Differential role of HAMP-like linkers in regulating the functionality of the group III histidine kinase DhNik1p

J Biol Chem. 2014 Jul 18;289(29):20245-58. doi: 10.1074/jbc.M114.554303.

Abstract

Nik1 orthologs are sensor kinases that function upstream of the high osmolarity glycerol/p38 MAPK pathway in fungi. They contain a poly-HAMP module at their N terminus, which plays a pivotal role in osmosensing as well as fungal death upon exposure to fludioxonil. DhNik1p is a typical member of this class that contains five HAMP domains and four HAMP-like linkers. We investigated the contribution of each of the HAMP-like linker regions to the functionality of DhNik1p and found that the HAMP4b linker was essential as its deletion resulted in the complete loss of activity. Replacement of this linker with flexible peptide sequences did not restore DhNik1p activity. Thus, the HAMP-like sequence and possibly structural features of this linker region are indispensable for the kinase activity of DhNik1p. To gain insight into the global shape of the poly-HAMP module in DhNik1p (HAMP1–5), multi-angle laser light and small angle x-ray scattering studies were carried out. Those data demonstrate that the maltose-binding protein-tagged HAMP1–5 protein exist as a dimer in solution with an elongated shape of maximum linear dimension ∼365 Å. Placement of a sequence similarity based model of the HAMP1–5 protein inside experimental data-based models showed how two chains of HAMP1–5 are entwined on each other and the overall structure retained a periodicity. Normal mode analysis of the structural model is consistent with the H4b linker being a key to native-like collective motion in the protein. Overall, our shape-function studies reveal how different elements in the HAMP1–5 structure mediate its function.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Debaryomyces / drug effects
  • Debaryomyces / enzymology*
  • Debaryomyces / genetics
  • Dioxoles / pharmacology
  • Fungal Proteins / chemistry*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • Fungicides, Industrial / pharmacology
  • Genes, Fungal
  • Histidine Kinase
  • Intracellular Signaling Peptides and Proteins / chemistry
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Kinases / chemistry*
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Protein Multimerization
  • Protein Serine-Threonine Kinases / chemistry
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism
  • Protein Structure, Tertiary
  • Pyrroles / pharmacology
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequence Deletion
  • Structural Homology, Protein

Substances

  • Dioxoles
  • Fungal Proteins
  • Fungicides, Industrial
  • Intracellular Signaling Peptides and Proteins
  • Pyrroles
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • Protein Kinases
  • HSL1 protein, S cerevisiae
  • Protein Serine-Threonine Kinases
  • Histidine Kinase
  • SLN1 protein, S cerevisiae
  • fludioxonil