The Cytosolic Zinc Finger Domain Structure of the CCHFV Glycoprotein n Is Maintained in Its Membrane-Bound Form

The structural analysis of the ecto- or cytosolic domains of membrane proteins is frequently realized through the analysis of isolated domains, i.e., by using a divide-and-conquer approach, as membrane protein expression remains a laborious task. However, the membrane environment can influence the structural features of the protein. Here, we investigated the membrane-bound form of the cytosolic zinc finger domain of the Crimean-Congo hemorrhagic fever virus glycoprotein n (Gn^TMcyto), which had previously been studied in isolation. We obtained the membrane-reconstituted protein by cell-free protein synthesis and analyzed it using ¹H-detected solid-state NMR. We show that the overall structure of the zinc finger motif is conserved in Gn^TMcyto, but interestingly observe that the membrane-bound version has two major conformations, as revealed by peak doubling. This likely indicates that the cytosolic domain forms a multimer in its membrane-bound form, with an at least partially asymmetric conformation. NMR relaxation measurements further reveal that the protein includes dynamic components, providing a possible rationale for the absence of Gn^cyto in cryo-EM structures of the envelope glycoproteins in other Bunyaviricetes.