ADP-2Ho as a phasing tool for nucleotide-containing proteins.
Trivalent holmium ions were shown to isomorphously replace magnesium ions to form an ADP-2Ho complex in the nucleotide-binding domain of Bacillus subtilis 5-methylthioribose (MTR) kinase. This nucleotide-holmium complex provided sufficient phasing power to allow SAD and SIRAS phasing of this previously unknown structure using the L(III) absorption edge of holmium. The structure of ADP-2Ho reveals that the two Ho ions are approximately 4 A apart and are likely to share their ligands: the phosphoryl O atoms of ADP and a water molecule. The structure determination of MTR kinase using data collected using Cu Kalpha X-radiation was also attempted. Although the heavy-atom substructure determination was successful, interpretation of the map was more challenging. The isomorphous substitution of holmium for magnesium in the MTR kinase-nucleotide complex suggests that this could be a useful phasing tool for other metal-dependent nucleotide-containing proteins.
Ku
Shao-Yang
SY
Program in Molecular Structure and Function, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.
Smith
G David
GD
Howell
P Lynne
PL
D000244:Adenosine Diphosphate / Q000737:chemistry*; D001412:Bacillus subtilis / Q000201:enzymology*; D003300:Copper / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray; D006695:Holmium / Q000737:chemistry*; D008274:Magnesium / Q000737:chemistry*; D008958:Models, Molecular; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation / Q000528:radiation effects; D013455:Sulfur / Q000737:chemistry
D000244:Adenosine Diphosphate; D013455:Sulfur; D003300:Copper; D017853:Phosphotransferases (Alcohol Group Acceptor); C021787:5-methylthioribose kinase; D008274:Magnesium; D006695:Holmium