Volume 393, Issue 10

No abstract available.

The astacins are a family of multi-domain metallopeptidases with manifold functions in metabolism. They are either secreted or membrane-anchored and are regulated by being synthesized as inactive zymogens and also by co-localizing protein inhibitors. The distinct family members consist of N-terminal signal peptides and pro-segments, zinc-dependent catalytic domains, further downstream extracellular domains, transmembrane anchors, and cytosolic domains. The catalytic domains of four astacins and the zymogen of one of these have been structurally characterized and shown to comprise compact ∼200-residue zinc-dependent moieties divided into an N-terminal and a C-terminal sub-domain by an active-site cleft. Astacins include an extended zinc-binding motif (HEXXHXXGXXH) which includes three metal ligands and groups them into the metzincin clan of metallopeptidases. In mature, unbound astacins, a conserved tyrosine acts as an additional zinc ligand, which is swung out upon substrate or inhibitor binding in a ‘tyrosine switch’ motion. Other characteristic structural elements of astacin catalytic domains are three large α-helices and a five-stranded β-sheet, as well as two or three disulfide bonds. The N-terminal pro-segments are variable in length and rather unstructured. They inhibit the catalytic zinc following an ‘aspartate-switch’ mechanism mediated by an aspartate embedded in a conserved motif (FXGD). Removal of the pro-segment uncovers a deep and extended active-site cleft, which in general shows preference for aspartate residues in the specificity pocket (S 1 ′). Furthermore, astacins undergo major rearrangement upon activation within an ‘activation domain,’ and show a slight hinge movement when binding substrates or inhibitors. In this review, we discuss the overall architecture of astacin catalytic domains and their involvement in function and zymogenic activation.

Protein protease inhibitors are the tools of nature in controlling proteolytic enzymes. They come in different shapes and sizes. The β-trefoil protease inhibitors that come from plants, first discovered by Kunitz, were later complemented with representatives from higher fungi. They inhibit serine (families S1 and S8) and cysteine proteases (families C1 and C13) as well as other hydrolases. Their versatility is the result of the plasticity of the loops coming out of the stable β-trefoil scaffold. For this reason, they display several different mechanisms of inhibition involving different positions of the loops and their combinations. Natural diversity, as well as the initial successes in de novo protein engineering, makes the β-trefoil proteins a promising starting point for the generation of strong, specific, multitarget inhibitors capable of inhibiting multiple types of hydrolytic enzymes and simultaneously interacting with different protein, carbohydrate, or DNA molecules. This pool of knowledge opens up new possibilities for the exploration of their naturally occurring as well as modified properties for applications in many fields of medicine, biotechnology, and agriculture.

Phytopathogen transcription activator-like effectors (TALEs) bind DNA in a sequence specific manner in order to manipulate host transcription. TALE specificity correlates with repeat variable diresidues in otherwise highly stereotypical 34–35mer repeats. Recently, the crystal structures of two TALE DNA-binding domains have illustrated the molecular basis of the TALE cipher. The structures show that the TALE repeats form a right-handed superhelix that is wound around largely undistorted B-DNA to match its helical parameters. Surprisingly, repeat variable residue 1 is not in contact with the bases. Instead, it is involved in hydrogen bonding interactions that stabilize the overall structure of the protein. Repeat variable residue 2 contacts the top strand base and forms sequence-specific hydrogen bonds and/or van der Waals contacts. Very unexpectedly, bottom strand bases are exposed to solvent and do not make any direct contacts with the protein. This review contains a summary of TALE biology and applications and a detailed description of the recent breakthroughs that have provided insights into the molecular basis of the TALE code.

The tetranuclear Cu Z cluster is the unique active site of nitrous oxide reductase, the enzyme that catalyzes the reduction of nitrous oxide to dinitrogen as the final reaction in bacterial denitrification. Three-dimensional structures of orthologs of the enzyme from a variety of different bacterial species were essential steps in the elucidation of the properties of this center. However, while structural data first revealed and later confirmed the presence of four copper ions in spectroscopically distinct forms of Cu Z , the exact structure and stoichiometry of the cluster showed significant variations. A ligand bridging ions Cu Z1 and Cu Z2 was initially assigned as a water or hydroxo species in the structures from Pseudomonas nautica (now Marinobacter hydrocarbonoclasticus ) and Paracoccus denitrificans . This ligand was absent in a structure from ‘ Achromobacter cycloclastes ’, and could be reconstituted by iodide that acted as an inhibitor of catalysis. A recent structure of anoxically isolated nitrous oxide reductase from Pseudomonas stutzeri revealed the bridging ligand to be sulfide, S 2- , and showed an unprecedented side-on mode of nitrous oxide binding to this form of Cu Z .

ATP binding cassette proteins are a large and diverse family of molecular machines and include transmembrane transporter, chromosome maintenance and DNA repair proteins, and translation factors. However, the function of the ABCE1, the only member of subfamily E of ABC proteins, remained mysterious for over a decade, even though it is perhaps the most conserved ABC protein in eukaryotes and archaea. Recent results have now identified ABCE1 as the ribosome-recycling factor of eukaryotes and archaea. Thus, two iron-sulfur clusters – the hallmark feature of ABCE1 – help catalyze an integral step of the translational cycle at the core of the protein synthesis machinery.

[NiFe] hydrogenases catalyze reversible hydrogen production/consumption. The active site of [NiFe] hydrogenases contains a complex NiFe(CN) 2 CO center, and the biosynthesis/maturation of these enzymes is a complex and dynamic process, primarily involving six Hyp proteins (HypABCDEF). HypA and HypB are involved in the Ni insertion, whereas the other four Hyp proteins (HypCDEF) are required for the biosynthesis, assembly and insertion of the Fe(CN) 2 CO group. Over the last decades, a large number of functional and structural studies on maturation proteins have been performed, revealing detailed functions of each Hyp protein and the framework of the maturation pathway. This article will focus on recent advances in structural studies of the Hyp proteins and on mechanistic insights into the [NiFe] hydrogenase maturation.

The 20S proteasome core particle (CP) is the proteolytically active key element of the ubiquitin proteasome system that directs the majority of intracellular protein degradation in eukaryotic cells. Over the past decade, the CP has emerged as an anticancer therapy target after approval of the first-in-class drug bortezomib (Velcade ® ) by the US Food and Drug Administration. However, bortezomib and all second-generation CP inhibitors that are currently explored in clinical phase studies react covalently and most often irreversibly with the proteolytic sites of the CP, hereby causing permanent CP blockage. Furthermore, reactive head groups result in unspecific binding to proteasomal active centers and in substantial enzymatic off-target activities that translate to severe side effects. Thus, reversible proteasome inhibitors might be a promising alternative, overcoming these drawbacks, but are challenging with respect to their urge for thorough enthalpic and entropic optimization. This review describes developments in the hitherto neglected field of reversible proteasome inhibitors focusing on insights gained from crystal structures, which provide valuable knowledge and strategies for future directions in drug development.

The era of structure-based protein kinase inhibitor design began in the early 1990s with the determination of crystal structures of protein kinase A (PKA, or cyclic AMP-dependent kinase). Although many other protein kinases have since been extensively characterized, PKA remains a prototype for studies of protein kinase active conformations. It serves well as a model for the structural properties of AGC subfamily protein kinases, clarifying inhibitor selectivity profiles. Its reliable expression, constitutive activity, simple domain structure, and reproducible crystallizability have also made it a useful surrogate for the discovery of inhibitors of both established and emerging AGC kinase targets.

Splicing of pre-mRNA requires the activity of at least eight different DEAD/H-box proteins that are involved in distinct steps of the splicing process. These proteins are driving the spliceosomal machinery by ATP-dependent unwinding of dsRNA and/or disrupting protein-RNA complexes. The spliceosomal DEAH-box proteins Prp2, Prp16, Prp22 and Prp43 share homologous C-terminal domains (CTD). We have determined the crystal structure of the CTD of human Prp22 by means of MAD. The fold of the human Prp22-CTD closely resembles that of the yeast Prp43-CTD. The similarity of these helicase-associated CTDs to the winged-helix and ratchet domains of the DNA helicase Hel308 suggests an analogous function in dsRNA binding and unwinding. Here, we also demonstrate that the CTD has a significant impact on the ATPase activity of yPrp22 in vitro . Homology modeling of the CTDs of hPrp2, hPrp16 and hPrp43 suggests that the CTDs of spliceosomal helicases contain conserved positively charged patches on their surfaces representing a common RNA-binding surface as well as divergent regions most likely mediating specific interactions with different proteins of the spliceosome.

Annexin A2 (AnxA2), a Ca 2+ -regulated phospholipid binding protein involved in membrane-cytoskeleton contacts and membrane transport, exists in two physical states, as a monomer or in a heterotetrameric complex mediated by S100A10. Formation of the AnxA2-S100A10 complex is of crucial regulatory importance because only the complex is firmly anchored in the plasma membrane, where it functions in the plasma membrane targeting/recruitment of certain ion channels and receptors. The S100A10 binding motif is located in the first 12 residues of the AnxA2 N-terminal domain, but conflicting reports exist as to the importance of N-terminal AnxA2 acetylation with regard to S100A10 binding. We show here that AnxA2 is subject to N-terminal modification when expressed heterologously in Escherichia coli . Met1 is removed and Ser2 is acetylated, yielding the same modification as the authentic mammalian protein. Bacterially expressed and N-terminally acetylated AnxA2 binds S100A10 with an affinity comparable to AnxA2 from porcine tissue and is capable of forming the AnxA2-S100A10 heterotetramer. Complex formation is competitively inhibited by acetylated but not by non-acetylated peptides covering the N-terminal AnxA2 sequence. These results demonstrate that N-terminal acetylation of AnxA2 is required for S100A10 binding and that this common eukaryotic modification is also obtained upon expression in bacteria.

Annexin A2 (AnxA2), a 38-kDa member of the Ca 2+ -binding annexin family, has been implicated in numerous cancer pathways. Withaferin A (WithfA), a natural plant compound, has been reported previously to bind covalently to Cys133 of the AnxA2 core domain leading to a reduction of the invasive capabilities of cancer cells by altering their cytoskeleton. We show here that AnxA2 has an inhibitory effect on actin polymerization, and a modification with WithfA significantly increases this inhibitory role of AnxA2. Using mass spectrometry and single-site mutants, we localized the WithfA-AnxA2 interaction to the N-terminal domain of AnxA2 where WithfA binds covalently to Cys9. Whereas binding to F-actin filaments has been mapped to the C terminus of AnxA2, our results suggest that the N-terminal domain modified by WithfA may also play a role in the AnxA2-actin interaction. The binding of WithfA may regulate the AnxA2-mediated actin dynamics in two distinct ways: (i) the increase of F-actin bundling activity by the Anx2/p11 heterotetramer and (ii) the decrease of actin polymerization as a result of the increased affinity of AnxA2 to the barbed end of actin microfilaments. We demonstrate the susceptibility of Cys9 of AnxA2 to chemical modifications and exclude Cys133 as a binding site for WithfA.

The PhoR-PhoB phosphorelay is a bacterial two-component system that activates the transcription of several genes involved in phosphate uptake and assimilation. The response begins with the autophosphorylation of the sensor kinase PhoR, which activates the response regulator PhoB. Upon binding to the pho box DNA sequence, PhoB recruits the RNA polymerase and thereby activates the transcription of specific genes. To unveil hitherto unknown molecular mechanisms along the activation pathway, we report biochemical data characterizing the PhoB binding to promoters containing multiple pho boxes and describe the crystal structure of two PhoB DNA-binding domains bound in tandem to a 26-mer DNA.

Human phospholipid scramblase 4 (hPLSCR4), an isoform of the scramblase family, is a type II single-pass transmembrane protein whose function remains unknown. To understand its role, recombinant hPLSCR4 was obtained by cloning the ORF into a pET28 a(+) vector and overexpressed in Escherichia coli . Functional assay showed that Ca 2+ , Mg 2+ , and Zn 2+ activate hPLSCR4 and mediate scrambling activity independent of the phospholipid head group. Far-UV-CD and fluorescence spectroscopy revealed that Ca 2+ and Mg 2+ binding induces conformation change in hPLSCR4, exposing hydrophobic patches of the protein, and Ca 2+ has more affinity than Mg 2+ and Zn 2+ . Stains-all studies further confirm that hPLSCR4 is a Ca 2+ -binding protein. Point mutation (Asp 290 →Ala) in hPLSCR4 decreased the Ca 2+ -binding affinity as well as Tb 3+ luminescence, suggesting residues of the predicted Ca 2+ -binding motif are involved in Ca 2+ binding. Functional reconstitution with (Asp 290 →Ala) mutant led to ∼50% and ∼40% decrease in scramblase activity in the presence of Ca 2+ and Mg 2+ , respectively.

The Wnt/β-catenin and the Ras/mitogen-activated protein kinase (MAPK) pathways play important roles in cancer development. Both pathways have been studied discretely, but the mechanisms of possible crosstalk are still not fully understood. We have previously shown that β-catenin and MAPK signaling interfere with each other in murine liver in vivo and in vitro . Here, we show that dual specificity phosphatases (Dusps) 6 and 14, known to play an essential role in regulating MAPK pathway activity via feedback mechanisms, are up-regulated by activation of β-catenin in murine liver cells, whereas the epidermal growth factor receptor, an upstream effector in the Ras/MAPK cascade, is down-regulated by β-catenin. In addition, we identified a β-catenin-binding site within the Dusp6 promoter, which is responsible for the activation of the promoter by β-catenin signaling, and demonstrated reduced inducibility of MAPK signaling in cultured mouse hepatoma cells following β-catenin activation. Thus, β-catenin is able to inhibit activation of the Egfr/Ras/MAPK signaling cascade, both at the receptor level and by interfering with MAPK activity via Dusps.

ASP is a serine protease secreted by Aeromonas sobria . ASP cleaves various plasma proteins, which is associated with onset of sepsis complications, such as shock and blood coagulation disorder. To investigate a host defense mechanism against this virulence factor, we examined the plasma for ASP inhibitor(s). Human plasma inhibited ASP activity for azocasein, which was almost completely abolished by treating plasma with methylamine, which inactivates α 2 -macroglobulin (α 2 -MG). The ASP-inhibitor complex in ASP-added plasma was not detected by immunoblotting using anti-ASP antibody; however, using gel filtration of the plasma ASP activity for an oligopeptide, the ASP substrate was eluted in the void fraction ( M w >200 000), suggesting ASP trapping by α 2 -MG. Indeed, human α 2 -MG inhibited ASP azocaseinolytic activity in a dose-dependent manner, rapidly forming a complex with the ASP. Fibrinogen degradation by ASP was completely inhibited in the presence of α 2 -MG. α 1 -Protease inhibitor, antithrombin, and α 2 -plasmin inhibitor neither inhibited ASP activity nor formed a complex with ASP. Surprisingly, ASP degraded these plasma serine protease inhibitors. Thus, α 2 -MG is the major ASP inhibitor in the human plasma and can limit ASP virulence activities in A. sobria infection sites. However, as shown by fluorescence correlation spectroscopy, slow ASP inhibition by α 2 -MG in plasma may indicate insufficient ASP control in vivo .