Crystal structure and ligand binding properties of the truncated hemoglobin from Geobacillus stearothermophilus
Section snippets
General molecular biology techniques
Methods for plasmid isolation, agarose gel electrophoresis, use of restriction and DNA-modification enzymes, DNA ligation, PCR, and electroporation of Escherichia coli cells were performed according to standard protocols. Chromosomal DNA was prepared from G. stearothermophilus strain 10 essentially according to standard protocols. E. coli strains TOP10 cells (mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 deoR recA1 araD139 Δ(ara-leu)7697 galU galK rpsL endA1 nupG) and TUNER(DE3) (F−ompT hsdSB
Overall fold
The three-dimensional structure of oxygenated Gs-trHb was solved at 1.5 Å and compared with those of other truncated hemoglobins characterized by the presence of the Trp-G8 residue within the heme pocket (group II, according to the classification of Wittenberg et al. [1]), namely Mt-trHbO from M. tuberculosis, Bs-trHb from B. subtilis and Tf-trHb from T. fusca. The overall fold and the relative position of the heme contacting residues are similar. Thus, the superposition of the Cα carbons
Discussion
The X-ray diffraction data collected so far on truncated hemoglobins provide a clear structural frame to test biochemical and functional hypotheses. The structures of the active sites of group II truncated hemoglobins are largely overlapping and highlight a strong conservation of the overall geometry of the distal heme pocket thus suggesting similar biochemical, if not physiological, functions. The heme pocket structure of these proteins indicates that the “ligand inclusive network of hydrogen
Protein data bank accession number
The atom coordinates and structure factors (2BKM) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunwick, NJ (http://www.rcsb.org/).
Acknowledgments
We thank Prof. Bruce Roe, University of Oklahoma, for providing G. stearothermophilus strain 10. This work was funded by Grant FIRB 2003 from MIUR (Ministero dell’Università e della Ricerca) and local grants to E.C. and A.B., COFIN 2004 to A.B., Crafoord Foundation and the Carl Tryggers Foundation to C.V.W.. P.K. was supported by a fellowship from the Research School of Pharmaceutical Sciences (FLÄK).
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