Background: A native Kir channel in a distant relative of vertebrates interacts weakly with PIP 2 . Results: Mutagenesis restores the vertebrate channel sensitivity to PIP 2 in sponge channels. Conclusion: A basic residue in the tether helix of Kir is required for high affinity PIP 2 regulation. Significance: Evolution conferred a high affinity interaction of vertebrate Kir channels with PIP 2 , which is lacking in a distant relative.

Approximately one million people die from malaria each year, and thus this disease is a prevalent and pressing matter. Plasmodium falciparum, the protozoan parasite responsible for malaria has been subject to intensive study with the goal of developing specific drugs for malaria treatment. Plasmodium falciparum has a distinctive Malate Dehydrogenase (MDH), being tetrameric compared to the canonical dimeric forms of MDH, yet shares the same catalytic and substrate binding site motives as human mitochondrial and cytosolic forms. To investigate the possible existence of a cryptic allosteric site in the P falciparum enzyme, we have used sequence and structural bioinformatics and identified several short sequences,(G175‐L176, Q211‐M216) each containing highly conserved (in Plasmodium and related species) amino acids that are different in the Plasmodium falciparum enzyme than the human mitochondrial or cytosolic forms. To investigate the possibility that these regions form a cryptic allosteric site in Plasmodium falciparum we expressed, purified, using NiNTA Affinity chromatography, and characterized all three forms (Plasmodium falciparum, human cytosolic and human mitochondrial malate dehydrogenases). In terms of kinetic parameters the Plasmodium falciparum enzyme resembled the mammalian mitochondrial isoform, exhibiting similar substrate inhibition with oxaloacetate and weaker affinity for NADH than the mammalian cytosolic isoform. To determine the potential that the Plasmodium falciparum enzyme had a cryptic allosteric site we designed two mutations, one in each of the two significant sequence differences between the human isoforms and the Plasmodium falciparum enzyme (.D176N and R214E ). Each mutation was constructed using Quikchange mutagenesis, transformed into XL Gold cells and expressed. The purified mutant proteins were characterized using enzyme kinetics and size exclusion chromatography and their overall secondary structures compared to the wildtype enzyme using circular dichroism spectroscopy. Stability was compared using CD thermal melts at 222nm and a Fluorescence based Thermal Shift assay. While both mutations showed altered specific activities compared to wildtype, the D176N mutant showed a 90% lower activity than wildtype Plasmodium falciparum malate dehydrogenase. Both mutations significantly lowered affinity for the cofactor NADH, >20fold decrease in affinity. Taken together these results suggest that Plasmodium falciparum malate dehydrogenase may have a highly conserved cryptic allosteric site distinct from any such site in the mammalian isoforms. This Cryptic Allosteric Site could be exploited to develop inhibitors of Plasmodium falciparum malate dehydrogenase that would not impact the human isoforms. Support or Funding Information This work was supported by NSF Grants 1726932 and 0448905.

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Suppressor of IKKepsilon (SIKE), a protein first identified in the antiviral innate immune response associated with TANK‐binding kinase 1 (TBK1), is associated with multiple, distinct protein complexes including TBK1, STRIPAK (striatin interacting phosphatase and kinase) and cytoskeletal proteins, tubulin and actinin. Although SIKE's function is not fully defined in these complexes, SIKE does function as a high‐affinity substrate of TBK1 with phosphorylation occurring at up to six SIKE serine residues, S133, S185, S187, S188, S190, and S198. The accompanying figure shows a cartoon diagram of SIKE dimer model with the phosphorylation sites shown in spheres (red 187, 190; orange 133; 187 green; 188 and 190 cyan). Using a phosphomimetic mutant (S133/185/187/188/190/198E) of SIKE, size exclusion chromatography and chemical crosslinking studies showed primarily a monomeric species, whereas unmodified SIKE separated as a dimeric species. These observations suggested that SIKE could undergo a phosphorylation‐induced change in quaternary structure, but how many of the six phosphorylation sites were necessary? To explore this idea, conservation of the phosphorylation sites was explored. 133 SIKE sequences (Animalia, Fungi) from OrthoDB and Protein Blast were compiled and aligned using Clustal Omega. Positions 187 and 190 were conserved as serine in >90% of sequences whereas position 133 was ~60% serine and 25% aspartate or glutamate with a 9:10 bias towards aspartate. The remaining positions had 75% or less retaining serine with position 188 registering ~32% lysine. Using a model of the SIKE dimer where the phosphorylation sites line part of the dimer interface, phosphorylated S187/190 in each subunit would be positioned to repulse one another. To gain further insight into the role of individual phosphorylation sites on the theoretical dimer interface, phosphomimetic mutants at each phosphorylation site were computationally created (PyMOL), energy minimized (GalaxyRefineComplex) to yield 10 models, and the interface stability of each model for each mutant assessed on a per residue basis (HawkDock MM/GBSA). These mutant data were then compared to a similar analysis of the wild type SIKE model to identify residues with significant changes in total free energy of binding (dimer interface). S133E showed no significant changes from WT whereas S185/187/188/190/198E individually revealed a similar pattern of residues with altered total free energy of binding adjacent to the phosphorylation sites that differed in the number (19‐30) of residues affected in the region. The sequence conservation suggested that positions 187 and 190 could serve as key phosphorylation sites to trigger a dimer to monomer transition. Although the computational analyses has not resolved the effect of multiple phosphorylation events, the primary alteration to dimer stability is localized to the regions adjacent to the phosphorylation sites.

Wound inducible transcript 3.0 (WIT3.0) is a novel cytoskeleton protein that regulates fibroblast migration and initiates rapid wound closure. WIT3 shares ~50% sequence identity with an innate immune protein of unknown function, Suppressor of IKKepsilon (SIKE), that also associates with cytoskeletal proteins. We hypothesize that SIKE and WIT3 will have similar biochemical and biophysical characteristics that contribute to a shared function. To assess these similarities, we undertook characterization of WIT3 structure utilizing circular dichroism, fluorescence‐based thermal shift assays, ligand binding studies using an ANS reporter, and crosslinking with BS3. Using PHYRE2, a WIT3 model was predicted that consisted of an alpha helical coil with 30% disordered regions. The secondary structure of WIT3 was assessed via circular dichroism. Wavelength spectra is consistent with an alpha helical protein and thermal melt data showed a linear unfolding pattern with Tm of 37°C, suggesting a minimally stable protein. As part of the PHYRE2 model, 3DLigandSite proposed a zinc binding site. To assess an interaction between WIT3 and divalent cations, WIT3‐ANS titration and fluorescence‐based thermal shift assays were completed +/‐ Mg, Mn, Ca, Ni and Zn. An interaction between WIT3 and Zn was confirmed where, in the presence of zinc, WIT3's affinity for ANS was 2 fold higher than in the absence of zinc. The initial comparison suggests that WIT3 and SIKE share similar helical structure and similar FTS melt curve topography indicating exposed hydrophobic residues prior to denaturation, but different structural stability and a potential divalent cation binding site on WIT3. Together, these data suggest that WIT3 and SIKE share several biochemical characteristics that may allow these two proteins to have complimentary function.Support or Funding InformationStudies were funded in part by USD SURE.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.