Structural characterization of core-bradavidin in complex with biotin

PLoS One. 2017 Apr 20;12(4):e0176086. doi: 10.1371/journal.pone.0176086. eCollection 2017.

Abstract

Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 ("Brad-tag") act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin-Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin.

MeSH terms

  • Affinity Labels
  • Binding Sites
  • Biotin / chemistry*
  • Carrier Proteins / chemistry*
  • Crystallography, X-Ray
  • Models, Molecular
  • Protein Conformation

Substances

  • Affinity Labels
  • Carrier Proteins
  • bradavidin protein, Bradyrhizobium japonicum
  • Biotin

Grants and funding

This work was supported by grant funding from the Academy of Finland (257814 (MSJ), 272283 (MSJ), 290506 (VPH), 261285 (MSK), and 272288 (MSK)); Sigrid Juselius Foundation; Joe Pentti, and Tor Borg Memorial Fund; Academy of Finland FIRI program (141398) and Åbo Akademi Center of Excellence in Cell Stress and Aging. The funders had no influence in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Fimlab Laboratories (Tampere, Finland) provided support in the form of salaries for author VPH and in the form of research materials for the group lead by VPH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.