activity during evolution

Pyridoxal kinase catalyses the phosphorylation of pyridoxal, pyridoxine and pyridoxamine to their 5' phosphates and plays an important role in the pyridoxal 5' phosphate salvage pathway. The crystal structure of a dimeric pyridoxal kinase from Bacillus subtilis has been solved in complex with ADP to 2.8 A resolution. Analysis of the structure suggests that binding of the nucleotide induces the ordering of two loops, which operate independently to close a flap on the active site. Comparisons with other ribokinase superfamily members reveal that B. subtilis pyridoxal kinase is more closely related in both sequence and structure to the family of HMPP kinases than to other pyridoxal kinases, suggesting that this structure represents the first for a novel family of "HMPP kinase-like" pyridoxal kinases. Moreover this further suggests that this enzyme activity has evolved independently on multiple occasions from within the ribokinase superfamily.     

Hydrogen-exchange mass spectrometry reveals activation-induced changes in the conformational mobility of p38alpha MAP kinase

Hydrogen-deuterium exchange measurements represent a powerful approach to investigating changes in conformation and conformational mobility in proteins. Here, we examine p38α MAP kinase (MAPK) by hydrogen-exchange (HX) mass spectrometry to determine whether changes in conformational mobility may be induced by kinase phosphorylation and activation. Factors influencing sequence coverage in the HX mass spectrometry experiment, which show that varying sampling depths, instruments, and peptide search strategies yield the highest coverage of exchangeable amides, are examined. Patterns of regional deuteration in p38α are consistent with tertiary structure and similar to deuteration patterns previously determined for extracellular-signal-regulated kinase (ERK) 2, indicating that MAPKs are conserved with respect to the extent of local amide HX. Activation of p38α alters HX in five regions, which are interpreted by comparing X-ray structures of unphosphorylated p38α and X-ray structures of phosphorylated p38γ. Conformational differences account for altered HX within the activation lip, the P + 1 site, and the active site. In contrast, HX alterations are ascribed to activation-induced effects on conformational mobility, within substrate-docking sites (αF-αG, β7-β8), the C-terminal core (αE), and the N-terminal core region (β4-β5, αL16, αC). Activation also decreases HX in a 3-10 helix at the C-terminal extension of p38α. Although this helix in ERK2 forms a dimerization interface that becomes protected from HX upon activation, analytical ultracentrifugation shows that this does not occur in p38α because both unphosphorylated and diphosphorylated forms are monomeric. Finally, HX patterns in monophosphorylated p38α are similar to those in unphosphorylated kinase, indicating that the major activation lip remodeling events occur only after diphosphorylation. Importantly, patterns of activation-induced HX show differences between p38α and ERK2 despite their similarities in overall deuteration, suggesting that although MAPKs are closely related with respect to primary sequence and tertiary structure, they have distinct mechanisms for dynamic control of enzyme function.     

The regulation of Bax by c-Jun N-terminal protein kinase (JNK) is a prerequisite to the mitochondrial-induced apoptotic pathway

The signaling mechanism by which JNK affects mitochondria is critical to initiate apoptosis. Here we show that the absence of JNK provides a partial resistance to the toxic effect of the heavy metal cadmium. Both wild type and jnk−/− fibroblasts undergoing death exhibit cytosolic cytochrome c but, unlike wild type cells, the JNK-deficient fibroblasts do not display increased caspase activity and DNA fragmentation. The absence of apoptotic death correlates with a specific defect in activation of Bax. We conclude that JNK-dependent regulation of Bax is essential to mediate the apoptotic release of cytochrome c regardless of Bid and Bim activation.     

The crystal structure of UMP kinase from Bacillus anthracis (BA1797) reveals an allosteric nucleotide-binding site

Uridine monophosphate (UMP) kinase is a conserved enzyme that catalyzes the ATP-driven conversion of uridylate monophosphate into uridylate diphosphate, an essential metabolic step. In prokaryotes, the enzyme exists as a homohexamer that is regulated by various metabolites. Whereas the enzymatic mechanism of UMP kinase (UK) is well-characterized, the molecular basis of its regulation remains poorly understood. Here we report the crystal structure of UK from Bacillus anthracis (BA1797) in complex with ATP at 2.82 Å resolution. It reveals that the cofactor, in addition to binding in the active sites, also interacts with separate binding pockets located near the center of the hexameric structure. The existence of such an allosteric binding site had been predicted by biochemical studies, but it was not identified in previous crystal structures of prokaryotic UKs. We show that this putative allosteric pocket is conserved across different bacterial species, suggesting that it is a feature common to bacterial UKs, and we present a structural model for the allosteric regulation of this enzyme.     

Role of bundle helices in a regulatory crosstalk in the trimeric betaine transporter BetP

The Na⁺-coupled betaine symporter BetP regulates transport activity in response to hyperosmotic stress only in its trimeric state, suggesting a regulatory crosstalk between individual protomers. BetP shares the overall fold of two inverted structurally related five-transmembrane (TM) helix repeats with the sequence-unrelated Na⁺-coupled symporters LeuT, vSGLT, and Mhp1, which are neither trimeric nor regulated in transport activity. Conformational changes characteristic for this transporter fold involve the two first helices of each repeat, which form a four-TM-helix bundle. Here, we identify two ionic networks in BetP located on both sides of the membrane that might be responsible for BetP's unique regulatory behavior by restricting the conformational flexibility of the four-TM-helix bundle. The cytoplasmic ionic interaction network links both first helices of each repeat in one protomer to the osmosensing C-terminal domain of the adjacent protomer. Moreover, the periplasmic ionic interaction network conformationally locks the four-TM-helix bundle between the same neighbor protomers. By a combination of site-directed mutagenesis, cross-linking, and betaine uptake measurements, we demonstrate how conformational changes in individual bundle helices are transduced to the entire bundle by specific inter-helical interactions. We suggest that one purpose of bundle networking is to assist crosstalk between protomers during transport regulation by specifically modulating the transition from outward-facing to inward-facing state.     

The role of p38 MAP kinase and c-Jun N-terminal protein kinase signaling in the differentiation and apoptosis of immortalized neural stem cells

The two distinct members of the mitogen-activated protein (MAP) kinase family c-Jun N-terminal protein kinase (JNK) and p38 MAP kinase, play an important role in central nervous system (CNS) development and differentiation. However, their role and functions are not completely understood in CNS. To facilitate in vitro study, we have established an immortal stem cell line using SV40 from fetal rat embryonic day 17. In these cells, MAP kinase inhibitors (SP600125, SB202190, and PD98059) were treated for 1, 24, 48, and 72 h to examine the roles of protein kinases. Early inhibition of JNK did not alter phenotypic or morphological changes of immortalized cells, however overexpression of Bax and decrease of phosphorylated AKT was observed. The prolonged inhibition of JNK induced polyploidization of immortalized cells, and resulted in differentiation and inhibition of cell proliferation. Moreover, JNK and p38 MAP kinase but not ERK1/2 was activated, and p21, p53, and Bax were overexpressed by prolonged inhibition of JNK.

These results indicate that JNK and p38 MAP kinase could play dual roles on cell survival and apoptosis. Furthermore, this established cell line could facilitate study of the role of JNK and p38 MAP kinase on CNS development or differentiation/apoptosis.     

The crystal structure of l-sorbose reductase from Gluconobacter frateurii complexed with NADPH and l-sorbose

l-Sorbose reductase from Gluconobacter frateurii (SR) is an NADPH-dependent oxidoreductase. SR preferentially catalyzes the reversible reaction between d-sorbitol and l-sorbose with high substrate specificity. To elucidate the structural basis of the catalytic mechanism and the substrate specificity of SR, we have determined the structures of apo-SR, SR in complex with NADPH, and the inactive mutant (His116Leu) of SR in complex with NADPH and l-sorbose at 2.83 Å, 1.90 Å, and 1.80 Å resolutions, respectively. Our results show that SR belongs to the short-chain dehydrogenase/reductase (SDR) family and forms a tetrameric structure. Although His116 is not conserved among SDR family enzymes, the structures of SR have revealed that His116 is important for the stabilization of the proton relay system and for active-site conformation as a fourth catalytic residue. In the ternary complex structure, l-sorbose is recognized by 11 hydrogen bonds. Site-directed mutagenesis of residues around the l-sorbose-binding site has shown that the loss of almost full enzymatic activity was caused by not only the substitution of putative catalytic residues but also the substitution of the residue used for the recognition of the C4 hydroxyl groups of l-sorbose (Glu154) and of the residues used for the construction of the substrate-binding pocket (Cys146 and Gly188). The recognition of the C4 hydroxyl group of l-sorbose would be indispensable for the substrate specificity of SR, which recognizes only l-sorbose and d-sorbitol but not other sugars. Our results indicated that these residues were crucial for the substrate recognition and specificity of SR.     

Crystal structures of MKK4 kinase domain reveal that substrate peptide binds to an allosteric site and induces an auto-inhibition state

MKK4 activates both JNKs and p38s. We determined the crystal structures of human non-phosphorylated MKK4 kinase domain (npMKK4) complexed with AMP-PNP (npMKK4/AMP) and a ternary complex of npMKK4, AMP-PNP and p38α peptide (npMKK4/AMP/p38). These crystal structures revealed that the p38α peptide-bound npMKK4 at the allosteric site rather than at the putative substrate binding site and induced an auto-inhibition state. While the activation loop of the npMKK4/AMP complex was disordered, in the npMKK4/AMP/p38 complex it configured a long α-helix, which prevented substrate access to the active site and αC-helix movement to the active configuration of MKK4.     

Denaturational stress induces formation of zinc-deficient monomers of Cu,Zn superoxide dismutase: implications for pathogenesis in amyotrophic lateral sclerosis

Mutations in the Cu,Zn superoxide dismutase (SOD1) cause a subset of amyotrophic lateral sclerosis cases. SOD1 is a homodimer in which each monomer binds one copper atom and one zinc atom. Mutation is believed to increase the conformational flexibility of SOD1, giving rise to a misfolded SOD1 population with novel cytotoxic properties. While SOD1's metal ligands affect its stability greatly, little is known about the role these metals play in the folding, unfolding, and misfolding processes. Here, we present a method by which we were able to measure the rates of metal release during SOD1 unfolding in guanidine hydrochloride. Rates of dimer dissociation, measured by a time-resolved cross-linking assay, and conformational changes in SOD1's β-barrel core, monitored by tryptophan fluorescence intensity, were compared with the rates of copper release and zinc release. Correlations were observed across a range of denaturant concentrations, giving rise to a more detailed model of the SOD1 unfolding process than was previously available. According to this model, the major unfolding pathway involves simultaneous dimer dissociation and zinc release as an early step that is followed by a slow conformational change in the protein's core, which, in turn, is followed by rapid copper release. This model establishes a zinc-deficient, copper-loaded SOD1 monomer as a well-populated SOD1 unfolding intermediate and a species likely to be populated under conditions of denaturational stress. Because the cytotoxicity of zinc-deficient SOD1 has been demonstrated previously, this species is a good candidate for the cytotoxic species in SOD1-associated amyotrophic lateral sclerosis.     

Crystal structure of the catalytic domain of human MAP kinase phosphatase 5: structural insight into constitutively active phosphatase

MAP kinase phosphatase 5 (MKP5) is a member of the mitogen-activated protein kinase phosphatase (MKP) family and selectively dephosphorylates JNK and p38. We have determined the crystal structure of the catalytic domain of human MKP5 (MKP5-C) to 1.6 A. In previously reported MKP-C structures, the residues that constitute the active site are seriously deviated from the active conformation of protein tyrosine phosphatases (PTPs), which are accompanied by low catalytic activity. High activities of MKPs are achieved by binding their cognate substrates, representing substrate-induced activation. However, the MKP5-C structure adopts an active conformation of PTP even in the absence of its substrate binding, which is consistent with the previous results that MKP5 solely possesses the intrinsic activity. Further, we identify a sequence motif common to the members of MKPs having low catalytic activity by comparing structures and sequences of other MKPs. Our structural information provides an explanation of constitutive activity of MKP5 as well as the structural insight into substrate-induced activation occurred in other MKPs.     

High-resolution crystal structure of activated Cyt2Ba monomer from Bacillus thuringiensis subsp. israelensis

The Cyt family of proteins consists of δ-endotoxins expressed during sporulation of several subspecies of Bacillus thuringiensis. Its members possess insecticidal, hemolytic, and cytolytic activities through pore formation and attract attention due to their potential use as vehicles for targeted membrane destruction. The δ-endotoxins of subsp. israelensis include three Cyt species: a major Cyt1Aa and two minor proteins, Cyt2Ba and Cyt1Ca. A cleaved Cyt protein that lacks the N- and C-terminal segments forms a toxic monomer. Here, we describe the crystal structure of Cyt2Ba, cleaved at its amino and carboxy termini by bacterial endogenous protease(s). Overall, its fold resembles that of the previously described volvatoxin A2 and the nontoxic form of Cyt2Aa. The structural similarity between these three proteins may provide information regarding the mechanism(s) of membrane-perforating toxins.     

The 2.7 A crystal structure of the autoinhibited human c-Fms kinase domain

c-Fms, a member of the Platelet-derived Growth Factor (PDGF) receptor family of receptor tyrosine kinases (RTKs), is the receptor for macrophage colony stimulating factor (CSF-1) that regulates proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. Abnormal expression of c-fms proto-oncogene is associated with a significant number of human pathologies, including a variety of cancers and rheumatoid arthritis. Accordingly, c-Fms represents an attractive therapeutic target. To further understand the regulation of c-Fms, we determined the 2.7 A resolution crystal structure of the cytosolic domain of c-Fms that comprised the kinase domain and the juxtamembrane domain. The structure reveals the crucial inhibitory role of the juxtamembrane domain (JM) that binds to a hydrophobic site immediately adjacent to the ATP binding pocket. This interaction prevents the activation loop from adopting an active conformation thereby locking the c-Fms kinase into an autoinhibited state. As observed for other members of the PDGF receptor family, namely c-Kit and Flt3, three JM-derived tyrosine residues primarily drive the mechanism for autoinhibition in c-Fms, therefore defining a common autoinhibitory mechanism within this family. Moreover the structure provides an understanding of c-Fms inhibition by Gleevec as well as providing a platform for the development of more selective inhibitors that target the inactive conformation of c-Fms kinase.     

Functional implications of the conformational switch in AICD peptide upon binding to Grb2-SH2 domain

It has been hypothesized previously that synergistic effect of both amyloid precursor protein intracellular C-terminal domain (AICD) and Aβ aggregation could contribute to Alzheimer's disease pathogenesis. Structural studies of AICD have found no stable globular fold over a broad range of pH. Present work is based on the premises that a conformational switch involving the flipping of C-terminal helix of AICD would be essential for effective binding with the Src homology 2 (SH2) domain of growth factor receptor binding protein-2 (Grb2) and subsequent initiation of Grb2-mediated endo-lysosomal pathway. High-resolution crystal structures of Grb2-SH2 domain bound to AICD peptides reveal a unique mode of binding where the peptides assume a noncanonical conformation that is unlike other structures of AICD peptides bound to protein-tyrosine-binding domains or that of its free state; rather, a flipping of the C-terminal helix of AICD is evident. The involvement of different AICD residues in Grb2-SH2 interaction is further elucidated through fluorescence-based assays. Our results reveal the significance of a specific interaction of the two molecules to optimize the rapid transport of AICD inside endosomal vesicles presumably to reduce the cytotoxic load.