EFFECTS OF MUTATION ON FUNCTIONAL MOVEMENTS OF KEOPS COMPLEX USING HYBRID SIMULATION METHODS AND HIGH-RESOLUTION CRYO-ELECTRON MICROSCOPY (CRYO-EM) MAPS

The KEOPS (Kinase, Endopeptidase, and Other Proteins of Small Size) complex plays an essential role in tRNA modification and the maintenance of translational fidelity in human cells. Mutations in genes encoding KEOPS subunits, including TP53RK and OSGEP, can disrupt this function and are associated with Galloway–Mowat syndrome (GAMOS). This work represents the initial stage of an ongoing research project and presents preliminary results for five GAMOS-associated mutations in the apo form of the KEOPS complex. In this initial stage, we used the wild-type (WT) KEOPS structure determined by cryo-electron microscopy (cryo-EM) and reported by a collaborating French research group. The corresponding mutant structures were generated from the WT model using the CHARMM-GUI interface and subsequently subjected to classical molecular dynamics (MD) simulations. We then performed an initial characterization of the structural dynamics of the WT and mutant complexes using Normal Mode Analysis (NMA). We explored their conformational landscapes using the hybrid VMOD approach. In particular, we seek to identify collective functional motions that may support the hypothesis that tRNA binding stabilizes a closed, catalytically competent conformation of KEOPS/OSGEP. Such a conformational transition may restrict the conformational freedom of the tRNA anticodon loop and favor conformations compatible with formation of the U32–U36–A38 base triple, thereby facilitating the proper positioning of A37 within the OSGEP catalytic site for t⁶A modification. The initial results indicate that one studied mutation altered movement and reduced flexibility. The final cryo-EM map of the KEOPS–tRNA complex was subjected to 3D variability analysis (3DVA) in cryoSPARC v4.7.0, using the mask from the final non-uniform refinement and a 5 Å filter resolution. Three variability components were calculated, and 20 frames were generated for each component using the 3DVA Display tool. Component 2, which captured the conformational movement of OSGEP, was selected for further analysis. These frames were used to generate intermediate states of the complex KEOPS-RNAt-Tc-AMP using MDFF_NM.