Structure of Crimean-Congo hemorrhagic fever virus nucleoprotein: superhelical homo-oligomers and the role of caspase-3 cleavage.

Crimean-Congo hemorrhagic fever, a severe hemorrhagic disease found throughout Africa, Europe, and Asia, is caused by the tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is a negative-sense single-stranded RNA (ssRNA) virus belonging to the Nairovirus genus of the Bunyaviridae family. Its genome of three single-stranded RNA segments is encapsidated by the nucleocapsid protein (CCHFV N) to form the ribonucleoprotein complex. This ribonucleoprotein complex is required during replication and transcription of the viral genomic RNA. Here, we present the crystal structures of the CCHFV N in two distinct forms, an oligomeric form comprised of double antiparallel superhelices and a monomeric form. The head-to-tail interaction of the stalk region of one CCHFV N subunit with the base of the globular body of the adjacent subunit stabilizes the helical organization of the oligomeric form of CCHFV N. It also masks the conserved caspase-3 cleavage site present at the tip of the stalk region from host cell caspase-3 interaction and cleavage. By incubation with primer-length ssRNAs, we also obtained the crystal structure of CCHFV N in its monomeric form, which is similar to a recently published structure. The conformational change of CCHFV N upon deoligomerization results in the exposure of the caspase-3 cleavage site and subjects CCHFV N to caspase-3 cleavage. Mutations of this cleavage site inhibit cleavage by caspase-3 and result in enhanced viral polymerase activity. Thus, cleavage of CCHFV N by host cell caspase-3 appears to be crucial for controlling viral RNA synthesis and represents an important host defense mechanism against CCHFV infection.

The variable N-terminal region of DDX5 contains structural elements and auto-inhibits its interaction with NS5B of hepatitis C virus.

RNA helicases of the DEAD (Asp-Glu-Ala-Asp)-box family of proteins are involved in many aspects of RNA metabolism from transcription to RNA decay, but most of them have also been shown to be multifunctional. The DEAD-box helicase DDX5 of host cells has been shown to interact with the RNA-dependent RNA polymerase (NS5B) of HCV (hepatitis C virus). In the present study, we report the presence of two independent NS5B-binding sites in DDX5, one located at the N-terminus and another at the C-terminus. The N-terminal fragment of DDX5, which consists of the first 305 amino acids and shall be referred as DDX5-N, was expressed and crystallized. The crystal structure shows that domain 1 (residues 79-303) of DDX5 contains the typical features found in the structures of other DEAD-box helicases. DDX5-N also contains the highly variable NTR (N-terminal region) of unknown function and the crystal structure reveals structural elements in part of the NTR, namely residues 52-78. This region forms an extensive loop and an α-helix. From co-immunoprecipitation experiments, the NTR of DDX5-N was observed to auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1-51 residues in NTR and the NS5B-binding site in DDX5-N. Furthermore, NMR investigations reveal that there is a direct interaction between DDX5 and NS5B in vitro.

Expression, purification and preliminary crystallographic analysis of recombinant human DEAD-box polypeptide 5.

The DEAD-box RNA helicase DDX5 is involved in many aspects of RNA processing and has been implicated in a number of cellular processes involving alteration of RNA secondary structure. The N-terminal region of DDX5, which contains the conserved domain 1 of the DEAD-box helicases, has been cloned and expressed in Escherichia coli and purified. Here, the crystallization and preliminary diffraction analysis of this region is reported. X-ray diffraction data were processed to a resolution of 2.7 A. The crystals belonged to space group I222, with unit-cell parameters a = 66.18, b = 73.80, c = 104.00 A, alpha = beta = gamma = 90 degrees .

Bortezomib inhibits chikungunya virus replication by interfering with viral protein synthesis.

Chikungunya virus (CHIKV) is an alphavirus that causes a febrile illness accompanied by myalgia and arthralgia. Despite having re-emerged as a significant public health threat, there are no approved therapeutics or prophylactics for CHIKV infection. In this study, we explored the anti-CHIKV effects of proteasome inhibitors and their potential mechanism of antiviral action. A panel of proteasome inhibitors with different functional groups reduced CHIKV infectious titers in a dose-dependent manner. Bortezomib, which has been FDA-approved for multiple myeloma and mantle cell lymphoma, was further investigated in downstream studies. The inhibitory activities of bortezomib were confirmed using different cellular models and CHIKV strains. Time-of-addition and time-of-removal studies suggested that bortezomib inhibited CHIKV at an early, post-entry stage of replication. In western blot analysis, bortezomib treatment resulted in a prominent decrease in structural protein levels as early as 6 hpi. Contrastingly, nsP4 levels showed strong elevations across all time-points. NsP2 and nsP3 levels showed a fluctuating trend, with some elevations between 12 to 20 hpi. Finally, qRT-PCR data revealed increased levels of both positive- and negative-sense CHIKV RNA at late stages of infection. It is likely that the reductions in structural protein levels is a major factor in the observed reductions in virus titer, with the alterations in non-structural protein ratios potentially being a contributing factor. Proteasome inhibitors like bortezomib likely disrupt CHIKV replication through a variety of complex mechanisms and may display a potential for use as therapeutics against CHIKV infection. They also represent valuable tools for studies of CHIKV molecular biology and virus-host interactions.

Expression, purification and preliminary crystallographic analysis of Pseudomonas aeruginosa RocR protein.

Pseudomonas aeruginosa RocR, an EAL-domain protein which regulates the expression of virulence genes and biofilm formation, has been cloned and expressed in Escherichia coli and purified. Here, the crystallization and preliminary diffraction analysis of RocR are reported. The X-ray diffraction data were processed to a resolution of 2.50 A. The crystals belonged to space group P6(1)22 or P6(5)22, with unit-cell parameters a = 118.8, b = 118.8, c = 495.1 A, alpha = beta = 90, gamma = 120 degrees .

Structural and functional characterization of human SGT and its interaction with Vpu of the human immunodeficiency virus type 1.

The small glutamine-rich tetratricopeptide repeat protein (SGT) belongs to a family of cochaperones that interacts with both Hsp70 and Hsp90 via the so-called TPR domain. Here, we present the crystal structure of the TPR domain of human SGT (SGT-TPR), which shows that it contains typical features found in the structures of other TPR domains. Previous studies show that full-length SGT can bind to both Vpu and Gag of human immunodeficiency virus type 1 (HIV-1) and the overexpression of SGT in cells reduces the efficiency of HIV-1 particle release. We show that SGT-TPR can bind Vpu and reduce the amount of HIV-1 p24, which is the viral capsid, secreted from cells transfected with the HIV-1 proviral construct, albeit at a lower efficiency than full-length SGT. This indicates that the TPR domain of SGT is sufficient for the inhibition of HIV-1 particle release but the N- and/or C-terminus also have some contributions. The SGT binding site in Vpu was also identified by using peptide array and confirmed by GST pull-down assay.

Expression, purification and preliminary crystallographic analysis of recombinant human small glutamine-rich tetratricopeptide-repeat protein.

Human small glutamine-rich tetratricopeptide-repeat protein (hSGT) is a 35 kDa protein implicated in a number of biological processes that include apoptosis, cell division and intracellular cell transport. The tetratricopeptide-repeat (TPR) domain of hSGT has been cloned and expressed in Escherichia coli and purified. Here, the crystallization and preliminary diffraction analysis of the TPR domain of hSGT is reported. X-ray diffraction data were processed to a resolution of 2.4 A. Crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 67.82, b = 81.93, c = 55.92 A, alpha = beta = gamma = 90 degrees .

Comparative Study of Imprint Cytology and Histopathology of Soft Tissue Tumors.

BACKGROUND: The components of soft tissue are fibroblasts, collagen, vascular structures, fatty tissue, skeletal muscles, smooth muscles, and neural tissue. The real incidence of soft tissue tumors (STTs) is difficult to estimate because most of them are benign (Benign: Malignant-100:1). AIMS: The aim of the present study was undertaken to note the patterns of presentation of patients with STTs and to evaluate the findings of imprint cytology (IC) and histopathological examination (HPE) of STTs. MATERIALS AND METHODS: The present study was undertaken for 1 year. A total of 41 patients with clinically and radiologically diagnosed STTs were included in the study. Following surgery, imprint smear was taken for each tumor, before delivering the tissue to 10% formalin. HPE was subsequently performed. RESULTS: The age of the patients ranged from 4 months to 80 years with a mean of 35.6 ± 17.5 years. The ratio of males to females was 1.05:1. HPE revealed that 21 (51.2%) tumors were benign and 20 (48.8%) malignant. Imprint smears revealed 16 (39%) tumors to be benign and 20 (48.8%) malignant. IC was inconclusive in 5 (12.2%) cases. The sensitivity of IC was found to be 89.5% and specificity 82.35%. The positive predictive value of IC was 85%. The accuracy of IC for diagnosis of both benign and malignant tumors was found to be 75%. CONCLUSION: IC of STTs is a rapid and simple method of intraoperative diagnosis, and it can serve as a viable alternative to frozen section biopsy, particularly in rural settings.

Model Compounds for Iron Proteins. Structures and Magnetic, Spectroscopic, and Redox Properties of Fe(III)M(II) and [Co(III)Fe(III)](2)O Complexes with (&mgr;-Carboxylato)bis(&mgr;-phenoxo)dimetalate and (&mgr;-Oxo)diiron(III) Cores.

A series of heterobimetallic complexes of the type [Fe(III)M(II)L(&mgr;-OAc)(OAc)(H(2)O)](ClO(4)).nH(2)O (2-5) and [{Fe(III)Co(III)L(&mgr;-OAc)(OAc)}(2)(&mgr;-O)](ClO(4))(2).3H(2)O (6) where H(2)L is a tetraaminodiphenol macrocyclic ligand and M(II) = Zn(2), Ni(3), Co(4), and Mn(5) have been synthesized and characterized. The (1)H NMR spectrum of 6 exhibits all the resonances between 1 and 12 ppm. The IR and UV-vis spectra of 2-5 indicate that in all the cases the metal ions have similar coordination environments. A disordered crystal structure determined for 3 reveals the presence of a (&mgr;-acetate)bis(&mgr;-phenoxide)-Ni(II)Fe(III) core, in which the two metal ions have 6-fold coordination geometry and each have two amino nitrogens and two phenolate oxygens as the in-plane donors; aside from the axial bridging acetate, the sixth coordination site of nickel(II) is occupied by the unidentate acetate and that of iron(III) by a water molecule. The crystal structure determination of 6 shows that the two heterobinuclear Co(III)Fe(III) units are bound by an Fe-O-Fe linkage. 6 crystallizes in the orthorhombic space group Ibca with a = 17.577(4) Å, b = 27.282(7) Å, c = 28.647(6) Å, and Z = 8. The two iron(III) centers in 6 are strongly antiferromagnetically coupled, J = -100 cm(-1) (H = -2JS(1).S(2)), whereas the other two S(1) = S(2) = (5)/(2) systems, viz. [Fe(2)(III)(HL)(2)(&mgr;-OH)(2)](ClO(4))(2) (1) and the Fe(III)Mn(II) complex (5), exhibit weak antiferromagnetic exchange coupling with J = -4.5 cm(-1) (1) and -1.8 cm(-1) (5). The Fe(III)Ni(II) (3) and Fe(III)Co(II) (4) systems, however, exhibit weak ferromagnetic behavior with J = 1.7 cm(-1) (3) and 4.2 cm(-1) (4). The iron(III) center in 2-5 exhibits quasi-reversible redox behavior between -0.44 and -0.48 V vs Ag/AgCl associated with reduction to iron(II). The oxidation of cobalt(II) in 4 occurs quasi-reversibly at 0.74 V, while both nickel(II) and manganese(II) in 3 and 5 undergo irreversible oxidation at 0.85 V. The electrochemical reduction of 6 leads to the generation of 4.

Macrocyclic Cu(II)(2), Cu(II)(4), Ni(II)(3), and Ni(II)(4) Complexes: Magnetic Properties of Tetranuclear Systems.

A binuclear tetraprotonated macrocyclic complex [Mg(2)(L(2)-H(4))(NO(3))(2)](NO(3))(2).6H(2)O (1) has been obtained by template condensation of 4-methyl-2,6-diformylphenol and 1,2-diaminoethane in the presence of magnesium acetate and nitrate. Complex 1 on reduction with NaBH(4), followed by the removal of magnesium, yields the 36-membered octaaminotetraphenol macrocyclic ligand H(4)L(1). The replacement of magnesium in 1 with copper(II) leads to the formation of the binuclear complex [Cu(2)L(3)(ClO(4))(2)] (2) derived from the [2+2] cyclization product of 4-methyl-2,6-diformylphenol and 1,2-diaminoethane. From H(4)L(1) a series of tetranuclear nickel(II) complexes 5-8 with the core cation [Ni(4)L(1)(&mgr;(2)-X)(2)(&mgr;(2)-H(2)O)(2)](2+) (X = NCS, N(3), OAc, or Cl) have been synthesized and characterized. The trinuclear complex [Ni(3)L(1)(acac)(2)(H(2)O)(2).2H(2)O (9), obtained by reacting nickel(II) acetylacetonate with H(4)L(1), on treatment with nickel(II) perchlorate produces the tetranuclear compound [Ni(4)L(1)(acac)(2)(H(2)O)(4)](ClO(4))(2) (10). Variable-temperature (4-300 K) magnetic susceptibility measurements have been carried out for the tetracopper(II) complex [Cu(4)L(1)(H(2)O)(4)](ClO(4))(4) (3) and the tetranickel(II) complexes [Ni(4)L(1)(&mgr;(3)-OH)(&mgr;(2)-H(2)O)(2)(ClO(4))](ClO(4))(2).2CH(3)COCH(3).H(2)O (4), [Ni(4)L(1)(&mgr;(2)-NCS)(2)(&mgr;(2)-H(2)O)(2)](ClO(4))(2).2CH(3)CN (5), [Ni(4)L(1)(&mgr;(2)-N(3))(2)(&mgr;(2)-H(2)O)(2)](ClO(4))(2).2CH(3)OH (6), [Ni(4)L(1)(&mgr;(2)-OAc)(2)(&mgr;(2)-H(2)O)(2)](ClO(4))(2).2H(2)O (7), and [Ni(4)L(1)(&mgr;(2)-Cl)(2)(&mgr;(2)-H(2)O)(2)]Cl(2).4H(2)O (8). The X-ray structure of 5 has been determined. The complex (C(50)H(70)N(12)O(14)Cl(2)S(2)Ni(4)) crystallizes in the triclinic space group P&onemacr; with a = 11.794(6) Å, b = 12.523(4) Å, c = 12.794(5) Å, alpha = 117.28(5) degrees, beta = 96.38(4) degrees, gamma = 109.65(3) degrees, and Z = 1. In the asymmetric unit each of the nickel(II) centers with distorted octahedral geometry is triply-bridged by a phenoxide group, a water molecule, and a N-bonded thiocyanate and these metal centers are further bridged to their symmetry-related counterparts by another phenoxide group. The experimental susceptibility data have been analyzed using appropriate Heisenberg spin coupling models (H = -2J(ij)()S(i)().S(j)()) and the best-fit spin exchange parameters obtained are as follows: J = -288(3) cm(-)(1) (3); J(1) = -8.1(2) cm(-)(1), J(2) = -10.2(2) cm(-)(1) (4); J(1) = -34.5(1.0) cm(-)(1), J(2) = -9.5(2.0) cm(-)(1) (5); J(1) = -34(1) cm(-)(1), J(2) = 11(2) cm(-)(1) (6); J(1) = -30(1) cm(-)(1), J(2) = -7.0(1.5) cm(-)(1) (7); J(1) = -32(1) cm(-)(1), J(2) = -4(1) cm(-)(1) (8).

Cytodiagnosis of epithelioid malignant melanoma (amelanotic) and diagnostic dilemmas.

Melanoma is an aggressive neoplasm, and early diagnosis can reduce mortality in such patients. Diagnosis may be delayed in amelanotic tumors. We present one such case, a 35-year-old lady with a rapidly growing mass over the right angle of mandible. Fine-needle aspiration cytology was done, and smears showed discretely arranged large epithelioid cells with high N:C ratio, prominent single to multiple nucleoli. Occasional binucleated and multinucleated tumor giant cells were also noted. Poorly differentiated carcinoma, high-grade non-Hodgkin lymphoma, amelanotic melanoma, and pleomorphic sarcoma were included as differential diagnoses. Immunocytochemistry (ICC) revealed Melan-A/MART-1 positivity in some cells and S-100 positivity in most tumor cells. Desmin, pancytokeratin, and leukocyte common antigen were negative. Based on cytomorphological features and ICC findings, a diagnosis of epithelioid variant of amelanotic melanoma was rendered. Later on, true cut biopsy and histologic examination of excised specimen and adjunct immunohistochemistry with positive Melan-A and S-100 confirmed the diagnosis.

Regulation of NOXO1 activity through reversible interactions with p22 and NOXA1.

Reactive oxygen species (ROS) have been known for a long time to play important roles in host defense against microbial infections. In addition, it has become apparent that they also perform regulatory roles in signal transduction and cell proliferation. The source of these chemicals are members of the NOX family of NADPH oxidases that are found in a variety of tissues. NOX1, an NADPH oxidase homologue that is most abundantly expressed in colon epithelial cells, requires the regulatory subunits NOXO1 (NOX organizing protein 1) and NOXA1 (NOX activating protein 1), as well as the flavocytochrome component p22(phox) for maximal activity. Unlike NOX2, the phagocytic NADPH oxidase whose activity is tightly repressed in the resting state, NOX1 produces superoxide constitutively at low levels. These levels can be further increased in a stimulus-dependent manner, yet the molecular details regulating this activity are not fully understood. Here we present the first quantitative characterization of the interactions made between the cytosolic regulators NOXO1 and NOXA1 and membrane-bound p22(phox). Using isothermal titration calorimetry we show that the isolated tandem SH3 domains of NOXO1 bind to p22(phox) with high affinity, most likely adopting a superSH3 domain conformation. In contrast, complex formation is severely inhibited in the presence of the C-terminal tail of NOXO1, suggesting that this region competes for binding to p22(phox) and thereby contributes to the regulation of superoxide production. Furthermore, we provide data indicating that the molecular details of the interaction between NOXO1 and NOXA1 is significantly different from that between the homologous proteins of the phagocytic oxidase, suggesting that there are important functional differences between the two systems. Taken together, this study provides clear evidence that the assembly of the NOX1 oxidase complex can be regulated through reversible protein-protein interactions.

Potent suppression of agglutinability of human A and B type erythrocytes by KMnO(4)-EDTA concerted action.

PURPOSE OF THE STUDY: Study was focused to effectively suppress the agglutinability of human A and B type erythrocyte by concerted chemical oxidation approach. Consequently some major bio-physiological consequences of human erythrocytes were analyzed. MATERIALS AND METHODS: Concerted chemical oxidation of human A and B type erythrocyte were done by potassium permanganate (KMnO(4)) in presence of ethylene diamine tetra-acetic acid (EDTA). All the chemical reactions were carried out at 37°C temperature and pH 7.4 in phosphate buffer. Finally, suppression of agglutinability were determined by using anti-A and anti-B antisera. RESULTS: A marked suppression in agglutinability was observed after chemical treatment on human A and B type erythrocytes in vitro. Effective suppression of agglutinability was observed in 0.57 mM concentration of KMnO(4) in presence of 0.9 mM EDTA at optimum condition. CONCLUSIONS: Results showed that KMnO(4)-EDTA concerted action is feasible. The future work is directed towards the universal human erythrocytes preparation from A and B type human erythrocytes.

Gd-complexes of DTPA-bis(amide) conjugates of tranexamic acid and its esters with high relaxivity and stability for magnetic resonance imaging.

The synthesis and the characterization of a series of DTPA-bis(amide) conjugates of tranexamic acid (L1), its esters (L2-L6), and their Gd(III) complexes of the type [Gd(L)(H2O)].nH2O (L = L1-L6) are described. Except for the case of , all Gd-complexes exhibit greatly enhanced R1 relaxivity. Highest R1 reaches up to 12.9 mM(-1) s(-1) for [Gd(L2)(H2O)]. Such high relaxivity is reflected in the intensity enhancement of the in vivo MRI study on H-ras transgenic mice bearing hepatic tumor when employing [Gd(L2)(H2O)] as an MRI contrast agent. Thermodynamic stability constants, conditional stability constants, and the pM values demonstrate higher stability of [Gd(L)(H2O)].nH2O (L =L1-L6) than Omniscan under physiological conditions. The MTT assay performed on these complexes reveals cytotoxicity as low as that for Omniscan in the concentration range required to obtain intensity enhancement in the in vivo MRI study.

Synthesis, reactivities, and magnetostructural properties of FeIII, FeIII-O-FeIII, and ZnIIFeIII-O-FeIIIZnII complexes of a tetraiminodiphenolate macrocycle.

The mononuclear iron(III) complexes [Fe(LH2)(H2O)Cl](ClO4)2.2H2O (1) and [Fe(LH2)(H2O)2](ClO4)3.H2O (2) have been prepared by reacting [Pb(LH(2))](ClO4)2 with FeCl3.6H2O and Fe(ClO(4))(3).6H(2)O, respectively. Complex 2 upon treatment with 1 equiv of alkali produces the oxo-bridged dimer [{Fe(LH2)(H2O)}2(mu-O)](ClO4)4.2H2O (3). In these compounds, LH2 refers to the tetraiminodiphenol macrocycle in the zwitterionic form whose two uncoordinated imine nitrogens are protonated and hydrogen-bonded to the metal-bound phenolate oxygens. The aqua ligands of complexes 1-3 get exchanged in acetonitrile. Reaction equilibria involving binding and exchange of the terminal ligands (Cl-/H2O/CH3CN) in these complexes have been studied spectrophotometrically. The equilibrium constant for the aquation reaction (K(aq)) [1]2+ + H2O <==> [2]3+ + Cl- in acetonitrile is 8.65(5) M, and the binding constant (K(Cl)-) for the reaction [1]2+ + Cl- [1Cl]+ + CH3CN is 4.75(5) M. The pK(D) value for the dimerization reaction 2[2]3+ + 2OH- <==> [3]4+ + 3H(2)O in 1:1 acetonitrile-water is 9.38(10). Complexes 1-3 upon reaction with Zn(ClO4)(2).6H(2)O and sodium acetate (OAc), pivalate (OPiv), or bis(4-nitrophenyl)phosphate (BNPP) produce the heterobimetallic complexes [{FeLZn(mu-X)}2(mu-O)](ClO4)2, where X = OAc (4), OPiv (5), and BNPP (6). The pseudo-first-order rate constant (k(obs)) for the formation of 4 at 25 degrees C from either 1 or 3 with an excess of Zn(OAc)2.2H2O in 1:1 acetonitrile-water at pH 6.6 is found to be the same with k(obs) = 1.6(2) x 10(-4) s(-1). The X-ray crystal structures of 3, 4, and 6 have been determined, although the structure determination of 3 was severely affected because of heavy disordering. In 3, the Fe-O-Fe angle is 168.6(6) degrees, while it is exactly 180.0 degrees in 4 and 6. Cyclic and square-wave voltammetric (CV and SWV) measurements have been carried out for complexes 1-4 in acetonitrile. The variation of the solvent composition (acetonitrile-water) has a profound effect on the E(1/2) and DeltaE(p) values. The binding of an additional chloride ion to an iron(III) center in 1-3 is accompanied by a remarkable shift of E(1/2) to more negative values. The observation of quasi-reversible CV for complexes containing a Fe(III)-O-Fe(III) unit (3 and 4) indicates that in the electrochemical time scale unusual Fe(III)-O-Fe(II) is produced. The 1H NMR spectra of complexes 3-6 exhibit hyperfine-shifted signals in the range 0-90 ppm with similar features. The metal-hydrogen distances obtained from T(1) measurements are in good agreement with the crystallographic data. Variable-temperature (2-300 K) magnetic susceptibility measurements carried out for 3 and 4 indicate strong antiferromagnetic exchange interaction (H = -2JS1.S2) between the high-spin iron(III) centers in the Fe-O-Fe unit with J = -114 cm(-1) (3) and -107 cm(-1) (4).

Crystallization and preliminary crystallographic analysis of the kinase-recruitment domain of the PP2C-type phosphatase RsbU.

The general stress response of Bacillus subtilis provides a protective resistance to a variety of pressures. The key molecule is a subunit of RNA polymerase, sigma(B), which confers promoter specificity and is regulated by two signalling modules. Each module comprises protein kinases and phosphatases and 'switch' protein substrates for the kinase and phosphatase. The phosphorylation state of the switch molecules indirectly controls the activity of sigma(B). The binding of the kinase RsbT to the phosphatase RsbU stimulates its enzymatic activity towards its substrate, phosphorylated RsbV. To understand how these enzymes interact, thus regulating transcription, crystallization of the kinase-recruitment domain of RsbU in a form suitable for high-resolution structure determination is reported.

Functional and structural characterization of RsbU, a stress signaling protein phosphatase 2C.

RsbU is a positive regulator of the activity of sigmaB, the general stress-response sigma factor of Gram+ microorganisms. The N-terminal domain of this protein has no significant sequence homology with proteins of known function, whereas the C-terminal domain is similar to the catalytic domains of PP2C-type phosphatases. The phosphatase activity of RsbU is stimulated greatly during the response to stress by associating with a kinase, RsbT. This association leads to the induction of sigmaB activity. Here we present data on the activation process and demonstrate in vivo that truncations in the N-terminal region of RsbU are deleterious for the activation of RsbU. This conclusion is supported by comparisons of the phosphatase activities of full-length and a truncated form of RsbU in vitro. Our determination of the crystal structure of the N-terminal domain of RsbU from Bacillus subtilis reveals structural similarities to the regulatory domains from ubiquitous protein phosphatases and a conserved domain of sigma-factors, illuminating the activation processes of phosphatases and the evolution of "partner switching." Finally, the molecular basis of kinase recruitment by the RsbU phosphatase is discussed by comparing RsbU sequences from bacteria that either possess or lack RsbT.