Structure, dynamics and transferability of the metal-dependent polyhistidine tetramerization motif TetrHis for single-chain Fv antibodies

Abstract

The polyhistidine (6XHis) motif is one of the most ubiquitous protein purification tags. The 6XHis motif enables the binding of tagged proteins to various metals, which can be advantageously used for purification with immobilized metal affinity chromatography. Despite its popularity, protein structures encompassing metal-bound 6XHis are rare. Here, we obtained a 2.5 Å resolution crystal structure of a single chain Fv antibody (scFv) bearing a C-terminal sortase motif, 6XHis and TwinStrep tags (LPETGHHHHHHWSHPQFEK[G₃S]₃WSHPQFEK). The structure, obtained in the presence of cobalt, reveals a unique tetramerization motif (TetrHis) stabilized by 8 Co²⁺ ions. The TetrHis motif contains four 6 residues-long β-strands, and each metal center coordinates 3 to 5 residues, including all 6XHis histidines. By combining dynamic light scattering, small angle x-ray scattering and molecular dynamics simulations, We investigated the influence of Co²⁺ on the conformational dynamics of scFv 2A2, observing an open/close equilibrium of the monomer and the formation of cobalt-stabilized tetramers. By using a similar scFv design, we demonstrate the transferability of the tetramerization property. This novel metal-dependent tetramerization motif might be used as a fiducial marker for cryoelectron microscopy of scFv complexes, or even provide a starting point for designing metal-loaded biomaterials.

Fig. 1. Crystal structure of the cobalt-bound scFv 2A2 tetramer.

a Protein construct used for crystallization of scFv 2A2. The expressed sequence contains a (cleaved) N-terminal BiP signal sequence and a C-terminal tag composed of a sortase motif (LPETG), a 6XHis and a twinstrep tag (WSHPQFEK[G₃S]₃WSHPQFEK). b Overall architecture of the tetramer in 2 views rotated by 90°. The protein chains are shown in cartoon representation and colored in green, cyan, yellow, and purple. The complementarity determining regions (CDRs) are colored in orange. The 8 cobalt ions are shown as salmon-colored spheres. c Close up view of the cobalt-bound TetrHis motif showing the 4 types of metal binding sites related by the twofold symmetry axis. d Close up view of the dimeric V_L−V_L interface (defined as VL-11 in ref. ). e Hydrophobic interactions stabilizing the interface between the TetrHis motif and the scFv V_H and V_L domains. Framework residues belonging to the V_L domain are shown in gray. f, g Details of the V_L−V_L interface interactions, in 2 views rotated by 180°.

Fig. 2. Architecture of the cobalt-bound tetrameric polyhistidine (TetrHis) motif.

a Close up of the TetrHis motif, in two views rotated by 90°. The protein chains are shown in cartoon representation and the color code is the same as in Fig. 1. b Schematic representation of the metal-protein contacts within the tetrameric motif. c–f Close ups of the 4 cobalt binding sites showing the coordination geometries and distances.

Fig. 3. Survey of metal-bound polyhistidine tags in the PDB.

a–d Basic statistics extracted from the 27 unique PDB structures containing at least 4 consecutive histidines and a bound metal. The structures are classified according to the nature of the bound metal (a), the number of bound metal ions in the assembly (b), the number of protein chains that contribute coordinating residue sidechains (c), and whether the assembly involves a short linear sequence motif or a three-dimensional motif (with residues located far away within the protein sequence) (d). e, f Nickel-dependent dimeric assemblies of SlyD from Thermus thermophiles, observed in PDB entries 4odp and 3cgm. 3 His residues located at the C-terminus of the construct are involved in metal coordination, along with 2 His from a 6XHis tag located 3 residues downstream, and 1 His from the 6XHis tag of a symmetric-related molecule. g–i Nickel-dependent trimeric assemblies observed in PDB entries 2fg9, 6wxa, and 7c3a. In each case, 3 × 2 His residues from 6XHis tags assemble to form a octahedral coordination sphere around a single nickel ion.

Fig. 4. Cobalt titrations of scFv 2A2 and anti-ADIPOR scFv by dynamic light scattering (DLS) at 20 °C in 50 mM HEPES pH 7.5, 150 mM NaCl using a protein concentration of 1 mg/ml.

Each point corresponds to the average of 3 independent measurements, with standard error shown as a bar. The apparent hydrodynamic radii were calculated from the intensity autocorrelations using the Stokes–Einstein equation.

Fig. 5. SAXS characterization of scFv 2A2.

a SAXS profiles of scFv 2A2 in the presence of 5 mM EDTA (black curves), in regular buffer (50 mM HEPES pH 7.5 and 150 mM NaCl, blue curves), or in the presence of 5 mM NiSO₄ or 5 mM CoCl₂ (green and red curves, respectively). The data were measured at increasing protein concentrations of 2.1, 4.2, and 8.5 mg/ml for EDTA and NiSO₄, while additional curves were measured at 0.5 and 1 mg/ml in regular buffer and CoCl₂ conditions. b Kratky plots the EDTA and CoCl₂ data using the 2.1 mg/ml SAXS profiles. c Pair-distance distribution functions p(r) calculated from the SAXS profiles measured in the presence or absence of 5 mM Co²⁺ at different protein concentrations. The SAXS curves extracted from the SEC-SAXS profile is used as the cobalt-free reference. d SEC-SAXS profiles of scFv 2A2 in the presence (red) or absence of 5 mM CoCl₂ (black curve), and analysis of Radius of gyration versus frame number.

Fig. 6. Molecular dynamics simulations analysis of scFv 2A2 in its monomeric and tetrameric forms.

a Cα root mean square deviation (R.M.S.D) as a function of simulation time for two independent MD trajectories of the scFv monomer. Five representative conformers of the open and closed states sampled during the simulations are superimposed and shown as cartoon. b Cα R.M.S.D as a function of simulation time for the MD trajectories of the scFv tetramer. Representative conformers along the trajectories are shown in cartoon representation and colored by chain. The longer trajectory (black curve) was run without distance restraints on metal binding sites, which resulted in the sampling of partially dissociated tetrameric states.

Fig. 7. Ensemble optimization (EOM) analysis of the SAXS curves extracted from the SEC-SAXS profiles of scFv 2A2.

a Radius of gyration distributions for the MD-generated ensemble of monomeric models (gray area) and for the optimized ensemble (OE) that fits the SEC-SAXS data in the absence of cobalt (black curve). b Fitted SAXS profiles of scFv 2A2 measured in the presence or absence of 5 mM NiSO₄ or CoCl₂ in solution or in SEC-SAXS. The protein concentration for each curve is indicated on the figure panel. Experimental scattering curves are shown as black lines with OEs fits shown as red lines. c Radius of gyration distributions for the MD-generated ensemble of monomeric and tetrameric models (black area) and for the OEs that fits the solution-based or SEC-SAXS data (curves) in the presence of 5 mM CoCl₂ or 5 mM NiSO₄. d EOM Goodness-of-fit χ_exp as a function of the number of models in the OEs, in the presence (red) or absence (black) of 5 mM CoCl₂. e Histograms showing the percentage of tetramers, open and closed state monomers observed in the best fitting OEs for different experimental conditions. f Conformational equilibrium of monomeric scFv 2A2 in 50 mM HEPES pH 7.5 and 150 mM NaCl. For each state, 5 representative models extracted from the OEs are superimposed and shown as cartoon. The disordered C-terminal tail corresponding to the sortase-His6-Twinstrep sequence is colored in gray. g Conformational equilibrium of scFv 2A2 in the presence of 5 mM CoCl₂. For each state, 5 representative models extracted from the OEs are superimposed and shown as cartoon. The sortase-His6-Twinstrep tail encompassing the TetrHis motif is colored in gray.

Fig. 8. SAXS characterization and ensemble optimization of scFv 748.

a Fitted SAXS profiles of scFv 748 measured in the presence or absence of 5 mM CoCl₂ in solution at 0.5 mg/ml protein concentration. b Pair-distance distribution functions p(r) calculated from the SAXS profiles measured in the presence or absence of 5 mM CoCl₂. c Proportion of open and closed states monomers and tetramers in the OEs in the presence or absence of 5 mM CoCl₂. d Radius of gyration distributions for the MD-generated ensemble of monomeric and tetrameric models (black area) and for the OEs that fit the solution SAXS data (brown and orange curves) in the presence or absence of 5 mM CoCl₂.