Volume 9, Issue 11, Pages 1095-1106 (November 2001) Structure of Thermotoga maritima Stationary Phase Survival Protein SurE  R.-G. Zhang, T. Skarina, J.E. Katz, S. Beasley, A. Khachatryan, S. Vyas, C.H. Arrowsmith, S. Clarke, A. Edwards, A. Joachimiak, A. Savchenko  Structure  Volume 9, Issue 11, Pages 1095-1106 (November 2001) DOI: 10.1016/S0969-2126(01)00675-X

Figure 1 Structure of SurE (a) Structure of the monomer with the N-terminal domain in red and C-terminal domain in maroon. (b) Structure of the dimer with two-fold dyad in the plane of the figure. Color coding for one monomer is as in (a), and the second monomer is in yellow. N and C termini are labeled in the red/maroon monomer. (c) Specific interactions responsible for the formation of the tetramer in β hairpin region of blue and green monomers. (d) Structure of the SurE tetramer Structure 2001 9, 1095-1106DOI: (10.1016/S0969-2126(01)00675-X)

Figure 1 Structure of SurE (a) Structure of the monomer with the N-terminal domain in red and C-terminal domain in maroon. (b) Structure of the dimer with two-fold dyad in the plane of the figure. Color coding for one monomer is as in (a), and the second monomer is in yellow. N and C termini are labeled in the red/maroon monomer. (c) Specific interactions responsible for the formation of the tetramer in β hairpin region of blue and green monomers. (d) Structure of the SurE tetramer Structure 2001 9, 1095-1106DOI: (10.1016/S0969-2126(01)00675-X)

Figure 1 Structure of SurE (a) Structure of the monomer with the N-terminal domain in red and C-terminal domain in maroon. (b) Structure of the dimer with two-fold dyad in the plane of the figure. Color coding for one monomer is as in (a), and the second monomer is in yellow. N and C termini are labeled in the red/maroon monomer. (c) Specific interactions responsible for the formation of the tetramer in β hairpin region of blue and green monomers. (d) Structure of the SurE tetramer Structure 2001 9, 1095-1106DOI: (10.1016/S0969-2126(01)00675-X)

Figure 1 Structure of SurE (a) Structure of the monomer with the N-terminal domain in red and C-terminal domain in maroon. (b) Structure of the dimer with two-fold dyad in the plane of the figure. Color coding for one monomer is as in (a), and the second monomer is in yellow. N and C termini are labeled in the red/maroon monomer. (c) Specific interactions responsible for the formation of the tetramer in β hairpin region of blue and green monomers. (d) Structure of the SurE tetramer Structure 2001 9, 1095-1106DOI: (10.1016/S0969-2126(01)00675-X)

Figure 2 Diagram of the Secondary-Structural Elements in the SurE Monomer Structure 2001 9, 1095-1106DOI: (10.1016/S0969-2126(01)00675-X)

Figure 3 Multiple Sequence Alignment (CLUSTAL W [1.81]) of Proteins Belonging to the SurE Family Completely conserved residues are highlighted with blue or yellow. Amino acids forming the presumed active site are highlighted with yellow Structure 2001 9, 1095-1106DOI: (10.1016/S0969-2126(01)00675-X)

Figure 4 Enzymatic Analysis of SurE Phosphatase Activity on p-Nitrophenyl Phosphate (PNPP) Assays were done as described in the Experimental Procedures, and each data point reflects the average of three trials, with the error bars reflecting ±1 standard deviation when they are larger than the plotted point. (a) SurE catalyzes the hydrolysis of PNPP in a concentration-dependent manner. Data are shown from three experiments in which 0.1 μg of SurE protein (diamonds) or a buffer control (squares) was incubated in a 250 μl reaction with 100 mM MOPS (pH 6.0 at 75oC), 5% glycerol, and 10 mM MgSO4 at 75oC. (b) SurE phosphatase is activated by magnesium ion. SurE (0.1 μg protein, diamonds) or a buffer control (squares) was incubated at 76°C with increasing concentrations of magnesium sulfate in a 250 μl reaction mixture with 100 mM sodium MOPS (pH 6.1 at 76oC), 5% glycerol, and 15 mM disodium PNPP. (c) SurE is an acid phosphatase. SurE (0.1 μg protein, diamonds) or a buffer control (squares) was incubated at 76°C in a series of 100 mM sodium 2-[N-morpholino]ethanesulfonic acid (MES) buffers adjusted to different pH values at 76°C containing 5% glycerol, 6 mM MgSO4, and 15 mM disodium PNPP. Similar results were found when MOPS or phosphate buffers were used (data not shown). (d) SurE is activated at elevated temperatures and has minimal activity at 40oC. SurE (0.1 μg, diamonds) or a buffer control (squares) was incubated at various temperatures in a 100 mM MOPS buffer (adjusted to pH 6 at 76°C) with 5% glycerol, 10 mM MgSO4, and 15 mM disodium PNPP. The solid line represents the enzyme specific activity and shows a peak at 80oC Structure 2001 9, 1095-1106DOI: (10.1016/S0969-2126(01)00675-X)

Figure 5 Enzymatic Activity of SurE phosphatase on PNPP, Adenosine-5′-Monophosphate, and α-Napthyl Phosphate Phosphate release was measured with the EnzChek system as described in the Experimental Procedures. The non-SurE-dependent spontaneous hydrolytic activity was subtracted from the SurE activity, and the resultant enzyme specific activity is shown in squares for α-napthyl phosphate (α-NP), in diamonds for adenosine-5′-monophosphate (AMP), and triangles for PNPP. The error bars for each symbol represent ± 1 standard deviation. For each substrate, the best-fit Km curve (as determined by a least-squares fit) is superimposed through the points. The calculated Km and Vmax values for α-NP are 5.0 mM and 233 μmol/min/mg, respectively. For AMP, these values are 3.4 mM and 105 μmol/min/mg, while for PNPP the values are 31.5 mM and 51.6 μmol/min/mg. The kinetics for the PNPP substrate were determined with additional data points at higher concentrations up to 150 mM as shown in Figure 6a Structure 2001 9, 1095-1106DOI: (10.1016/S0969-2126(01)00675-X)

Figure 6 Presumed Active Site of SurE (a) A stereo view of conserved residues forming a well-defined cluster near the protein surface. Water molecules are represented as pink spheres. (b) Location of the presumed active site in the SurE dimer and its solvent accessibility. Protein is shown as a space-filling model viewed down the two-fold dyad. Residues that are conserved and form the presumed active site are in space-filling representation (red and green in two different subunits, respectively). The conserved Asp88 in both subunits is labeled for reference Structure 2001 9, 1095-1106DOI: (10.1016/S0969-2126(01)00675-X)

Figure 6 Presumed Active Site of SurE (a) A stereo view of conserved residues forming a well-defined cluster near the protein surface. Water molecules are represented as pink spheres. (b) Location of the presumed active site in the SurE dimer and its solvent accessibility. Protein is shown as a space-filling model viewed down the two-fold dyad. Residues that are conserved and form the presumed active site are in space-filling representation (red and green in two different subunits, respectively). The conserved Asp88 in both subunits is labeled for reference Structure 2001 9, 1095-1106DOI: (10.1016/S0969-2126(01)00675-X)