Structure solution and molecular dynamics refinement of the yeast Cu,Zn enzyme superoxide dismutase

Abstract

Cu,Zn yeast superoxide dismutase was crystallized from polyethylene glycol solutions. The crystals belong to the P2~212 space group, with cell dimensions a = 105.3, b = 143.0, c = 62-1 ]k; two dimers of Mr = 32000 each are contained in the asymmetric unit. Diffraction data at 2.5A resolution were collected with the image-plate system at the EMBL synchrotron radiation facility in Hamburg. The structure was determined by molecular replacement using as a search model the 'blue-green' dimer of the bovine Cu,Zn superoxide dismutase. The crystallographic refinement of the molecular replacement solution was performed by means of molecular dynamics techniques and resulted in an R factor of 0.268 for the data between 6-0 and 2.5 A. The model was subsequently subjected to conventional restrained crystallographic refinement of the coordi-* To whom correspondence should be addressed.0108-7681/91/060918-10503.00 nates and temperature factors. The current R value for the data between 6.0 and 2.5 A is 0.220. Owing to the large radius of convergence of the molecular dynamics-crystallographic refinement, the convergence of the refinement process was reached after 18.1 ps of simulation time. The geometry of the active site of the enzyme appears essentially preserved compared with the bovine superoxide dismutase. The fl-barrel structure in the yeast enzyme is closed at the upper part by an efficient hydrogen-bonding scheme.

The solution structure of reduced dimeric copper zinc superoxide dismutase. The structural effects of dimerization

“…NOE cross-peaks in three-dimensional 15 N and 13 C-NOESY-HSQC spectra and in two-dimensional NOESY spectra were integrated and converted into upper distance limits for interproton distances with the program CALIBA [41]. The calibration curves for this conversion were adjusted iteratively as the structure calculations proceeded.…”

“…In three-dimensional 15 N-and 13 C-NOESY-HSQC spectra and in two-dimensional 15 N-NOESY spectra, 3566 NOE cross peaks were assigned and converted into distance constraints. Forty-nine dihedral / angles constraints were obtained from the analysis of the HNHA spectrum, 52 dihedral w angles were obtained from the 15 N-NOESY-HSQC spectrum and 14 v 1 torsion angles from the HNHB spectrum.…”

“…However, most of the 1 H- 15 N cross peak degeneracies present in 15 N-HSQC spectrum were resolved in at least one of the HNCA, HN(CO)CA, HNCO and HN(CA)CO-TROSY-type spectra already performed for the backbone assignment, reported by us [30].…”

“…Forty-nine dihedral / angles constraints were obtained from the analysis of the HNHA spectrum, 52 dihedral w angles were obtained from the 15 N-NOESY-HSQC spectrum and 14 v 1 torsion angles from the HNHB spectrum. A total of 45 proton pairs were stereospecifically assigned with the program GLOMSA: 16 protons belonging to bCH 2 , 11 to aCH, 1 to cCH 2 , 1 to dCH 3 and 1 to cCH 3 ; and 15 bCH 2 were assigned by the analysis of the HNHB experiment.…”

“…Copper occurs in the oxidized and in the reduced state, both of which are necessary for the function. The X-ray structure of the oxidized form has been available since 1982 for the bovine enzyme [6,7] and several other structures have become available [8][9][10][11][12][13][14][15][16][17][18]. Reduced state structures are also available although the picture is less clear-cut around the copper-binding site [19][20][21].…”