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Nucleoside Triphosphates - From Synthesis to Biochemical Characterization
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ADP-2Ho as a phasing tool for nucleotide-containing proteins.

Shao-Yang Ku1, G David Smith, P Lynne Howell

  • 1Program in Molecular Structure and Function, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.

Acta Crystallographica. Section D, Biological Crystallography
|March 21, 2007
PubMed
Summary

Trivalent holmium ions successfully replaced magnesium in Bacillus subtilis 5-methylthioribose (MTR) kinase, enabling structure determination. This holmium-MTR kinase complex offers a novel phasing tool for similar metalloproteins.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Crystallography

Background:

  • Bacillus subtilis 5-methylthioribose (MTR) kinase is crucial for cellular metabolism.
  • Determining the structure of MTR kinase is essential for understanding its function.
  • Previous structural studies faced challenges in phasing.

Purpose of the Study:

  • To determine the previously unknown structure of MTR kinase.
  • To investigate the use of trivalent holmium ions as a phasing tool.
  • To explore the binding of nucleotide-holmium complexes in metalloproteins.

Main Methods:

  • Isomorphous replacement of magnesium ions with trivalent holmium ions in the nucleotide-binding domain.
  • X-ray crystallography using synchrotron radiation at the L(III) absorption edge of holmium.
  • Single-wavelength anomalous diffraction (SAD) and single isomorphous replacement with anomalous scattering (SIRAS) phasing.

Main Results:

  • Formation of an ADP-2Ho complex where holmium ions isomorphously replaced magnesium.
  • Successful SAD and SIRAS phasing of the MTR kinase structure using the holmium complex.
  • The structure revealed two holmium ions approximately 4 Å apart, sharing ligands with ADP and a water molecule.
  • Structure determination using Cu Kα radiation was more challenging.

Conclusions:

  • Trivalent holmium ions serve as an effective phasing tool for MTR kinase structure determination.
  • The holmium-MTR kinase complex provides a robust method for solving previously intractable structures.
  • This approach holds promise for phasing other metal-dependent, nucleotide-containing proteins.