Hen egg-white lysozyme crystallisation: protein stacking and structure stability enhanced by a Tellurium(VI)-centred polyoxotungstate

Abstract

As synchrotron radiation becomes more intense, detectors become faster and structure-solving software becomes more elaborate, obtaining single crystals suitable for data collection is now the bottleneck in macromolecular crystallography. Hence, there is a need for novel and advanced crystallisation agents with the ability to crystallise proteins that are otherwise challenging. Here, an Anderson-Evans-type polyoxometalate (POM), specifically Na6 [TeW6 O24 ]⋅22 H2 O (TEW), is employed as a crystallisation additive. Its effects on protein crystallisation are demonstrated with hen egg-white lysozyme (HEWL), which co-crystallises with TEW in the vicinity (or within) the liquid-liquid phase separation (LLPS) region. The X-ray structure (PDB ID: 4PHI) determination revealed that TEW molecules are part of the crystal lattice, thus demonstrating specific binding to HEWL with electrostatic interactions and hydrogen bonds. The negatively charged TEW polyoxotungstate binds to sites with a positive electrostatic potential located between two (or more) symmetry-related protein chains. Thus, TEW facilitates the formation of protein-protein interfaces of otherwise repulsive surfaces, and thereby the realisation of a stable crystal lattice. In addition to retaining the isomorphicity of the protein structure, the anomalous scattering of the POMs was used for macromolecular phasing. The results suggest that hexatungstotellurate(VI) has great potential as a crystallisation additive to promote both protein crystallisation and structure elucidation.

Keywords: Anderson-Evans; electrostatic interactions; hen egg-white lysozyme; polyoxometalates; protein structures.

Figure 1

Asymmetric unit of HEWL–TEW structure. HEWL monomers are depicted as cartoons (monomer A–D in different shades of green); TEW molecules are shown as ball and stick (tellurium, grey; tungsten, black; oxygen, red). The catalytic sites of HEWL monomers point to the centre of the asymmetric unit.

Figure 2

Anomalous difference map of HEWL–TEW structure. 2F_O−F_C map (contoured at 1σ, grey) and anomalous map (contoured at 3σ, yellow) after model building and several refinement steps. Protein side chains are represented as ball and stick. Below: TEW molecule (ball and stick representation) from two different perspectives (tellurium, grey; tungsten, black; oxygen, red).

Figure 3

Crystal packing of the HEWL–TEW structure. A) Crystal packing in a 1×1×1 supercell. The unit cell lattice is shown in blue. HEWL “tetramers” are depicted in surface representation to show the parallel “tetramer” pairs. Different colours (green and cyan) distinguish adjacent “tetramers”. TEWs are illustrated as red spheres. Inset: “tetramer” stacked on four other “tetramers” with its opposite side (i.e., not involved in pair formation); three of the four “tetramers” are from the adjacent unit cell (different colours) and therefore are not visible in the supercell illustration. Curved arrows represent the operations to convert one “tetramer” pair into another pair. Red lines indicate the “rostrum”-like arrangement in the unit cell. B) Asymmetric unit consisting of a tetramer (chain A–D). Rotation by 90° of the tetramer is depicted to show the side view of the asymmetric unit (used in the picture below). C) Crystal packing of HEWL–TEW in a 1×2×1 supercell and the tetragonal reference structure of HEWL (PDB ID: 194L, right).[38] Unit cell lattices are shown in blue, protein molecules from HEWL–TEW and the reference structure are represented as cartoons; TEW molecules are shown as ball and stick (tellurium, grey; tungsten, black; oxygen, red). HEWL–TEW colours (green and cyan) indicate the stacking to neighbouring protein layers mediated by TEW; colours (red, yellow, green, magenta, blue, pink and cyan) of the reference structure distinguish different asymmetric units consisting of one monomer.

Figure 4

Electrostatic (Coulomb) potential surface presentation of HEWL–TEW. TEW molecules are illustrated as ball and stick (tellurium, grey; tungsten, black; oxygen, red). The figure shows clearly the binding of TEW molecules to protein pockets exhibiting a positive potential (blue). An additional protein surface would also bind to POM with a positively charged pocket from the solvent exposed site (not illustrated).

Figure 5

TEW binding sites. A–H) Binding modes of all TEW molecules (TEW-1 to -8). Involved protein side chains are illustrated as sticks (nitrogen, blue; oxygen, red; sulfur, yellow); the rest of the protein structure is depicted as a cartoon (20 % transparency). Bonds are shown as purple dashed lines. Colours (green, cyan and yellow) of the protein backbones differentiate monomers from different “tetramers”; different shades of a colour represent distinct chains within the same “tetramer”; parentheses after residue numbers also indicate chain; */**/*** denote distinct “tetramers”. NT: N terminus.

Figure 6

HEWL–TEW crystals by two approaches. Both crystals were obtained under same conditions (130 mg mL⁻¹ HEWL, 0.1 m NaOAc pH 4.8, 5–9 % NaCl (w/v) and 20 mm TEW). A) Crystals grow although precipitation occurred; the amount of precipitation decreased with time. B) A visible LLPS formed (yellowish ring). The crystal is at the phase boundary.