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).