Frederick, McKinzie

Hurley, David

Mai, Elissa

Schoeneman, Laura

English

OpenRiver@Winona State University

OpenRiver Student Research and Creative Projects 2021-2022 Student Research & Creative Achievement Projects 9-1-2022 Investigation of Disease-Causing Point Variants of VRK1 McKinzie Frederick Winona State University David Hurley Winona State University Elissa Mai Winona State University Laura Schoeneman Winona State University Follow this and additional works at: https://openriver.winona.edu/studentgrants2022 Recommended Citation Frederick, McKinzie; Hurley, David; Mai, Elissa; and Schoeneman, Laura, "Investigation of Disease-Causing Point Variants of VRK1" (2022). Student Research and Creative Projects 2021-2022. 4. https://openriver.winona.edu/studentgrants2022/4 This Grant is brought to you for free and open access by the Student Research & Creative Achievement Projects at OpenRiver. It has been accepted for inclusion in Student Research and Creative Projects 2021-2022 by an authorized administrator of OpenRiver. For more information, please contact klarson@winona.edu. Abstract (Program Number 807.8)Vaccinia-related kinase 1 (VRK1) is aserine/threonine kinase that plays a variety of roles intranscription regulation and the cell cycle. Itssubstrates include histones, p53, and coilin. Thekinase activity of the VRK1 protein is largelycontrolled by its C-terminal tail, which interacts withthe enzyme active site. Several rare point mutationsin VRK1 (including L195V, R89Q, and Y213H) areassociated with neurodegenerative disorders, andquestions remain as to how these mutations affectVRK1’s intrinsic stability and activity.For this in vitro study, mutations were generated in aHis-tagged VRK1 kinase domain construct, andproteins were expressed in E. coli and purified. Themutant proteins’ folding, and stability were analyzedby circular dichroism, and ligand binding wasinvestigated using differential scanning fluorimetry.ATP hydrolysis kinetics were measured for wild-type,R89Q, and L195V mutants using ADP Quest activityassays. Protein modeling in PyMOL was also used tovisualize the kinase and its associated changes.Differential Scanning Fluorimetry (DSF)Circular Dichroism (CD)References1. El-Bazzal L, Rihan K, Bernard-Marissal N, et al. Loss of Cajal bodies in motor neurons from patients with novel mutations in VRK1. Hum Mol Genet. 2019;28(14):2378-2394. doi:10.1093/hmg/ddz0602. Marcos AT, Martín-Doncel E, Morejón-García P, et al. VRK1 (Y213H) homozygous mutant impairs Cajal bodies in a hereditary case of distal motor neuropathy. Ann Clin Transl Neurol. 2020;7(5):808-818. doi:10.1002/acn3.510503. Martín-Doncel, E., Rojas, A.M., Cantarero, L. et al. VRK1 functional insufficiency due to alterations in protein stability or kinase activity of human VRK1 pathogenic variants implicated in neuromotor syndromes. Sci Rep 9, 13381 (2019). https://doi.org/10.1038/s41598-019-49821-74. Stoll M, Teoh H, Lee J, et al. Novel motor phenotypes in patients with VRK1 mutations without pontocerebellar hypoplasia. Neurology. 2016;87(1):65-70. doi:10.1212/WNL.0000000000002813h5. Couñago, R.M., Allerston, C.K., Savitsky, P. et al. Structural characterization of human Vaccinia-Related Kinases (VRK) bound to small-molecule inhibitors identifies different P-loop conformations. Sci Rep 7, 7501 (2017). https://doi.org/10.1038/s41598-017-07755-yVRK1 StructureInvestigation of Disease-Causing Point Variants of VRK1McKinzie M. Frederick*, David P. Hurley*, Elissa N. Mai*, Laura L. Schoeneman*, Emily F. RuffWinona State UniversityWinona, MN 55987 * = these authors contributed equally to this workWild-type active state VRK1 (PDBID 6ac9). AMP-PNP (black) isshown as sticks in the active site.Mutated residues are labeled.ADP Quest Activity Assay ConclusionsElectrostatic maps are shown foractive state (6ac9) VRK1 WT andR89Q. Mutation was made inPyMOL and refined using 3Drefineand MolProbity. Maps weregenerated using the APBSElectrostatics plugin (red: negativesurface area, blue: positive surfacearea). Black arrows show themutation’s location.VRK1 VariantTm measured by CD (°C)Tm measured by DSF (°C)WT 48.8 ± 0.4 46 ± 1R89Q 48.5 ± 0.3 45.4 ± 1L195V 45 ± 1 38.8 ± 0.9Y213H 41.6 ± 0.5 40 ± 1Left: Representative thermal denaturation scans. Right: Comparison of meltingtemperatures (Tm) for VRK1 and variants. Scans in the left panel were fit to a4PL function using MyCurveFit, and Tm ± error in the table are for a singleexperiment. DSF Tm ± error are the average and standard deviation for 3-6independent experiments.Representative wavelength scans and corresponding HT voltage for VRK1variants. Protein samples were diluted to 0.05-0.1 mg/mL.PDB ID 5uvf shows BI-D1870 (pink) inthe active site of WT VRK1 (gray). H2Ois shown as a red sphere. Polar contactsto the inhibitor are shown as red dashedlines.00.511.522.525 75RFUTemperature (°C) -0.3-0.25-0.2-0.15-0.1-0.0500.050.125 75-d(RFU)/dTTemperature (°C)024681012400 900 1400 1900[ADP] µMTime (sec)0 uM ATP 10 uM ATP 25 uM ATP50 uM ATP 100 uM ATP 250 uM ATPCircular dichroism shows VRK1 L195V and Y213H mutants have significantlylower Tm than WT and R89Q. However, all mutants have similar foldedstructure. This agrees well with previous work.1-4A fluorescence-based assay was used to measure ATP affinity and hydrolysis. k2for the L195V mutant seems to be higher than for WT, but k2 for the R89Qvariant and Km values are comparable. This does not agree with previous work,1-4 but standard deviations are large and more trials are needed.DSF was used to measure Tm in the presence and absence of known VRK1inhibitors.5 Higher ΔTm values measured with L195V and inhibitors suggest themutant has a significantly more flexible active site than the wild-type enzyme.The electrostatic maps created in PyMOL show a significant change in charge inthe region around the mutation R89Q on the αC helix. This change, from verypositive to slightly positive in the mutant form, may impact protein-proteininteractions in vivo.Funding was provided by Winona State University andThe WSU Foundation.Asp132Phe134BI-D1870Leu195 Asp197Tyr87Glu83Arg89 Tyr213H2O-15000-10000-5000050001000015000190 290Molar EllipticityWavelength (nm)02004006008001000190 290HT (V)Wavelength (nm)Wild TypeY123HR89QL195V-10000-8000-6000-4000-2000020 30 40 50 60 70Molar Elipticity at 220nm Temperature (°C)00.00050.0010.00150.0020.00250 200 400µM [ADP]/sec[ATP] µM0.000.200.400.600.801.001.20k 2(min-1)050100150200250300350400Km(µM ATP)Left: Representative ADP Quest experiment with WT VRK1. Assays wereperformed according to kit protocol, with 200 nM VRK1, varying [ATP], and noprotein substrate. Signal may be due to ATP hydrolysis or autophosphorylation.Right: Example Michaelis-Menten plot of VRK1 mutants. Fit was determinedusing MyCurveFit.k2 and Km, valuesfor VRK1 variants.Bars and error barsrepresent theaverage of 2-3Michaelis-Mentenplots with R2 of atleast 0.80.VRK1 WT R89Q00.511.522.5ΔTm (°C)+ADP0246810ΔTm (°C)+BI-D1870Average ΔTm values for 2-4 experiments. Error bars are the standard deviation for the average.Representative DSF experiment with VRK1 WT with (dashed lines) and without(black, solid) ADP. DSF was run with 0.2-0.6 µM VRK1 and saturating ADP orinhibitor. SYPRO Orange dye fluoresces upon binding to unfolding protein, andfluorescence drops as the unfolded protein aggregates at high temperatures. Tmwas calculated as the minimum of –dRFU/dT.Arg89 Tyr213 Leu19501234567ΔTm (°C+PIK-75

Investigation of Disease-Causing Point Variants of VRK1

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Investigation of Disease-Causing Point Variants of VRK1

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