Enhancing ubiquitin crystallization through surface-entropy reduction

Abstract

Ubiquitin has many attributes suitable for a crystallization chaperone, including high stability and ease of expression. However, ubiquitin contains a high surface density of lysine residues and the doctrine of surface-entropy reduction suggests that these lysines will resist participating in packing interactions and thereby impede crystallization. To assess the contributions of these residues to crystallization behavior, each of the seven lysines of ubiquitin was mutated to serine and the corresponding single-site mutant proteins were expressed and purified. The behavior of these seven mutants was then compared with that of the wild-type protein in a 384-condition crystallization screen. The likelihood of obtaining crystals varied by two orders of magnitude within this set of eight proteins. Some mutants crystallized much more readily than the wild type, while others crystallized less readily. X-ray crystal structures were determined for three readily crystallized variants: K11S, K33S and the K11S/K63S double mutant. These structures revealed that the mutant serine residues can directly promote crystallization by participating in favorable packing interactions; the mutations can also exert permissive effects, wherein crystallization appears to be driven by removal of the lysine rather than by addition of a serine. Presumably, such permissive effects reflect the elimination of steric and electrostatic barriers to crystallization.

Keywords: surface-entropy reduction; ubiquitin.

Figure 1

Hit rates obtained for wild-type ubiquitin and the seven single-site lysine-to-serine mutations. The total number of crystalline hits obtained out of 384 conditions representing four 96-well commercial screens are shown. Hits are broken down on a per-screen basis in Supplementary Table S1.

Figure 2

Comparison of deviations in backbone position for wild-type ubiquitin and three mutants. In all three panels, the solid black line represents the r.m.s. deviation from the average C^α position for nine independent wild-type ubiquitin structures (Supplementary Table S2). (a) K11S mutant. Differences in C^α position are shown for the mutant versus the average wild-type structure after superposition; the four independent chains in the K11S asymmetric unit are shown as open circles, filled circles, filled squares and inverted triangles for chains A, B, C and D, respectively. (b) K33S mutant. Differences in C^α position are shown for the mutant versus the average wild-type structure after superposition; the mutant A and B chains are represented by filled circles and inverted triangles, respectively. (c) K11S/K63S double mutant. Differences in C^α position are shown for the mutant versus the average wild-type structure after superposition; the mutant A and B chains are represented by filled circles and inverted triangles, respectively.

Figure 3

Crystal packing in the K11S crystals. (a) The asymmetric unit of the K11S crystals, containing two dimers (blue/red and cyan/magenta). The Ser11 residues are shown as gold spheres. The orientation of the unit-cell axes is also shown. (b) Close-up view of the interaction between the two monomers making up one dimer. The hydroxyl groups of Thr9 and Ser11 on one molecule (cyan) form hydrogen bonds to the backbone atoms of Leu71 and Leu73 on the facing molecule (magenta). (c) Superposition of the K11S dimer (green) upon the Lys11-linked diubiquitin structure (purple; chains A and G from PDB entry 2xew are shown).

Figure 4

Crystal packing in the K33S crystals. (a) The crystal lattice contains stacked layers of protein molecules. The A and B chains are colored yellow and blue, respectively; the Ser33 residues are shown as red spheres, emphasizing their positioning at the junction between adjacent layers in the lattice. The outline of the triclinic unit cell is shown. (b) An orthogonal view of the lattice packing, showing a single layer. The larger and smaller interfaces are shown as green and red dashed lines. (c, d) Detailed views showing neighboring basic residues in the environment of the Ser33 side chain in the A chain (c) and B chain (d); a lysine residue at position 33 would cause electrostatic repulsion in each case.

Figure 5

Crystal packing in crystals of the K11S/K63S double mutant. (a) The two principal interfaces through which A and B chains interact. The larger interface relates the blue B chain and the green A chain; the smaller interface relates the same blue B chain to the violet A chain. The Ser11 residues are shown as yellow spheres and the Ser63 residues are shown as red spheres. (b) A detailed view of the smaller interface, showing the hydrogen bonds formed between Ser11 on the violet A chain and Thr9 on the blue B chain. A sulfate ion (shown in ball-and-stick representation) also helps to bridge these two protein molecules and interacts with threonine residues on both chains. Ser11 on the blue B chain forms a hydrogen bond to Gln62 on a different symmetry mate of the A chain (shown in cyan).