Atomic resolution structure of a protein prepared by non-enzymatic His-tag removal. Crystallographic and NMR study of GmSPI-2 inhibitor.

Purification of suitable quantity of homogenous protein is very often the bottleneck in protein structural studies. Overexpression of a desired gene and attachment of enzymatically cleavable affinity tags to the protein of interest made a breakthrough in this field. Here we describe the structure of Galleria mellonella silk proteinase inhibitor 2 (GmSPI-2) determined both by X-ray diffraction and NMR spectroscopy methods. GmSPI-2 was purified using a new method consisting in non-enzymatic His-tag removal based on a highly specific peptide bond cleavage reaction assisted by Ni(II) ions. The X-ray crystal structure of GmSPI-2 was refined against diffraction data extending to 0.98 Å resolution measured at 100 K using synchrotron radiation. Anisotropic refinement with the removal of stereochemical restraints for the well-ordered parts of the structure converged with R factor of 10.57% and Rfree of 12.91%. The 3D structure of GmSPI-2 protein in solution was solved on the basis of 503 distance constraints, 10 hydrogen bonds and 26 torsion angle restraints. It exhibits good geometry and side-chain packing parameters. The models of the protein structure obtained by X-ray diffraction and NMR spectroscopy are very similar to each other and reveal the same β2αβ fold characteristic for Kazal-family serine proteinase inhibitors

Direct protein-protein intermolecular hydrogen bonds in the <i>Gm</i>SPI2 crystal structure.

(a)<p>x +½, −y + ½, −z.</p>(b)<p>−x, y + ½, −z+½.</p>(c)<p>−x+½, −y, z+½.</p><p>Direct protein-protein intermolecular hydrogen bonds in the <i>Gm</i>SPI2 crystal structure.</p

Plot of <i>B</i>_eq averaged over main-chain (green) and side-chain (orange) atoms of the crystallographic model.

<p>Values of zero in the lower plot correspond to glycines.</p

Stereo C_α tracing of the crystallographic model of <i>Gm</i>SPI-2 (yellow) superimposed with the NMR structure of <i>Gm</i>SPI-2 in solution (conformer 11, magenta), <i>Cr</i>SPI-1-D1 (red, PDB code 3pis), OMTKY3 (blue, 2 gkr) and LDTI (green, 1an1).

<p>The superposition was calculated in <i>Coot</i> (Emsley & Cowtan, 2004) using the <i>SSM</i> algorithm and displayed in <i>PyMOL</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106936#pone.0106936-DeLano1" target="_blank">[47]</a>.</p

Comparison of protein–DNA contacts observed for the and IR-1 ds oligonucleotides bound by the UspDBD/EcRDBD heterocomplex

<p><b>Copyright information:</b></p><p>Taken from "Novel DNA-binding element within the C-terminal extension of the nuclear receptor DNA-binding domain"</p><p></p><p>Nucleic Acids Research 2007;35(8):2705-2718.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885670.</p><p>© 2007 The Author(s)</p> The crystallographic data obtained here for the UspDBD/EcRDBD- complex and published previously for the UspDBD/EcRDBD-IR-1 complex (PDB accession code 1R0O) () were analyzed in order to identify protein–DNA interactions. The DNA double helices are unwrapped to show schematically the base pairs and the respective contacts in the upstream (beige) and downstream (gold) half-sites of the and IR-1 elements. Gray circles represent phosphodiester groups, black circles are ordered water molecules that mediate protein-DNA contacts. Amino acid residues that contact the DNA using side chains are shown as red (EcRDBD) or blue (UspDBD) ovals. Asterisks denote residues which assume two well-defined conformations. Red solid lines indicate direct or water-mediated specific hydrogen bonding to base pairs. Blue dashed lines indicate hydrogen bonds to sugar-phosphate backbone

CTE of the EcRDBD folds into an α-helix which is part of the EcR-specific element supporting DNA-binding

<p><b>Copyright information:</b></p><p>Taken from "Novel DNA-binding element within the C-terminal extension of the nuclear receptor DNA-binding domain"</p><p></p><p>Nucleic Acids Research 2007;35(8):2705-2718.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885670.</p><p>© 2007 The Author(s)</p> () Secondary structure predictions of and A-box segments. The predictions were performed using the GORIV, PROF and PSIPRED algorithms available at the ExPASy Proteomics Server (). The predicted α-helices are depicted as cylinders. The seven-residue segment forming the short α-helix in the CTE A-box of the EcRDBD, observed in the present crystal structure, is boxed with broken line. The average confidence score values of the GORIV algorithm for the residues forming a potential α-helix within the and A-box segments are 736 and 661, respectively. The PROF algorithm gives a prediction and a confidence value between 0 and 1 for each position in the amino acid sequence. The average confidence values for the and sequences are 0.68 and 0.53, respectively. The PSIPRED algorithm gives a confidence value between 0 and 9. The average confidence scores for the and are 5.1 and 4.8, respectively. () Superposition of the corresponding Cα atoms of the TR (PDB accession code 2NLL) (), VDR (PDB accession code 1KB4) (), LRH-1 (PDB accession code 2A66) (), Rev-ERBα (PDB accession code 1A6Y) (), NGFI-B (PDB accession code 1CIT) () and EcRDBD (from the present UspDBD/EcRDBD- complex) proteins complexed to DNA. () A detailed view of the CTEs and DNA shown in (). C and N denote C- and N-termini of respective DBDs

Evaluation of the crystallographic data indicates bending in and IR-1 elements

<p><b>Copyright information:</b></p><p>Taken from "Novel DNA-binding element within the C-terminal extension of the nuclear receptor DNA-binding domain"</p><p></p><p>Nucleic Acids Research 2007;35(8):2705-2718.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885670.</p><p>© 2007 The Author(s)</p> () The ds oligonucleotides used in the crystallographic analysis of the UspDBD/EcRDBD heterodimer complexed with the IR-1 and response elements. The sequences of the response elements are shown in frames, and ovals represent localization of the respective DBDs. Models and parameters of the DNA molecules were generated using the 3DNA software (), data from this study and the atomic coordinate file of the UspDBD/EcRDBD-IR-1 structure (1R0O) (). DNA base pairs are shown as rectangular blocks, and the idealized helical axis based on the axis computed by the 3DNA software (dotted line) is shown in black. () The minor and major grove widths of the (solid line) and IR-1 (broken line) ds oligonucleotides. Only the heptameric half-sites are shown. The values were derived using the 3DNA software. The solid black line represent standard values for B-DNA