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Transcript
kinase crystallization trials. Discovery of modulating effects of ATP and ATP mimics on SCS binding will assist in choices of conditions for crystallization trials and also provides evidence for regions of the protein kinase involved in SCS binding prior to structure determination. In order to reduce surface entropy and to improve the chances of the crystallization, point mutations are introduced into SnRK2 sequence. Moreover, both AtSCS-A/B as well as AtSnRK2 have been subjected to limited proteolysis using different proteases (chymotrypsin, trypsin, proteinase K). When protein contains intrinsically disordered regions obtaining crystals may be impossible. Limited proteolysis may either excise these regions, allowing the remaining (well ordered) protein to crystallize, or identify flexible regions to help design new crystallization constructs. EGTA 55 40 35 25 Ca2+ 55 40 35 25 M 1 2 3 4 5 6 7 8 Limited proteolysis of AtSCS-A using elastase at 1:10000 concentration. Cleavage was carried out in the presence of 5 mM EGTA or 5 mM CaCl2 at room temperature. 1 – protein at 0 time point, 2 – 15’; 3 – 30’; 4 – 60’; 5 – 180’; 6 – 360’; 7 – overnight; 8 – protein incubated overnight without addition of protease measurements of interaction energies will provide the basis for structure based modeling to understand the details of complex formation. Additionally, the amino acids contributing in the interaction, selected based on the complex structure, will be verified by site-directed mutagenesis. AtSCS-B crystals macro seeded into a sparse-matrix screen Project supported by The Polish-Norwegian Research Programme implemented under The Norwegian Financial Mechanism 2009-2014 Contact WP 4. SnRK2 and SCS-A/B crystallization trials and structure solution The partnership brings together experts in different research fields using various scientific techniques: Polish group – biochemistry and molecular biology of protein kinases and different proteins involved in stress signal transduction in plants, Norway group – crystallization of protein kinases with their specific effectors and solving crystal structures. WP leader Prof. Richard Alan Engh Consortium: In order to obtain structures with good resolution, significant efforts is required to identify optimal crystallization conditions for studied proteins. Some of the parameters that lead to success cannot be predicted, and so must be attempted randomly. Some cannot be predicted, but may be prioritized based on other information. Thus, as many protein constructs as possible are tested for crystallization, but greatest efforts concentrate on those constructs that are most likely to possess stable conformations and, for complexes, stable complex formation. Some crystallization conditions such as the inclusion of calcium (to assure proper conformation of AtSCS-A/B); strontium (as a substitute to enable anomalous methods), or ATP site inhibitors (chosen based on previous tests of effects on conformation) are tested. The first task is to perform a limited series of further tests to narrow the range of conditions further, by testing precipitation behavior and monodispersity (e.g. DLS). To obtain the greatest amount of structural information possible, providing information not only on complexation surfaces but also on e.g. the structural plasticity of the proteins, we are optimizing crystallization conditions for AtSCS-A/B alone, fragments of AtSCS-A/B with truncated AtSnRK2, and finally whole AtSCS-A/B with truncated AtSnRK2 or whole AtSnRK2, as tasks 2 and 3. Data collection and structure solution is charted as task 4. Finally, structures, combined with new biophysical Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, 02-106 Warsaw, Poland Phone:+ 48 22 5925718; Fax: 4822 6584804; e-mail: [email protected] and The Norwegian Structural Biology Centre (Department of Chemistry, UiT The Arctic University of Norway), N-9037 Tromsø, Norway Phone: +47 77644073, e-mail: [email protected] Toward structural characterization of novel mechanism of inhibition of SnRK2s activity by calcium sensor (SCS) in plants (SnRK-SCS) Acronim: SnRK-SCS Project number: 203156 Duration: 2013-2016 Project Promotor ÂÂ Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland The research should provide important insights into the signaling pathways that control plant defenses in harmful environments. As demonstrated by the high priority of SnRK2 pathway studies in the agricultural sciences, detailed knowledge of these mechanisms may be crucial in the choice, breeding, or even design of crops and other plants to be available in response to global warming. Moreover, the realization of the project will extend our knowledge on structure of protein kinases and their specific effectors. Finally, the common project is to build a platform for establishment of a long-term Polish-Norwegian collaboration. Project Partner ÂÂ UiT The Arctic University of Norway, Department of Chemistry, The Norwegian Structural Biology Centre (NorStruct), Tromsø, Norway Description of the Project Plan WP 1. Determination of the smallest interacting fragments of SCS-A, SCS-B and SnRK2 in SCS-SnRK2 Complex WP leader Prof. Grażyna Dobrowolska By virtually all scientific estimates, climate change that is irreversible on human time scales has begun and will accelerate. While some local changes may be positive for agriculture, the net global effect is predicted to severely degrade conditions for plant growth and development, resulting in significant expansion of non-arable land areas. All plants possess facilities to sense changes of environmental conditions and respond with the initiation of various defense mechanisms, however, plants differ drastically in their sensitivity to stress and capabilities to survive in challenging conditions; the most sensitive are usually crop plants. Plants that evolved under natural (wild) conditions have developed numerous defense mechanisms—these require diverse signaling pathways for their initiation and regulation. Understanding these pathways will give us more chances to successfully combat negative effects of coming climate changes. The type 2 SNF1-related kinases (SnRK2s) are plant specific kinases, which are crucial signaling elements involved in plant defense against harmful environmental conditions, especially those which result from climate change, i.e. drought and salinity. Their activity is strictly controlled inside the cell by the clade A PP2C phosphatases which in turn are regulated by ABA receptors (RCAR /PYR/PYL). Recently, in the laboratory of Institute of Biochemistry and Biophysics PAS in Poland (the Principal Investigator of the project), plant specific calcium sensors that interact with the SnRK2s (SCSs), thereby inhibiting their activity in Ca2+-dependent manner were identified. We predict that is an important mechanism of inhibition of SnRK2 signaling pathways. The general goal of the project is to characterize the novel mechanism of inhibition of SnRK2 kinases via their interaction with SCSs, analogous to the investigation of the structural mechanisms behind the inhibition of SnRK2 activity by PP2C phosphatases. In this project, we plan to identify the core of the SnRK2 complex with SCS and to solve the structure of the complex and the mechanism of the inhibition of SnRK2 activity by SCSs. We use several methods to reach this goal including the determination of the X-ray crystal structure of the SCS-SnRK2 complex. The major goals of the project are: 1. Determination of smallest interacting fragments of AtSCSs with AtSnRK2s; 2. Crystallization trials of AtSCSs alone and in complex with AtSnRK2, using whole proteins or the interacting fragments consisting a core of the complex; 3. Elucidation of mechanism of inhibition of SnRK2s by SCSs. WP 2. Elaboration of the procedure(s) of production, purification and storage of proteins: AtSCS-A, AtSCS-B, SnRK2 selected and their fragments WP leader Prof. Grażyna Dobrowolska For crystallization the protein quality (homogeneity, stability, and purity) is extremely important. To get reasonable amounts of pure and highly concentrated proteins, conditions for protein production, methodology for purification and storage need to be carefully evaluated. Therefore, we test several different conditions for proteins expression (time, temperature, type of the medium for growing bacteria), chromatographic procedures, and proteins stability and select the best ones for each of the proteins studies. The proteins whose production or purification is not efficient and which are not stable will not be included in further studies. The quality of the proteins will be analyzed by SDS-PAGE and MS. Estimation of molecular mass and purity of AtSCS-A by SDS-PAGE and mass spectrometry kDa 70 45 Our data indicate that in Arabidopsis thaliana two AtSCS forms exist, AtSCS-A and AtSCS-B. According to Prosite, AtSCS-A contains at least two EF-hand motifs; one of them is a classical EF-hand motif with all the conserved residues needed for calcium binding, whereas the other(s) do not completely match the EF-hand consensus. AtSCS-B is a shorter version of AtSCS-A without the N-terminal canonical EF-hand motif. Both of them interact with SnRK2s in planta and inhibit the SnRK2 activity. Additionally, we have already shown that the truncated form of SnRK2, encompassing only kinase domain is still able to bind AtSCS-A protein. To define the core of the complex we determine the smallest interacting parts of SCS-A/B and SnRK2. The selected pairs of SnRK2 and SCS-A/B (the whole proteins and their fragments) will be set for crystallization (WP 4). In addition to the determination of the smallest interacting fragments of AtSCSs with AtSnRK2s, we plan to define the portion of the AtSCS protein responsible for inhibition of AtSnRK2 kinase activity. 35 SCS-A 25 WP 3. Elucidation of mechanism of inhibition of SnRK2 by SCS and obtaining of modified proteins selected for crystallographic trials Amino acid sequence of NpSCS from Nicotiana plumbaginifolia (GenBank accession number FJ882981), Solanum tuberosum (CAA04670), Vitis vinifera (CBI37946), Arabidopsis thaliana (AAL07098) (AtSCS-A), and Oryza sativa (AAL77132). Predicted EF-hand motifs are boxed; EF-hand-like motif is marked with star (*). Gaps introduced to maximize alignment are marked with dashes. Blue arrows indicate sites of deletions for obtaining truncated form of AtSCS-A (ΔNAtSCS-AΔC), red arrow indicates first methionine of AtSCS-B. WP leader Prof. Grazyna Dobrowolska Before crystallization trials we have to determine whether SCS-A/B binding to SnRK2 competes with ATP using ATP-competitive kinase inhibitors, since ligand induced SnRK2 conformation changes could abolish SCS-A/B-SnRK2 interaction. Alternatively, ATP could compete against SCS-A/B directly in the ATP-binding site. It is critical to verify SCS-A/B/ATP competition because ATP analogs are standard additives in