Structure and action of urocanase

Urocanase (EC 4.2.1.49) from Pseudomonas putida was crystallized after removing one of the seven free thiol groups. The crystal structure was solved by multiwavelength anomalous diffraction (MAD) using a seleno-methionine derivative and then refined at 1.14 A resolution. The enzyme is a symmetric homodimer of 2 x 557 amino acid residues with tightly bound NAD+ cofactors. Each subunit consists of a typical NAD-binding domain inserted into a larger core domain that forms the dimer interface. The core domain has a novel chain fold and accommodates the substrate urocanate in a surface depression. The NAD domain sits like a lid on the core domain depression and points with the nicotinamide group to the substrate. Substrate, nicotinamide and five water molecules are completely sequestered in a cavity. Most likely, one of these water molecules hydrates the substrate during catalysis. This cavity has to open for substrate passage, which probably means lifting the NAD domain. The observed atomic arrangement at the active center gives rise to a detailed proposal for the catalytic mechanism that is consistent with published chemical data. As expected, the variability of the residues involved is low, as derived from a family of 58 proteins annotated as urocanases in the data banks. However, one well-embedded member of this family showed a significant deviation at the active center indicating an incorrect annotation.     

The regulatory factor SipA is a highly stable β-II class protein with a SH3 fold

The small regulator SipA, interacts with the ATP-binding domain of non-bleaching sensor histidine kinase (NblS), the most conserved histidine kinase in cyanobacteria. NblS regulates photosynthesis and acclimation to a variety of environmental conditions. We show here that SipA is a highly stable protein in a wide pH range, with a thermal denaturation midpoint of 345 K. Circular dichroism and 1D ¹H NMR spectroscopies, as well as modelling, suggest that SipA is a β-II class protein, with short strands followed by turns and long random-coil polypeptide patches, matching the SH3 fold. The experimentally determined m-value and the heat capacity change upon thermal unfolding (ΔC_p) closely agreed with the corresponding theoretical values predicted from the structural model, further supporting its accuracy.     

Intramembrane-sensing histidine kinases: a new family of cell envelope stress sensors in Firmicutes bacteria

Two-component signal-transducing systems (TCS) consist of a histidine kinase (HK) that senses a specific environmental stimulus, and a cognate response regulator (RR) that mediates the cellular response. Most HK are membrane-anchored proteins harboring two domains: An extracytoplasmic input and a cytoplasmic transmitter (or kinase) domain, separated by transmembrane helices that are crucial for the intramolecular information flow. In contrast to the cytoplasmic domain, the input domain is highly variable, reflecting the plethora of different signals sensed. Intramembrane-sensing HK (IM-HK) are characterized by their short input domain, consisting solely of two putative transmembane helices. They lack an extracytoplasmic domain, indicative for a sensing process at or from within the membrane interface. Most proteins sharing this domain architecture are found in Firmicutes bacteria. Two major groups can be differentiated based on sequence similarity and genomic context: (1) BceS-like IM-HK that are functionally and genetically linked to ABC transporters, and (2) LiaS-like IM-HK, as part of three-component systems. Most IM-HK sense cell envelope stress, and identified target genes are often involved in maintaining cell envelope integrity, mediating antibiotic resistance, or detoxification processes. Therefore, IM-HK seem to constitute an important mechanism of cell envelope stress response in low G+C Gram-positive bacteria.     

Molecular cloning and expression of a cDNA encoding a hybrid histidine kinase receptor in tropical periwinkle Catharanthus roseus

Signalling pathways involving histidine kinase receptors (HKRs) are widely used by prokaryotes and fungi to regulate a large palette of biological processes. In plants, HKRs are known to be implicated in cytokinin, ethylene, and osmosensing transduction pathways. In this work, a full length cDNA named CRCIK was isolated from the tropical species CATHARANTHUS ROSEUS (L.) G. Don. It encodes a 1205 amino acid protein that belongs to the hybrid HKR family. The deduced amino acid sequence shows the highest homology with AtHK1, an osmosensing HKR in ARABIDOPSIS THALIANA. In return, CrCIK protein shares very low identity with the other 10 ARABIDOPSIS HKRs. Southern blot analysis indicates that the CRCIK corresponding gene is either present in multiple copies or has very close homologues in the genome of the tropical periwinkle. The gene is widely expressed in the plant. In C. ROSEUS C20D cell suspension, it is slightly induced after exposure to low temperature, pointing to a putative role in cold-shock signal transduction.     

Colocalization and identification of interaction sites between IGFBP-3 and GalNAc-T14

GalNAc-T14 was identified as a novel IGFBP-3 binding partner in previous studies. Here, we furtherly confirmed the interaction between them by confocal microscopy, and identified the binding domain and probable interaction sites of GalNAc-T14 with IGFBP-3. The result of subcellular localization indicated that GalNAc-T14 was distributed in the cytosol, whereas IGFBP-3 existed in the cytosol and nucleolus. Confocal analyses demonstrated that IGFBP-3 and GalNAc-T14 colocalized in the cytosol. The result from yeast two hybrid assay showed that the C terminus of GalNAc-T14 (408-552aa) was essential for the interaction between GalNAc-T14 and IGFBP-3, especially Tyr(408), Pro(409), and Glu(410) of GalNAc-T14 may play key roles in the interaction with IGFBP-3. In conclusion, these studies demonstrated that IGFBP-3 and GalNAc-T14 are colocalized in MCF-7 cells and confirmed the interaction between IGFBP-3 and GalNAc-T14. This interaction may play an important role in the functional regulation of IGFBP-3.     

A novel histidine kinase gene, ZmHK9, mediate drought tolerance through the regulation of stomatal development in Arabidopsis

Plants have developed complex signaling networks to regulate biochemical and physiological acclimation, environmental signals were perceived and transmitted to cellular machinery to activate adaptive responses. Here, a novel drought responsive histidine kinase gene was identified and designated as ZmHK9. Under normal conditions, ZmHK9 was predominantly expressed in roots, and the roots of ZmHK9-OX transgenic lines are markedly hypersensitive to ABA and ethylene, as compare to wild type. Consistent with its expression induced by PEG and exogenous ABA treatment, promoter sequence of this gene possessed drought and ABA responsive element. Moreover, the transgenic plants were much less affected by drought stress and recovered quickly after rewatering, stomatal complex size and stomatal density in the transgenic plants are significantly smaller and lower than those of the wild-type plants. In addition, ABA induced stomatal closure and the stomatal aperture of ZmHK9-OX lines was smaller than that of wild type. Collectively, it can be concluded that ZmHK9 regulates root elongation, stomatal development and drought tolerance through ABA dependent signaling pathway in Arabidopsis.     

Crystal structure of a complex between the phosphorelay protein YPD1 and the response regulator domain of SLN1 bound to a phosphoryl analog

The crystal structure of the yeast SLN1 response regulator (RR) domain bound to both a phosphoryl analog [beryllium fluoride (BeF₃ ⁻)] and Mg^2 +, in complex with its downstream phosphorelay signaling partner YPD1, has been determined at a resolution of 1.70 Å. Comparisons between the BeF₃ ⁻-activated complex and the unliganded (or apo) complex determined previously reveal modest but important differences. The SLN1-R1·Mg^2 +·BeF₃ ⁻ structure from the complex provides evidence for the first time that the mechanism of phosphorylation-induced activation is highly conserved between bacterial RR domains and this example from a eukaryotic organism. Residues in and around the active site undergo slight rearrangements in order to form bonds with the essential divalent cation and fluorine atoms of BeF₃ ⁻. Two conserved switch-like residues (Thr1173 and Phe1192) occupy distinctly different positions in the apo versus BeF₃ ⁻-bound structures, consistent with the “Y-T” coupling mechanism proposed for the activation of CheY and other bacterial RRs. Several loop regions and the α4-β5-α5 surface of the SLN1-R1 domain undergo subtle conformational changes (∼ 1-3 Å displacements relative to the apo structure) that lead to significant changes in terms of contacts that are formed with YPD1. Detailed structural comparisons of protein-protein interactions in the apo and BeF₃ ⁻-bound complexes suggest at least a two-state equilibrium model for the formation of a transient encounter complex, in which phosphorylation of the RR promotes the formation of a phosphotransfer-competent complex. In the BeF₃ ⁻-activated complex, the position of His64 from YPD1 needs to be within ideal distance of and in near-linear geometry with Asp1144 from the SLN1-R1 domain for phosphotransfer to occur. The ground-state structure presented here suggests that phosphoryl transfer will likely proceed through an associative mechanism involving the formation of a pentacoordinate phosphorus intermediate.     

Leishmania infantum: prime boost vaccination with C-terminal extension of cysteine proteinase type I displays both type 1 and 2 immune signatures in BALB/c mice

The aims of current study are to describe the immunogenicity and protective efficacy of prime boost vaccine using C-terminal extension (CTE) of cysteine proteinase type I of Leishmania infantum in BALB/c mice. Group I as vaccinated group primed with 100 microg of pcDNA-CTE and 3 weeks later boosted with combination of 30 microg rCTE, 50 microg of CpG and Montanide 720. Groups II and III were served as control groups. Although, this vaccination regimen did not protect mice against the infectious challenge but it was highly immunogenic. IgG2a has been raised strongly against rCTE in contrast to IgG1 and remains high at every time point under study. By analysis of CTE synthetic peptides (CTE100) before challenge, both IgG1 and IgG2a were produced and for all overlapping synthetic peptides (CTE 1-8) IgG1 raised significantly. This statue is changed at 7 weeks after challenge and only CTE2 and CTE3 have shown to induce considerable amount of IgG1. In all groups, the level of IL-5 started to increase with high concentration shortly passing only 3 weeks after infectious challenge. In compare with two control groups, the vaccinated group produced significantly higher level of IL-5 at 7 weeks post-infection. The parasite burden of all groups is similar at 4 weeks post-challenge in both liver and spleen. In contrast, at 8 weeks post challenge, the spleen of the vaccinated group showed significantly higher level of parasite load in compare with two control groups. This study demonstrated that immunization with CTE display both type 1 and 2 immune signatures in experimental murine model of L. infantum infection.     

The importance of conserved amino acid residues in p94 protease sub-domain IIb and the IS2 region for constitutive autolysis

p94/calpain 3, a skeletal muscle-specific member of calpain protease family, is characterized by apparent Ca(2+)-independence during exhaustive autolysis and concomitant proteolysis of non-self substrates. The purpose of our study was to comprehensively profile the structural basis of p94 enabling activation in the cytosol without an extra Ca(2+). Ca(2+)-dependent p94 mutants were screened using "p94-trapping", which is an application of yeast genetic reporter system called "proteinase-trapping". Several amino acids were revealed as critical for apparent Ca(2+)-independent p94 activity. These results highlight the importance of conserved amino acids in domain IIb as well as in the p94-specific IS2 region.