Can the propensity of protein crystallization be increased by using systematic screening with metals?

Abstract

Protein crystallization is one of the major bottlenecks in protein structure elucidation with new strategies being constantly developed to improve the chances of crystallization. Generally, well-ordered epitopes possessing complementary surface and capable of producing stable inter-protein interactions generate a regular three-dimensional arrangement of protein molecules which eventually results in a crystal lattice. Metals, when used for crystallization, with their various coordination numbers and geometries, can generate such epitopes mediating protein oligomerization and/or establish crystal contacts. Some examples of metal-mediated oligomerization and crystallization together with our experience on metal-mediated crystallization of a putative rRNA methyltransferase from Sinorhizobium meliloti are presented. Analysis of crystal structures from protein data bank (PDB) using a non-redundant data set with a 90% identity cutoff, reveals that around 67% of proteins contain at least one metal ion, with ∼14% containing combination of metal ions. Interestingly, metal containing conditions in most commercially available and popular crystallization kits generally contain only a single metal ion, with combinations of metals only in a very few conditions. Based on the results presented in this review, it appears that the crystallization screens need expansion with systematic screening of metal ions that could be crucial for stabilizing the protein structure or for establishing crystal contact and thereby aiding protein crystallization.

Keywords: combination of metals in crystallization; crystallization screens; metals in crystallization; protein crystallization.

Figure 1

Crystals of the putative rRNA methyltransferase from Sinorhizobium meliloti, (A) in the condition 5 mM CoCl₂, 5 mM NiCl₂, 5 mM CdCl₂, 5 mM MgCl₂, 0.1 M HEPES pH 7.5, 12% wt/vol PEG 3,350 (B) in the condition 5 mM NiCl₂, 5 mM CdCl₂, 5 mM MgCl₂, 0.1 M HEPES pH 7.5, 9% wt/vol PEG 3,350 (C) in the condition 5 mM CoCl₂, 0.1 M HEPES pH 7.5, 9% wt/vol PEG 3,350 and (D) in the condition 5 mM NiCl₂, 0.1 M HEPES pH 7.5, 9% wt/vol PEG 3,350. In (C) and (D) the protein was incubated with MgCl₂.The best crystals so far seem to be in the condition with 5 mM CoCl₂, 0.1 M HEPES pH 7.5, 9% wt/vol PEG 3,350 with MgCl₂ incubated protein.

Figure 2

Crystal contact mediated by the metal ion in Sm_Mtase. Residues H40 and E43 from one molecule and H129* from a symmetry related molecule coordinate with the metal forming intermolecular crystal contacts. 2F_o – F_c map is represented as a blue mesh and anomalous map as a red mesh; a different colour (yellow) is used for the protein chain from symmetry related molecule. The figure was generated using PyMOL.45

Figure 3

2F_o – F_c density for Sm_Mtase after initial refinement. The L30 and SPOUT domains (marked with ellipses and indicated by labelled arrows), including a symmetry related SPOUT are involved in metal‐mediated interaction. The presence of anomalous peak between the L30 domain and an adjacent SPOUT domain from a symmetry related molecule, represented in red, clearly indicates that the metal involved in crystal contact is not Mg²⁺. 2F_o – F_c map is drawn at 1.5σ level and the anomalous map is drawn at 3.0σ level. The figure was generated using PyMOL.

Figure 4

Metal mediated conformational change observed in the crystal structure of a membrane fusion protein ZneB from Cupriavidus metallidurans. Zinc is represented as a cyan sphere. Of the two molecules in the asymmetric unit one of them has metal bound (Chain A) and the other one does not (Chain B). One disordered domain is missing in Chain B and on binding to metal the same domain is ordered as seen in Chain A, illustrating metal‐mediated conformational change and stabilization. The figure was generated using PyMOL.

Figure 5

Representative examples of metal‐mediated oligomerization and/or crystal contact formation from protein crystal structures in PDB: (A) Metal mediated symmetrization in the crystal structure of mouse C1QL1 globular domain (PDB entry 4D7Y). Three different metal ions are assigned in this structure, represented as colored spheres, Cd (red), Ni (grey) and Mg (magenta). The Cd and Ni ions are clearly seen to mediate the symmetrization and hence play role in the formation of crystals. (B) observation of both metal mediated symmetrization and crystal contact formation in the crystal structure of glutathione S‐transferase‐III from Zea mays var. mutin (PDB entry 1AW9). In this structure, Cd ions mediate both oligomerization (represented as yellow spheres) and crystal contact in all three directions (represented as red spheres). The figure was generated using PyMOL.