Crystal structure and ligand binding properties of the truncated hemoglobin from Geobacillus stearothermophilus

https://doi.org/10.1016/j.abb.2006.09.033Get rights and content

Abstract

A novel truncated hemoglobin has been identified in the thermophilic bacterium Geobacillus stearothermophilus (Gs-trHb). The protein has been expressed in Escherichia coli, the 3D crystal structure (at 1.5 Å resolution) and the ligand binding properties have been determined. The distal heme pocket displays an array of hydrogen bonding donors to the iron-bound ligands, including Tyr-B10 on one side of the heme pocket and Trp-G8 indole nitrogen on the opposite side. At variance with the highly similar Bacillus subtilis hemoglobin, Gs-trHb is dimeric both in the crystal and in solution and displays several unique structural properties. In the crystal cell, the iron-bound ligand is not homogeneously distributed within each distal site such that oxygen and an acetate anion can be resolved with relative occupancies of 50% each. Accordingly, equilibrium titrations of the oxygenated derivative in solution with acetate anion yield a partially saturated ferric acetate adduct. Moreover, the asymmetric unit contains two subunits and sedimentation velocity ultracentrifugation data confirm that the protein is dimeric.

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) (FompT 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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