Identification of the Vitamin K Hydroquinone binding site in GGCX
Since no knowledge exists regarding KH2 binding, we docked KH2 ligand on our simulation equilibrated model that revealed 29 docking poses (Figure S5). Out of these, only the docking pose 6 had the correct orientation of KH2 (with respect to membrane embedding) and is proximal to known active site residue K218 (Rishavy et al., 2006). Therefore, this docking pose was chosen for further analysis, in which residue F299 undergoes both Pi-Pi and hydrophobic interaction with the KH2 head group (Figure 4a,b and Figure S6). Hence, the substitution of F299 with a polar serine residue can be expected to disrupt its interactions with KH2, resulting into loss-of-function (Figure 4f). To evaluate the effect of GGCX:p.(S300F) and the loss-of-function variant GGCX:p.(M174R) on GGCX structure and KH2 binding, variant models were simulated. The production phase of S300F mutated complex´s simulation showed higher root mean square deviation (RMSD) for receptor and ligand movement when compared to wt (Figure 4c). The substitution to phenylalanine breaks a small helix between residues 299-300, disturbing the spatial orientation and Pi-Pi interaction of F299 to the KH2 head group, thereby leading to markedly decreased values of γ-carboxylation for all investigated VKD proteins. The production phase for the simulation of the M174R mutated docked complex also showed an increase in RMSD, although not as high as S300F (Figure 4c). We assume that M174R most likely alters the structural stability of GGCX which is in line with previous findings (Tie et al., 2016), than interfering with KH2 binding. Further immunofluorescent (IF) staining confirmed that GGCX:p.(F299S) and GGCX:p.(S300F) are co-localizing to the ER similar to wt GGCX with a Pearson´s correlation coefficient of +0.74 and +0.76, respectively (Figure 4d,e). GGCX:p.(M174R) co-localizes to the ER with a significantly reduced Pearson´s correlation coefficient of +0.53 when compared to wt (Figure 4e). The nonsense variant GGCX:p.(W315X) served as negative control and showed no expression. The positive control GGCX:p.(K218A) , where the active site is mutated results into loss-of-function, but is expressed as high as wt and co-localizes in the ER (Figure 4e and Figure S7).