Differential Role of HAMP-like Linkers in Regulating the Functionality of the Group III Histidine Kinase DhNik1p

Nik1 orthologs are sensor kinases that function upstream of the high osmolarityglycerol/p38 MAPK pathway in fungi. They contain a poly-HAMP module at their Nterminus, which plays a pivotal role in osmosensing as well as fungal death uponexposure to fludioxonil. DhNik1p is a typical member of this class that contains fiveHAMP domains and four HAMP-like linkers. We investigated the contribution of each ofthe HAMP-like linker regions to the functionality of DhNik1p and found that theHAMP4b linker was essential as its deletion resulted in the complete loss ofactivity. Replacement of this linker with flexible peptide sequences did not restoreDhNik1p activity. Thus, the HAMP-like sequence and possibly structural features ofthis linker region are indispensable for the kinase activity of DhNik1p. To gaininsight 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 proteinexist as a dimer in solution with an elongated shape of maximum linear dimension∼365 Å. Placement of a sequence similarity based model of theHAMP1–5 protein inside experimental data-based models showed how two chains ofHAMP1–5 are entwined on each other and the overall structure retained aperiodicity. Normal mode analysis of the structural model is consistent with the H4blinker being a key to native-like collective motion in the protein. Overall, ourshape-function studies reveal how different elements in the HAMP1–5 structuremediate its function.