Fusion crystallization reveals the behavior of both the 1TEL crystallization chaperone and the TNK1 UBA domain.

Human thirty-eight-negative kinase-1 (TNK1) is implicated in cancer progression. The TNK1-UBA domain binds polyubiquitin and plays a regulatory role in TNK1 activity and stability. Sequence analysis suggests an unusual architecture for the TNK1 UBA domain, but an experimentally-validated molecular structure is undetermined. To gain insight into TNK1 regulation, we fused the UBA domain to the 1TEL crystallization chaperone and obtained crystals diffracting as far as 1.53 Å. A 1TEL search model enabled solution of the X-ray phases. GG and GSGG linkers allowed the UBA to reproducibly find a productive binding mode against its host 1TEL polymer and to crystallize at protein concentrations as low as 0.1 mg/mL. Our studies support a mechanism of TELSAM fusion crystallization and show that TELSAM fusion crystals require fewer crystal contacts than traditional protein crystals. Modeling and experimental validation suggest the UBA domain may be selective for both the length and linkages of polyubiquitin chains.

Fusion crystallization reveals the behavior of both the 1TEL crystallization chaperone and the TNK1 UBA domain.

Human thirty-eight-negative kinase-1 (TNK1) is implicated in cancer progression. The TNK1 ubiquitin-associated (UBA) domain binds polyubiquitin and plays a regulatory role in TNK1 activity and stability. No experimentally determined molecular structure of this unusual UBA domain is available. We fused the UBA domain to the 1TEL variant of the translocation ETS leukemia protein sterile alpha motif (TELSAM) crystallization chaperone and obtained crystals diffracting as far as 1.53 Å. GG and GSGG linkers allowed the UBA to reproducibly find a productive binding mode against its host 1TEL polymer and crystallize at protein concentrations as low as 0.2 mg/mL. Our studies support a mechanism of 1TEL fusion crystallization and show that 1TEL fusion crystals require fewer crystal contacts than traditional protein crystals. Modeling and experimental validation suggest the UBA domain may be selective for both the length and linkages of polyubiquitin chains.

Crystals of TELSAM-target protein fusions that exhibit minimal crystal contacts and lack direct inter-TELSAM contacts.

While conducting pilot studies into the usefulness of fusion to TELSAM polymers as a potential protein crystallization strategy, we observed novel properties in crystals of two TELSAM-target protein fusions, as follows. (i) A TELSAM-target protein fusion can crystallize more rapidly and with greater propensity than the same target protein alone. (ii) TELSAM-target protein fusions can be crystallized at low protein concentrations. This unprecedented observation suggests a route to crystallize proteins that can only be produced in microgram amounts. (iii) The TELSAM polymers themselves need not directly contact one another in the crystal lattice in order to form well-diffracting crystals. This novel observation is important because it suggests that TELSAM may be able to crystallize target proteins too large to allow direct inter-polymer contacts. (iv) Flexible TELSAM-target protein linkers can allow target proteins to find productive binding modes against the TELSAM polymer. (v) TELSAM polymers can adjust their helical rise to allow fused target proteins to make productive crystal contacts. (vi). Fusion to TELSAM polymers can stabilize weak inter-target protein crystal contacts. We report features of these TELSAM-target protein crystal structures and outline future work needed to validate TELSAM as a crystallization chaperone and determine best practices for its use.