Fig 4.

Mapping of VKORC1v2 residues interacting with vIL-6. (A) Coprecipitation assays were employed to identify the region(s) of VKORC1v2 required for its interaction with vIL-6; CBD-tagged VKORC1v2 and deleted derivatives were utilized in chitin-mediated precipitations (IP) from transfected cell lysates. The region of VKORC1v2 immediately following the TM domain was found to be required for binding. Dialanine mutations within this region (residues 31 to 39) were introduced into VKORC1v2-CBD and tested for interaction with vIL-6; the DY₃₉AA mutation inhibited binding. IB, immunoblotting; WT, wild type. (B) Residues 31 to 39, constituting the vIL-6 binding domain of VKORC1v2, were transferred to a heterologous protein, KDEL-tagged hIL-6, to test their sufficiency for binding to vIL-6. The nonopeptide sequence was able to confer vIL-6 binding to hIL-6-KDEL in transfected cells. (C) In vitro coprecipitation assay using media-derived vIL-6–CBD and bacterially expressed thioredoxin (Trx)/His₆-fused VKORC1v2 C tail (residues 31 to 92), demonstrating direct interaction between the two proteins. Trx-His₆ (negative control) was unable to bind vIL-6–CBD, and VKORC1v2 protein could not be coprecipitated with control hIL-6-CBD.