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.

Increasing the bulk of the 1TEL-target linker and retaining the 10×His tag in a 1TEL-CMG2-vWa construct improves crystal order and diffraction limits.

TELSAM-fusion crystallization has the potential to become a revolutionary tool for the facile crystallization of proteins. TELSAM fusion can increase the crystallization rate and enable crystallization at low protein concentrations, in some cases with minimal crystal contacts [Nawarathnage et al. (2022), Open Biol. 12, 210271]. Here, requirements for the linker composition between 1TEL and a fused CMG2 vWa domain were investigated. Ala-Ala, Ala-Val, Thr-Val and Thr-Thr linkers were evaluated, comparing metrics for crystallization propensity and crystal order. The effect on crystallization of removing or retaining the purification tag was then tested. It was discovered that increasing the linker bulk and retaining the 10×His purification tag improved the diffraction resolution, likely by decreasing the number of possible vWa-domain orientations in the crystal. Additionally, it was discovered that some vWa-domain binding modes are correlated with scrambling of the 1TEL polymer orientation in crystals and an effective mitigation strategy for this pathology is presented.

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.

Decreasing the flexibility of the TELSAM-target protein linker and omitting the cleavable fusion tag improves crystal order and diffraction limits.

TELSAM crystallization promises to become a revolutionary tool for the facile crystallization of proteins. TELSAM can increase the rate of crystallization and form crystals at low protein concentrations without direct contact between TELSAM polymers and, in some cases, with very minimal crystal contacts overall (Nawarathnage et al ., 2022). To further understand and characterize TELSAM-mediated crystallization, we sought to understand the requirements for the composition of the linker between TELSAM and the fused target protein. We evaluated four different linkers Ala-Ala, Ala-Val, Thr-Val, and Thr-Thr, between 1TEL and the human CMG2 vWa domain. We compared the number of successful crystallization conditions, the number of crystals, the average and best diffraction resolution, and the refinement parameters for the above constructs. We also tested the effect of the fusion protein SUMO on crystallization. We discovered that rigidification of the linker improved diffraction resolution, likely by decreasing the number of possible orientations of the vWa domains in the crystal, and that omitting the SUMO domain from the construct also improved the diffraction resolution. Synopsis: We demonstrate that the TELSAM protein crystallization chaperone can enable facile protein crystallization and high-resolution structure determination. We provide evidence to support the use of short but flexible linkers between TELSAM and the protein of interest and to support the avoidance of cleavable purification tags in TELSAM-fusion constructs.

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.