Generation of human GGCX structural model and in silicodocking of vitamin K
Due to the absence of biophysical structure or adequate homologous structural templates, a hGGCX model was generated on the I-TASSER threading modeling server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) (Roy, Kucukural, & Zhang, 2010) by submitting the GGCX protein sequence (NP_000812.2) under default conditions. The model1 with the best C-score was used for further analysis but its N/C termini had the same orientation. Since this N/C-terminal orientation of GGCX contradicts previous findings (Tie, Wu, Jin, Nicchitta, & Stafford, 2000), where opposite orientations have been proposed, N-terminal amino acid residues 1-120 were re-modeled separately in ITASSER and manually replaced onto the previous model. A membrane embedded simulation was performed on the final re-modeled structure in a phosphatidylcholine bilayer in order to equilibrate the model using md_memsim macro embedded in YASARA. The equilibrated model was then docked with K1 hydroquinone downloaded from Pubchem (PUBCHEM ID: 5280585) using the AutoDock module embedded as a macro in YASARA. The top docking poses were analyzed and one docking pose was selected as the final complex based on rational elimination. The selected docked complex was further subjected to membrane embedded simulation for a total of 250 ns including equilibration and production phase (>100ns). Selected variants S300F and M174R were introduced into the equilibrated docked complex model and both the mutated complexes were simulated in parallel for >100 ns in order to understand the structural effect of the variants on GGCX. All simulations, visual rendering and structural analysis were performed on the YASARA version 18.2.24 platform (Land & Humble, 2018) (Figure S1).