Structural mechanism of SDS-induced enzyme activity of scorpion hemocyanin revealed by electron cryomicroscopy.

Purification of the scorpion

Pandinus imperator Hc The Hc of the scorpion Pandinus imperator (animals from Tropenhaus Marxen, Hamburg, Germany) was collected by dorsal punctuation of the membrane between the fifth and sixth mesosomal tergites. The emanating hemolymph was collected into an eppendorf cap with a stabilisation buffer to prevent hemolymph coagulation and protein precipitation. The buffer contained 100 mM TRIS/HCl at pH 7.8, 10 mM CaCl 2 and 10 mM MgCl 2 , was degassed and sterilely filtered. The hemolymph / buffer solution was centrifuged at 32,000×g in a cooling centrifuge Universal 16R (Hettich Zentrifugen, Tuttlingen, Germany) for 30 minutes to get rid of cellular debris and tissue contaminations. Hereafter, the blue supernatant was subjected to an analytical size exclusion chromatography using a Biologic-FPLC system (Bio-Rad, Munich, Germany) with a Sephacryl S 300 column (HiPrep 26/60, Pharmacia Biotech, Freiburg, Germany) to purify the Hc and to separate it from other hemolymph proteins. The elution was carried out using the same stabilisation buffer at a constant flow rate of 0.4ml·min -1 . The fractions containing pure Hc were joined and concentrated with centrifugal filter units (Biomax 30K, Millipore, Eschborn, Germany) in the same centrifuge at 4,000×g and 4°C. Protein concentration was estimated spectroscopically at 278nm using the standard value for proteins: ε 278 = 1.0 ml·mg −1 ·cm −1 . The resulting stock solution had a concentration of 23 mg·ml −1 and was kept at 4°C until further use. SDS activation of Hc For activation of the Hc we used 2mM SDS. A solution of Hc from the stock (~ 23 mg·ml −1 ) was diluted with an SDS-buffer-solution (0.1 M TRIS/HCl, pH 7.8 and 2.5 mM SDS) at a ratio of at least 1:30 (v/v). This also avoids the precipitation of Ca-SDS. The Hc will be activated after a very short time and will remain so for several hours ( Baird et al., 2007 ; Jaenicke and Decker, 2008 ; Nillius et al., 2008 ). Electron cryo-microscopy Hc samples were prepared for cryo-EM studies by dilution of the purified Hc to 0.5 mg·ml −1 . Resting state samples were frozen immediately after dilution. Activated state Hc samples were achieved by the procedure described above with SDS final concentration at ~2mM followed by ~5 minutes activation, which were then frozen within ~15 minutes. 3ul of sample was deposited onto a glow-discharged 400-mesh Quantifoil holey grid with 1.2×1.3 µm hole size (Quantifoil Micro Tools GmbH, Jena Germany). The grid was flash frozen in liquid ethane using a Vitrobot (FEI, Hillsboro, Oregon, USA). Data was recorded on a JEM 3200FSC microscope equipped with a field-emission gun operated at 300 kV and an in-column energy filter (using a 20eV slit). The specimen temperature was at 101K during data collection. Images were recorded at 50,000× nominal magnification on Kodak SO-163 films. The specimen dose was 18 electrons·Å −2 . The images were digitized on a Nikon Super CoolScan 9000 ED scanner with 6.35µ m per pixel scanning interval. Micrographs with a defocus range of 1~3.5µ m were used in the final reconstructions. Image processing, 3-D reconstruction, segmentation and variance map calculation The particles were boxed out using EMAN ( Ludtke et al., 1999 ; Ludtke et al., 2004 ) and SwarmPS ( Woolford et al., 2007 ). Contrast transfer function parameter estimation was carried out using the EMAN program ctfit . All 3-D reconstructions were completed using EMAN 1.8 with C2 symmetry imposed. Such symmetry imposition was based on previous evidence produced by negative stain electron microscopy ( Boisset et al., 1990 ; van Bruggen et al., 1980 ; van Heel and Dube, 1994 ) and other X-ray crystallography and cryo-EM studies on arthropod Hcs ( de Haas and van Bruggen, 1994 ; Fiser et al., 2000 ; Martin et al., 2007 ). In addition, a recently developed 2-D fast rotational matching (FRM2D) algorithm ( Cong et al., 2005 ; Cong et al., 2003 ) for image alignment, available in EMAN 1.8+, was adopted in the final refinement steps. The resolutions of the resting and activated state maps were evaluated to be 6.8 Å and 8.0 Å ( Figure S2 ), respectively, using the 0.5 FSC criterion ( Harauz and van Heel, 1986 ). There were 17,500 and 13,400 particles in the final reconstructions in the resting and activated states, respectively. Segmentations of the individual 12 subunits within an asymmetric unit (a dodecamer) for each state were done utilizing Amira ( http://www.amiravis.com ; TGS) and CHIMERA ( http://www.cgl.ucsf.edu/chimera/ ) ( Pettersen et al., 2004 ). The segmentation is straightforward and without ambiguity at this resolution. All visualizations were done with CHIMERA ( Pettersen et al., 2004 ). The correlation score, reflecting the similarity between the model and map, was measured using a normalized correlation coefficient after the pseudo atomic model was filtered to the same resolution of the map. For each state, we normalized and aligned the 12 segmented subunit maps within an asymmetric unit using EMAN program proc3d and CHIMERA “ fit into map ” module, respectively. Then a 3D variance map as well as an average map were calculated among these 12 subunit maps using EMAN program mapvariance . Comparative model building To build a pseudo atomic model for a single subunit of Hc, comparative modelling was performed using MODELLER 9v1 ( Sali and Blundell, 1993 ). Since no amino acid sequence was available for the Hc from scorpion Pandinus imperator , we chose the sequence of the Hc As6 subunit from scorpion Androctonus australis ( Buzy et al., 1995 ) , which is very close to our system based on sequence analysis of As6 with other typ3 copper protein subunits ( Burmester, 2001 ), as well as immunological crossreactivity ( Markl et al., 1984 ). Moreover, the 2.18 Å crystal structure of oxygenated Limulus polyphemus subunit II Hc (PDB ID: 1NOL ( Hazes et al., 1993 ), Figure S1 ) was used as template, which has ~60% sequence identity to As6. We used the standard single template modelling protocol implemented in the ‘ model ’ module of MODELLER 9v1. 50 models were generated, and the best one was selected based on the multivariate model assessment score ( Eramian et al., 2006 ). In addition, for the missing loop regions in the template structure 1NOL (including residues 21–29, 134–138, 527–530, and 569–572 as indicated by arrows in Figure S1 ), 200 loop models were generated by the loopmodel class in MODELLER 9v1 ( Sali and Blundell, 1993 ). The best loop model was chosen by a combination of the DOPE statistical potential score ( Shen and Sali, 2006 ) and the cross-correlation score between the loop and the density map ( Topf et al., 2006 ).

Complete 24-mer pseudo atomic model building by flexible fitting

To build a complete model for our 24-meric Hc, using the CHIMERA ( Pettersen et al., 2004 ) “ fit into map ” module, we first rigid-body fitted the comparative model of a single subunit into the segmented subunit (i.e. the linker subunit C1) for both states. There are noticeable conformational changes between the model and the map especially for the activated state. To capture the conformational changes revealed by the cryo-EM maps, SITUS ( Wriggers and Birmanns, 2001 ) flexible fitting procedure was used to flexibly fit the comparative model into the map. Taking the resting state C1 subunit as an example, flexible fitting by energy minimization was carried out using X-PLOR ( Brunger, 1992 ) with the default parameters of the CHARMM united-atom force field ( Brooks et al., 1983 ), version 19. In the energy minimization we included six pairs of positional markers determined by SITUS ( Wriggers and Birmanns, 2001 ), with the starting markers from the model and the ending markers from the map. We used the same procedure as described in ( Cong et al., 2008 ): by adding a constraining energy function using landmarks as if they were NOE constraints in X-PLOR so as to force the model to move towards the ending conformation as in the cryo-EM map. The constrained structure was subjected to 10000 steps of Powell energy minimization. Hookean potentials with force constants of 310 kcal· mol −1 ·Å −2 were employed ( Wriggers and Birmanns, 2001 ). In this way, the comparative model was flexibly fitted into the map of the C1 subunit. We repeated the same flexible fitting procedure to build all the 12 pseudo atomic models in one asymmetric unit in the resting state. Afterwards, by applying the 2-fold symmetry of this Hc structure we then obtained a complete 24-mer Hc pseudo atomic model for the resting state ( Figure S3A ). Still, in this complete model, several side chain clashes occurred in the interfaces of adjacent subunits. To eliminate those clashes, a local refinement procedure by energy minimization utilizing X-PLOR was carried out. The clashing regions were subjected to 2000 steps of Powell energy minimization while all other residues were maintained in their original coordinates. As to the activated state, the same flexible fitting procedure described above was applied to obtain a complete 24-mer pseudo atomic model.

Supplementary Material 01 SUPPLEMENTAL DATA Supplemental Data include five additional figures, two tables and three movies are available online at ***. The cryo-EM density maps and corresponding models are available from the EMDB and PDB. Their accession numbers are EMD-5100 and PDB ID 3IXV (for the Hc resting state) and EMD-5101 and PDB ID 3IXW (for the activated state), respectively. 02 Movie 1 Structural organization of the 24-meric Hc, exemplified by the resting state Hc cryo-EM map with different subunits in different colors. The representative views of the 24-mer, dodecamer, hexamer and monomer are illustrated. The B2 subunit map together with the corresponding pseudo atomic model are shown in the last portion of the movie, with the well resolved loop region in the cryo-EM map (not resolved in the crystal template structure 1NOL, discussed in the text) highlighted by ellipsoid. 03 Movie 2 Conformational change of Hc induced by SDS activation, illustrated by the Hc density maps as well as the pseudo atomic models. Pseudo atomic model morph between the two states clearly elucidates that the 24-oligomeric Hc complex undergoes a tertiary to collective quaternary conformational change upon SDS activation. There is a slight decrease in distance (~4 Å) between the two dodecamers due to SDS activation as illustrated during time period 0:08~0:17. 04 Movie 3 Averaged Hc monomer Cα model morph between the resting and the activated states. The most mobile domain I is illustrated in deep pink, while the other two domains in yellow and light green. The cyan and magenta spheres demonstrate the putative locations of the Cu and oxygen atoms forming the active site, and the blue sphere illustrates the Cα of the placeholder residue PHE49. This movie clearly demonstrates that Hc domain I has a more pronounced movement than domains II and III; more importantly, its moving direction is to twist away from the other two domains leaving the active site more exposed.