Polymer-driven crystallization

Abstract

Obtaining well-diffracting crystals of macromolecules remains a significant barrier to structure determination. Here we propose and test a new approach to crystallization, in which the crystallization target is fused to a polymerizing protein module, so that polymer formation drives crystallization of the target. We test the approach using a polymerization module called 2TEL, which consists of two tandem sterile alpha motif (SAM) domains from the protein translocation Ets leukemia (TEL). The 2TEL module is engineered to polymerize as the pH is lowered, which allows the subtle modulation of polymerization needed for crystal formation. We show that the 2TEL module can drive the crystallization of 11 soluble proteins, including three that resisted prior crystallization attempts. In addition, the 2TEL module crystallizes in the presence of various detergents, suggesting that it might facilitate membrane protein crystallization. The crystal structures of two fusion proteins show that the TELSAM polymer is responsible for the majority of contacts in the crystal lattice. The results suggest that biological polymers could be designed as crystallization modules.

Figure 1.

E80 TELSAM forms a helical polymer in a pH-dependent fashion. (A) Recombinant WT-TELSAM protein aggregates when expressed, but E80 TELSAM only aggregates when the pH is below 7. (B) E80 TELSAM forms a helical polymer when crystallized. Alternating units are shaded differently to distinguish each TELSAM monomer. Six monomers form one helical turn in the polymer with a pitch of 53 Å.

Figure 2.

The 2TEL module crystallized as a helical polymer. (A) The 2TEL module was composed of an E80 TELSAM connected to a WT TELSAM via a Gly₄Ser linker. (B) 2TEL polymerization behavior. At high pH, deprotonated Glu80 prevents 2TEL polymerization. Conversely, protonation at low pH permits polymerization. (C) The crystal structure of the 2TEL module forms a double helix. One polymer is illustrated in a surface representation while the other is shown as a ribbon diagram.

Figure 3.

(A) Examples of crystals of 2TEL fused to various soluble proteins of known structures. (B) Crystals of 2TEL fused to novel soluble protein: 2TEL–hPhZn (left) and 2TEL-hRING2 (right). (C) Highly mosaic diffraction pattern of 2TEL-hRING2.

Figure 4.

Crystal structures and packing of the 2TEL module (sticks model) fused to T4 lysozyme (ribbon diagram) viewed down the helical polymer axis. Six helical polymers are included for each structure. Lysozyme molecules fused to 2TEL on neighboring hexagonal tubes are deleted for clarity. Lateral contacts (see text) between neighboring polymers are highlighted.