Elucidation of haem-binding sites in the actinobacterial protein HbpS

Abstract

The extracellular haem-binding protein from Streptomyces reticuli (HbpS) has been shown to be involved in redox sensing and to bind haem. However, the residues involved in haem coordination are unknown. Structural alignments to distantly related haem-binding proteins from Mycobacterium tuberculosis were used to identify a candidate haem-coordinating residue, and site-directed mutagenesis with UV/Vis spectroscopy was used to assess haem binding in vivo and in vitro. We present strong evidence that HbpS belongs to the small set of proteins, which do not use histidine to coordinate the metal in the haem group. Further spectroscopic evidence strongly indicates that threonine 113 is actively involved in coordination of haem. Subsequent protein/haem titration experiments show a 1 : 2, protein/haem stoichiometry. We also present data showing the degradation of haem by HbpS in vivo. Because HbpS is conserved in many Actinobacteria, the presented results are applicable to related species.

The Extracellular Heme-binding Protein HbpS from the Soil Bacterium Streptomyces reticuli Is an Aquo-cobalamin Binder

“…Such a K d value in heme-sensing proteins might be an indication of a flexible heme-binding pocket that is required during sensing of heme. From structural comparisons (27), one knows that HbpS is similar to the heme binding domains within DosS and DosT from Mycobacterium tuberculosis. These are two-component membrane-bound kinases that are involved in heme sensing (61,62).…”

“…Escherichia coli strains BL21(DE3)pLysS and DH5␣ were cultivated in LB medium. The plasmid vector pETM11 as well as the plasmid constructs pETHbpS, pETHbpS-H28A, pETHbpS-H51A, pETHbpS-H156A, pETHbpS-T113A, and pETHbpS-T113H (27,28,32) were used.…”

“…This multifunctional protein sequesters large quantities of ferrous iron ions that might protect S. reticuli from the effects of peroxide-and iron-based oxidative stress (25). HbpS is also an unusual heme-binding protein in which a threonine residue (Thr-113) apparently binds to the tetrapyrrole macrocycle (26,27). In vitro and in vivo studies have also shown that HbpS can degrade the heme group.…”

“…This oligomerization is essential for the interaction with iron ions as well as with the sensor kinase SenS but not for the interaction with heme (25,27,28,32). Sequence comparisons using the S. reticuli HbpS protein showed a large number of related proteins from both Gram-positive and Gram-negative bacteria, including species from the genera of Streptomyces, Arthrobacter, Rhodococcus, Nocardia, Leifsonia, Vibrio, Klebsiella, Pseudomonas, and Agrobacterium.…”

Iron Binding at Specific Sites within the Octameric HbpS Protects Streptomycetes from Iron-Mediated Oxidative Stress

“…Protein-DNA interaction studies revealed that the phosphorylated response regulator SenR activates the transcription of furS , cpeB , hbpS , senS and senR [32], [33]. HbpS is a novel type of haem-binding protein and is secreted via the twin-arginine translocation (Tat) pathway [34], [35]. High resolution 3D crystal structures (PDB: 3FPV and 3FPW) of HbpS reveal an octameric assembly; analyses of electron density maps followed by mutagenesis and biochemical studies showed that a serine (Ser-26) and a histidine (His-28) within the N-terminal domains from neighboring subunits are crucial for oligomerization as well as for interaction with SenS-SenR [36].…”