Of the vulnerability of orphan proteins: The case study of the Arabidopsis thaliana p70 ribosomal S6 Kinase 2

Plants have developed a diverse and multi-layered range of mechanisms to perceive, respond and adapt to changing environmental conditions, understanding of which is vital for global food production and security. One of the most conserved cellular machineries involved in stress signalling is the Target of Rapamycin (TOR) kinase, which acts as an “energy status” sensor, connecting nutrient and energy signalling to growth and homeostasis, under normal and stress conditions. The plant p70 ribosomal S6 kinase (AtS6K) family, composed of two members (AtS6K1 and AtS6K2), is a critical downstream effector of TOR involved in environmental stress response. Despite earlier studies which showed that AtS6K2, but not AtS6K1, is activated in response to cold and salt stresses, many key aspects of its regulation and biological role are poorly understood. Indeed, AtS6K2 is an orphan protein in search of a function. Here, to gain structural information indispensable to increase our understanding of S6Ks activity, function and regulation, an extensive range of experiments aiming to express, purify and crystallise Arabidopsis thaliana S6K2 were attempted. A combination of sequence alignment, modelling and molecular dynamics simulation approaches were employed to predict the protein structure and dynamics, and guide design and optimisation of the expression constructs. A total of 33 different expression experiments, about 26 different purification condition tested, for the 23 constructs cloned, failed to produce samples of active protein amenable for structural studies. Specifically, KL_123_471_NC_C-His_N-T7; KL_123_471_NC_C-His; KD_123_414_NC_C-His_N-T7; KD_123_414_C_C-His_N-T7; and KD_123_414_C_N-GST constructs were expressed in soluble form, purified and used for crystallisation screening. Constructs for co-expression with λ-phosphatase were extremely promising for both crystallisation (KDN_133_414_NC_C-His_λ and KDN_133_414_C_C-His_λ) and activity assays (FL_1_471_NC_N-His_λ both WT and mutant), however they need to be optimised for larger scale production. Despite the lack of experimental structural data, this case study represents one of the largest, compared to published work on recombinant plant proteins, systematic recombinant protein expression and purification campaign, which provides clear directions for future studies on AtS6K and, in general, plant AGC kinases (Pearce et al., 2010; Rademacher and Offringa, 2012).