User:Oleg Kovalevskiy/Engineered mutants of HlyIIR

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Engineered mutants of HlyIIR

Abstract

B.cereus HlyIIR[1] belongs to the TetR family of dimeric transcriptional regulators. HlyIIR contains an insert between α-helices α8 and α9, which is located at the subunit-subunit interface. N-terminal segment of this insert (amino acids Pro161-Ser169) forms a short α-helix α8* that occupies a complementary cavity on the surface of the adjacent subunit, while the C-terminal segment comprising 16 amino acids (Leu170-Glu185) is disordered. To understand whether this disordered segment is important for protein’s function we determined crystal structures of two engineered HlyIIR proteins where this segment was either substituted by a 7-residue flexible Ser-Gly linker (dS-HlyIIR) or replaced by a cleavable peptide containing proteolytic sites at both ends (dThr-HlyIIR). Unexpectedly, alteration or proteolytic removal of the disordered segment resulted in changes in protein’s conformation and in a remarkable rearrangement at the subunit-subunit interface. X-ray structures of the two engineered proteins revealed an unusual plasticity at the dimerisation interface of HlyIIR enabling it to form dimers stabilised by different sets of interactions. Structural comparison indicates that in spite of the flexible nature of the disordered segment, it is critical for maintaining the native structure as it influences the position of α8*. The data demonstrate how disordered loops on protein surfaces may affect folding and subunit-subunit interactions.


Supplementary figures

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Wild type HlyIIR subunits are shown in blue and red, dS-HlyIIR in yellow and dThr-HlyIIR in green

Orientation of subunits within the dimers of wild type and engineered HlyIIR

  • Ribbon diagram of the wild type HlyIIR. Disordered segment 170-185 is indicated by dotted curve.
  • Superposition of dimers of HlyIIR variants. Dimers are shown as Cα models, overlaid using single subunit of the dimer.
  • Contacts at the dimerisation interface in the wild type and mutant proteins illustrating positional shifts induced by mutagenesis. You can view superposition of all three structures or details of the dimerisation interface of wild type HlyIIR, dS-HlyIIR and dThr-HlyIIR separately.

Conformational differences between wild type and mutant HlyIIR.

  • Overlaid models of single subunits shown as Cα; the disordered segment 170-185 is indicated by dotted curve. The most striking conformational differences are observing between heices α6 and α7; position of α8* changed as well.
  • Hydrophobic interactions between α8* residues F163 and F166 of one subunit and α6/α7 residues of another subunit. These interactions are critical for maintaining protein conformation in wild type HlyIIR and are disrupted in dS-HlyIIR and dThr-HlyIIR.





References

  1. ↑ Kovalevskiy OV, Lebedev AA, Surin AK, Solonin AS, Antson AA. Crystal structure of Bacillus cereus HlyIIR, a transcriptional regulator of the gene for pore-forming toxin hemolysin II. J Mol Biol. 2007 Jan 19;365(3):825-34. Epub 2006 Oct 26. PMID:17097673 doi:10.1016/j.jmb.2006.10.074

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Oleg Kovalevskiy

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