In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors

Abstract

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotidecontrolled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.

Rapid screening of in cellulo grown protein crystals via a small-angle X-ray scattering/X-ray powder diffraction synergistic approach

“…Highresolution structural information on several recombinant proteins has already been obtained from diffraction of in cellulo crystals, e.g. for the coral Dipsastraea favus derived fluorescent protein Xpa (Tsutsui et al, 2015), the metazoanspecific human kinase PAK4 in complex with Inka1 (Baskaran et al, 2015) and the BinAB larvicide from Lysinibacillus sphaericus (Colletier et al, 2016), as well as of cathepsin B (CatB; Redecke et al, 2013) and IMP dehydrogenase (IMPDH;Nass et al, 2020) from the parasite Trypanosoma brucei.…”

“…Moreover, they indicate that in cellulo protein crystallization is able to offer exciting possibilities complementary to conventional crystallization techniques (Chayen & Saridakis, 2008). The approach is particularly important for proteins that were/are not accessible for crystallization using established in vitro screening strategies, as shown for T. brucei IMPDH (Nass et al, 2020) and fully glycosylated T. brucei CatB (Redecke et al, 2013). In cellulo crystallization provides an alternative to the time-consuming optimization of protein purification and extensive crystal screening steps.…”

“…In cellulo crystallization provides an alternative to the time-consuming optimization of protein purification and extensive crystal screening steps. Additionally, the quasinative conditions in host cells prevent crystal distortion that could arise from non-physiological conditions imposed by recrystallization and provide the opportunity to identify native co-factors present in the highly versatile natural reservoir of compounds within living cells (Nass et al, 2020). However, exploiting the tremendous potential of in cellulo protein crystallization requires a more detailed understanding of the cellular processes involved in crystal formation.…”

“…Moreover, high local protein concentrations seem to be required, which might result from a preceding protein phase separation event (Hasegawa, 2019). In insect cells, crystals occurred in the endoplasmic reticulum (CatB; Koopmann et al, 2012) and in peroxisomes (IMPDH, luciferase; Nass et al, 2020;Schö nherr et al, 2015), depending on the native translocation signals harboured in the sequence of the recombinant proteins. Furthermore, a cytosolic localization of crystals was observed [calcineurin, avian reovirus nonstructural protein fused to green fluorescent protein (GFP-NS), IMPDH; Fan et al, 1996;Schö nherr et al, 2015;Nass et al, 2020].…”

“…In insect cells, crystals occurred in the endoplasmic reticulum (CatB; Koopmann et al, 2012) and in peroxisomes (IMPDH, luciferase; Nass et al, 2020;Schö nherr et al, 2015), depending on the native translocation signals harboured in the sequence of the recombinant proteins. Furthermore, a cytosolic localization of crystals was observed [calcineurin, avian reovirus nonstructural protein fused to green fluorescent protein (GFP-NS), IMPDH; Fan et al, 1996;Schö nherr et al, 2015;Nass et al, 2020]. Thus, different cellular environments may represent the basis for developing a more systematic in cellulo crystallization screening approach that would exploit living cells as crystallization factories for a large number of recombinant proteins.…”