Function and structure of the novel haem-binding protein HbpS from streptomycetes
Microbial Ecology and Applied Microbiology
Final Report Abstract
We have discovered the Streptomyces three-component system HbpS-SenS-SenR in which the extracellular protein HbpS has an octameric assembly and modulates the activity of the sensor kinase SenS. Having purified the wild-type HbpS and a designed mutant proteins, we obtained high resolution 3D crystal structures showing an octameric assembly of HbpS. Analyses of HbpS-heme binding kinetics support the role HbpS as a heme-sensor and suggested a role in heme transport. Sequence comparisons suggested that HbpS may bind a cobalamin. UV-visible spectroscopy confirmed binding to aquo-cobalamin, but not to other cobalamins. Additional mutational studies allowed the identification of the histidine residue interacting with this compound. HbpS and corresponding close and distant homologues (>10000x) comprise the DUF336 domain either alone or in fusion with other protein domain(s), i.e. PduO. This is a two-domain protein, involved in 1,2-propanediol utilization in the pathogenic Gram-negative bacterium Salmonella enterica, is an ATP:Cob(I)alamin adenosyltransferase but this is a function of the N-terminal domain alone. The role of its C-terminal domain (PduOC) was until now unknown. We showed that PduOC binds heme in vivo. The structure of PduOC co-crystallised with heme was solved (1.9 Å resolution) showing an octameric assembly with four heme moieities. The four heme groups are highly solvent-exposed and the heme iron is hexacoordinated with bis-His ligation by histidines from different monomers. Static light scattering confirmed the octameric assembly in solution, but a mutation of the heme-coordinating histidine caused dissociation into dimers. Isothermal titration calorimetry using the PduOC apoprotein showed strong heme binding (Kd = 1.6 × 10^-7 M). Additional, physiological studies suggest that PduOC:heme plays an important role in the set of cobalamin transformations required for effective catabolism of 1,2-propanediol. The HbpS protein family includes redox-active proteins that interact with the tetrapyrroles heme and cobalamin. Are these protein able to interact with other pyrrole-based secondary metabolites? Are these interactions part of novel signalling networks? Finding of answers these questions will be highly relevant for Streptomycetes as well as for other microorganisms.
Publications
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(2014) The extracellular heme-binding protein HbpS from the soil bacterium Streptomyces reticuli is an aquo-cobalamin binder. J Biol Chem 289: 34214-28
Ortiz de Orué Lucana, Darío; Fedosov, Sergey N.; Wedderhoff, Ina; Che, Edith N. & Torda, Andrew E.
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(2016) Complete genome sequence of Streptomyces reticuli, an efficient degrader of crystalline cellulose. J Biotechnol.
Wibberg, Daniel; Al-Dilaimi, Arwa; Busche, Tobias; Wedderhoff, Ina; Schrempf, Hildgund; Kalinowski, Jörn & Ortiz de Orué Lucana, Darío
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(2016) Deciphering the transcriptional response mediated by the redox-sensing system HbpS-SenS-SenR from Streptomycetes. PLoS One
Busche, Tobias; Winkler, Anika; Wedderhoff, Ina; Rückert, Christian; Kalinowski, Jörn & Ortiz de Orué Lucana, Darío
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(2016) The Crystal Structure of the C-terminal domain of the PduO protein from Salmonella enterica: An old fold with a new heme-binding mode. Front Microbiol
Ortiz de Orué Lucana, Darío; Hickey, Neal; Hensel, Michael; Klare, Johann P.; Geremia, Silvano; Tiufiakova, Tatiana & Torda, Andrew E.
