Abstract
The polyhistidine (6XHis) tag is one of the most ubiquitous fusion tags used for the puri cation of recombinant proteins. The 6XHis motif enables the tagged protein to bind metal ions such as Co 2+ , Ni 2+ , Zn 2+ and Cu 2+ , which can be advantageously used for protein puri cation with immobilized metal a nity chromatography (IMAC). Despite its widespread use, high-resolution protein structures encompassing a metal-bound 6XHis motif rarely occur in the worldwide Protein Data Bank (PDB). Here, we obtained a 2.5 Å resolution crystal structure of a single chain variable fragment (scFv) antibody bearing a C-terminal tag composed of a sortase motif (LPETG), followed by a 6XHis and a TwinStrep tag (WSHPQFEK[G 3 S] 3 WSHPQFEK). The protein crystallized in the presence of cobalt (II) and its structure reveals a novel tetramerization motif (TetrHis) stabilized by 8 Co 2+ ions clustered within a small region of space. The TetrHis motif contains two 6 residues-long β-strands related by a twofold symmetry axis, and the 8 metal centers coordinate 3 to 5 protein residues each, including all of the 6XHis histidines, a glutamate residue from the sortase motif and an additional histidine residue from the TwinStrep tag. We reviewed metal-bound homopolymeric histidine tracts in deposited PDB structures and found no similar motifs. Using a combination of small angle x-ray scattering (SAXS), molecular dynamics (MD) simulations and ensemble analysis, we studied the structural dynamics of the tagged scFv in the absence and presence of Co 2+ ions, demonstrating an open/close equilibrium of the monomer and the formation of a cobalt-stabilized tetramer in solution. The structure of the novel metal-dependent tetramerization motif reported here might provide a useful starting point for designing metal-loaded biomaterials, with potential applications in the elds of biosensors, bioanalytical devices, or biocatalysts.