Download Toward structural characterization of novel mechanism of inhibition

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
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Ca2+
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M
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2 3
4 5 6
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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
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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.
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SCS-A
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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