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

Abstract

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.

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

“…7c). This result confirms our earlier hypothesis (Nawarathnage et al, 2022) that fusion to 1TEL allows the formation of crystals with a comparable diffraction resolution but increased molecular motion in the crystal lattice. Concomitant with this, the solvent content of the highest-resolution crystal of the vWa domain crystallized on its own was 47.22% (PDB entry 1shu), while the solvent content of His-1TEL-TV-vWa crystals was 49.46% (PDB entry 8ft8).…”

“…This construct also featured an N-terminal 10�His-SUMO domain that was proteolytically removed during protein purification. We discovered that TELSAM fusion could increase the rate of crystallization, form crystals without direct contact between TELSAM polymers and enable crystallization at low protein concentrations, in some cases using only very minimal crystal contacts overall (Nawarathnage et al, 2022). Replicating our previous results with 1TEL-flex-vWa uncovered potential areas for improvement of TELSAMmediated protein crystallization.…”

“…The 1TEL-AA-vWa (formerly 1TEL-flex-vWa) construct from Nawarathnage et al (2022) was used as the starting point for the design of the 1TEL-vWa variants used in this study. This construct placed residues 40-217 of the human anthrax toxin receptor 2 vWa domain (ANTXR2; also known as capillary morphogenesis gene 2, CMG2; UniProt P58335) downstream of residues 47-123 (the SAM domain) of human transcription factor ETV6 (ETS variant transcription factor 6, also known as translocation ETS leukaemia protein, TEL; UniProt P41212).…”

“…By comparison, the batch 1 1TEL-AA-vWa (SUMO) crystals gave an average resolution of 2.9 A ˚(range 2.8-3.0 A åcross three crystals), indexed 57% of non-ice reflections (range 43-67%), exhibited an average mosaicity of 0.47 � (range 0.38-0.60 � ) and had an average ISa of 16 (range 15-17) (Nawarathnage et al, 2022). These results suggest that the Thr-Val linker performed slightly better than the Ala-Ala linker, which in turn performed on a par with the Ala-Val linker, while the Thr-Thr linker abrogated crystal formation.…”

“…Our structures reveal that substitutions of the amino acids in the linker do not make it fully rigid, but rather appear to reduce the number of highly flexible residues that are present. We note that in all 1TEL-vWa structures solved to date there is always at least one amino acid in the linker that is not part of the 1TEL C-terminal �-helix or the vWa N-terminal �-sheet, such as in batch 1 (PDB entry 7n1o; Nawarathnage et al, 2022) or batch 2 1TEL-AA-vWa (SUMO) chain A (Fig. 7d).…”