Site-directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron-transfer complex with a copper-containing nitrite reductase from Alcaligenes faecalis S-6

AbstractKinetic analysis of electron transfer between azurin from Pseudomonas aeruginosa and copper-containing nitrite reductase (NIR) from Alcaligenes faecalis S-6 was carried out to investigate the specificity of electron transfer between copper-containing proteins. Apparent values of kcat and Km of NIR for azurin were 300-fold smaller and 172-fold larger than those for the physiological redox partner, pseudoazurin from A. faecalis S-6, respectively, suggesting that the electron transfer between azurin and NIR was less specific than that between pseudoazurin and NIR. One of the major differences in 3-D structure between these redox proteins, azurin and pseudoazurin, is the absence and presence of lysine residues near their type 1 copper sites, respectively. Three mutated azurins, D11K, P36K, and D11K/P36K, were constructed to evaluate the importance of lysine residues in the interaction with NIR. The redox potentials of D11K, P36K, and D11K/P36K azurins were higher than that of wild-type azurin by 48, 7, and 55 mV, respectively. As suggested by the increase in the redox potential, kinetic analysis of electron transfer revealed reduced ability of electron transfer in the mutated azurins. On the other hand, although each of the single mutations caused modest effects on the decrease in the Km value, the simultaneous mutations of D11K and P36K caused significant decrease in the Km value when compared to that for wild-type azurin. These results suggest that the introduction of two lysine residues into azurin facilitated docking to NIR

Structural Insight into TNIK Inhibition

TRAF2- and NCK-interacting kinase (TNIK) has emerged as a promising therapeutic target for colorectal cancer because of its essential role in regulating the Wnt/β-catenin signaling pathway. Colorectal cancers contain many mutations in the Wnt/β-catenin signaling pathway genes upstream of TNIK, such as the adenomatous polyposis coli (APC) tumor suppressor gene. TNIK is a regulatory component of the transcriptional complex composed of β-catenin and T-cell factor 4 (TCF4). Inhibition of TNIK is expected to block the aberrant Wnt/β-catenin signaling caused by colorectal cancer mutations. Here we present structural insights into TNIK inhibitors targeting the ATP-binding site. We will discuss the effects of the binding of different chemical scaffolds of nanomolar inhibitors on the structure and function of TNIK

Crystal structure of the probable haloacid dehalogenase PH0459 from Pyrococcus horikoshii OT3

PH0459, from the hyperthermophilic archaeon Pyrococcus horikoshii OT3, is a probable haloacid dehalogenase with a molecular mass of 26,725 Da. Here, we report the 2.0 Å crystal structure of PH0459 (PDB ID: 1X42) determined by the multiwavelength anomalous dispersion method. The core domain has an α/β structure formed by a six-stranded parallel β-sheet flanked by six α-helices and three 310-helices. One disulfide bond, Cys186–Cys212, forms a bridge between an α-helix and a 310-helix, although PH0459 seems to be an intracellular protein. The subdomain inserted into the core domain has a four-helix bundle structure. The crystal packing suggests that PH0459 exists as a monomer. A structural homology search revealed that PH0459 resembles the l-2-haloacid dehalogenases l-DEX YL from Pseudomonas sp. YL and DhlB from Xanthobacter autotrophicus GJ10. A comparison of the active sites suggested that PH0459 probably has haloacid dehalogenase activity, but its substrate specificity may be different. In addition, the disulfide bond in PH0459 probably facilitates the structural stabilization of the neighboring region in the monomeric form, although the corresponding regions in l-DEX YL and DhlB may be stabilized by dimerization. Since heat-stable dehalogenases can be used for the detoxification of halogenated aliphatic compounds, PH0459 will be a useful target for biotechnological research

Crystal structure of a predicted phosphoribosyltransferase (TT1426) from Thermus thermophilus HB8 at 2.01 Å resolution

TT1426, from Thermus thermophilus HB8, is a conserved hypothetical protein with a predicted phosphoribosyltransferase (PRTase) domain, as revealed by a Pfam database search. The 2.01 Å crystal structure of TT1426 has been determined by the multiwavelength anomalous dispersion (MAD) method. TT1426 comprises a core domain consisting of a central five-stranded β sheet surrounded by four α-helices, and a subdomain in the C terminus. The core domain structure resembles those of the type I PRTase family proteins, although a significant structural difference exists in an inserted 43-residue region. The C-terminal subdomain corresponds to the “hood,” which contains a substrate-binding site in the type I PRTases. The hood structure of TT1426 differs from those of the other type I PRTases, suggesting the possibility that TT1426 binds an unknown substrate. The structure-based sequence alignment provides clues about the amino acid residues involved in catalysis and substrate binding

Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody–antigen interaction

<div><p>Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody–antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab) of the anti-EGFR antibody 059–152 were rapidly determined in a day by using a 30-μl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355^EGFR, Gln384^EGFR, H409^EGFR, and Lys465^EGFR), so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.</p></div

Lobe A-mediated DOCK2 dimerization is required to activate Rac effectively during cell migration.

<p>(A, B) FRET analyses for Rac activation in BW5147α^–β^– cells stably expressing HA-tagged WT or mutant DOCK2 (V1538A or Δlobe A). Cells were retrovirally transduced with Raichu-Rac, and were loaded on stromal cells prepared from the lymph nodes. After 4 hours of incubation, images were taken every 30 seconds, and the emission ratio of 527 nm/475 nm (FRET/CFP ratio) was used to represent the FRET efficiency. The FRET/CFP ratios at the plasma membrane were normalized by dividing by the lowest value in the cells, and cells were judged FRET-positive when the ratio was above 1.6. Data indicate the percentage of FRET-positive cells (B) with representative images (A). For each category of cells, 12–19 cells were analyzed. Scale bar, 5 µm. (C) The association of DOCK2 with Rac in BW5147α^–β^– cells stably expressing HA-tagged WT or mutant DOCK2 (V1538A or Δlobe A). Cell extracts were incubated with anti-HA affinity matrix in the presence of 10 mM EDTA, and bound Rac was detected with anti-Rac1 antibody.</p

DOCK2 forms homodimer via lobe A of DHR-2 domain in cells.

<p>(A) Following expression of FLAG-tagged WT or mutant DOCK2 (Δlobe A or 3A) in HEK-293T cells in combination with GFP-tagged WT or mutant DOCK2 (Δlobe A or 3A) or GFP alone, cell extracts were immunoprecipitated with anti-FLAG M2 antibody or anti-GFP antibody. Immunoblotting was carried out to detect homodimerization using the relevant antibodies. (B) Extracts from HEK-293T cells expressing FLAG-tagged WT DOCK2 or Δlobe A were pulled down with GST-fusion Rac1. Assays were done in Tris-buffered saline-Tween-20 supplemented with 10 mM EDTA (E) or 10 mM MgCl₂ plus 30 µM GTPγS (M). (C) Following expression of FLAG-tagged WT DOCK2 or Δlobe A in HEK-293T cells, Rac activation was analyzed. In (A-C), data are representative of, at least, three independent experiments.</p

The lobe A is required for dimerization, but not for the Rac GEF activity of DHR-2 domain.

<p>(A) Ribbon diagram of the human DOCK2^DHR-2-Rac1 complex. Overall structure (left) and dimerization interface (right) are shown. The coordinates and structure factors have been deposited in the Protein Data Bank (<a href="http://www.pdb.org" target="_blank">www.pdb.org</a>) under accession code 3B13. (B) Size-exclusion chromatography analysis for DHR-2^WT and DHR-2^{Δlobe A}. For protein size estimation, their elution volumes were compared with those of protein standards, thyroglobulin (670 kDa), immunoglobulin G (158 kDa), ovalbumin (43 kDa), and myoglobulin (17 kDa). (C) The Rac GEF activity was compared among DHR-2^WT, DHR-2^{Δlobe A}, and DHR-2^V1538A using BODIPY-FL-GTP.</p

Crystal structure of the 059-152-Fv•EGFR-ECD complex.

<p>(A) Ribbon representation of the 059-152-Fv•EGFR-ECD complex. VH and VL domains of 059-152-Fv are respectively colored cyan and green. External region of the EGFR is shown with domain I in yellow, domain II in orange, domain III in red, and domain IV in purple. (B, C) Close up-view of the interactions between CDR loops of 059–152 and domain III. For clarity, interactions of CDR-H and CDR-L are separately shown. CDR-H loops, CDR-L loops, and domain III are colored cyan, green, and gray, respectively. Residues that make key interactions are shown in the stick models. Hydrogen bonds are indicated by gray dotted lines.</p