Group III hybrid histidine kinase (HHK3) is universally present in all pathogenic fungi and regulates various biological processes.1, 3 Like Sln1, a group VI HHK, HHK3 is involved in the HOG MAPK pathway as an osmotic stress sensor.4, 7 HHK3 orthologs complement the sln1 mutation in S. cerevisiae and negatively regulate the HOG pathway through Ssk1-Ypd1 phosphotransfer which is similar to Sln1,.8, 9 The defining characteristic feature of HHK3 is the presence of poly-HAMP domains (Histidine kinases, Adenylyl cyclases, Methyl-accepting chemotaxis proteins, and Phosphatases) at their N-terminal.10 Structurally each HAMP monomer contains two amphipathic α-helices (AS1 and AS2) separated by a flexible connector loop of ∼14 residues. AS1 and AS2 dimerize to form a parallel 4-helix bundle with a conserved heptad repeat pattern,.11, 13 Different models ranging from static on/off conformations to dynamic bundles have been proposed to explain HAMP-mediated signaling.14, 15 In bacterial HKs, the HAMP domain acts as a signal transducer that mediates signal transmission from the sensor domain to the kinase domain whereas the poly-HAMP domain acts as both sensor and signal transducer in HHK3.10, 16

HHK3 is an important target for developing novel antifungal agents. It is a molecular target for fludioxonil, a widely used agricultural fungicide. A recent study done on Drk1, an HHK3 orthologue from B. dermatitidis, showed that fludioxonil indirectly causes covalent modification of cysteine residues present in the poly-HAMP domain of Drk1 thereby modulating its activity from kinase to phosphatase which causes constitutive activation of the HOG pathway.17 Many bacterial histidine kinases show bifunctional activity by performing both phosphorylation and dephosphorylation of the downstream effector protein.18, 19, 20, 21 In contrast to the phosphotransfer reaction, which involves direct transfer of the phosphoryl group from the HK His to the RR Asp, HK-mediated RR dephosphorylation is thought to involve a water molecule,22 .24, 26 Like these kinases, fungal HHK3 appeared to perform kinase and phosphatase activity towards its downstream phosphotransfer protein Ypd1. Several studies showed that point mutations in poly-HAMP domains affect fludioxonil sensitivity and osmosensing functions of HHK3 orthologs.3, 26, 27, 28 Thus, the poly-HAMP domain appeared to play a very important role in regulating the bifunctional activity of HHK3.

The number of repeats in the poly-HAMP domain varies in HHK3 orthologs across fungal phyla. Orthologs from yeast e.g. DhNik1 of Debaryomyces hansenii contain five HAMP domains and four HAMP-like linkers (known as divergent HAMP) in its poly-HAMP domain. Orthologs from filamentous ascomycetes or basidiomycetes harbour a higher number of HAMP domain repeats at their N-terminal region. A few HHK3 orthologs from yeast and filamentous fungi have been studied earlier which indicated that HAMP domains played an important role in the functionality of HHK3.8, 29, 30, 31 The first structural understanding of HHK3 came from the SAXs study of DhNik1 which, in fact, is one of the most well-studied HHK3. It appears the poly-HAMP domain is a dynamic structure and the concerted motions of HAMP and HAMP-like linker domains regulate the histidine kinase activity through an in-line mechanism in DhNik1.27 Correct positioning of the HAMP domain is necessary for the proper functioning of DhNik1, as the reshuffling of the HAMP domain leads to inactive protein.8 HAMP domains and HAMP-like linker domains appeared to have a differential role and the deletion of individual domains results in distinct active forms in DhNik1 as well as in Hik1, a HHK3 ortholog from Magneporthe oryzae,28 Some of the deletion mutants showed complete loss of activity while some others became constitutively active kinases which were nonresponsive to fludioxonil or osmolarity changes.28 These studies indicated that poly-HAMP regulates multiple signal output conformations rather than just an on-off switch. HHK3 orthologs are also converted to a cytotoxic phosphatase form in vivo when cells are exposed to fludioxonil17 however this form of HHK3 has not been isolated or studied in vitro. A structural understanding of the conversion of HHK3 into this cytotoxic phosphatase form will be important for antifungal drug development. This study reports the isolation of a cytotoxic phosphatase LOCK-IN mutant of DhNik1 (DhNik1CT) that recapitulates the antifungal action of fludioxonil. DhNik1CT harbours an in-frame deletion in the poly-HAMP domain. The fusion of the poly-HAMP domain of DhNik1CT with the histidine kinase and receiver domain of another fungal hybrid histidine kinase also creates a hybrid that is cytotoxic to the fungal cell. We generated the atomic structure of wild-type DhNik1 and DhNik1CT using AlphaFold multimer.32 MD simulation of the modelled structure showed a difference in the movement of the residues involved in the opening and closing of the ATP lid in DhNik1CT bringing the kinase domain and the receiver domain in close proximity as well as increasing solvent accessibility of the kinase domain in DhNik1CT favouring reverse phospho-transfer reaction and converting it from histidine kinase to a phosphatase.