Séverine Jansen

Structure and binding specificity of the receiver domain of sensor histidine kinase CKI1 from Arabidopsis thaliana.

Multistep phosphorelay (MSP) signaling mediates responses to a variety of important stimuli in plants. In Arabidopsis MSP, the signal is transferred from sensor histidine kinase (HK) via histidine phosphotransfer proteins (AHP1-AHP5) to nuclear response regulators. In contrast to ancestral two-component signaling in bacteria, protein interactions in plant MSP are supposed to be rather nonspecific. Here, we show that the C-terminal receiver domain of HK CKI1 (CKI1(RD) ) is responsible for the recognition of CKI1 downstream signaling partners, and specifically interacts with AHP2, AHP3 and AHP5 with different affinities. We studied the effects of Mg²⁺, the co-factor necessary for signal transduction via MSP, and phosphorylation-mimicking BeF₃⁻ on CKI1(RD) in solution, and determined the crystal structure of free CKI1(RD) and CKI1(RD) in a complex with Mg²⁺. We found that the structure of CKI1(RD) shares similarities with the only known structure of plant HK, ETR1(RD) , with the main differences being in loop L3. Magnesium binding induces the rearrangement of some residues around the active site of CKI1(RD) , as was determined by both X-ray crystallography and NMR spectroscopy. Collectively, these results provide initial insights into the nature of molecular mechanisms determining the specificity of MSP signaling and MSP catalysis in plants.

Conformational dynamics as a key factor of signaling mediated by the receiver domain of sensor histidine kinase from Arabidopsis thaliana.

Multistep phosphorelay (MSP) cascades mediate responses to a wide spectrum of stimuli, including plant hormonal signaling, but several aspects of MSP await elucidation. Here, we provide first insight into the key step of MSP-mediated phosphotransfer in a eukaryotic system, the phosphorylation of the receiver domain of the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1RD) from Arabidopsis thaliana. We observed that the crystal structures of free, Mg2+-bound, and beryllofluoridated CKI1RD (a stable analogue of the labile phosphorylated form) were identical and similar to the active state of receiver domains of bacterial response regulators. However, the three CKI1RD variants exhibited different conformational dynamics in solution. NMR studies revealed that Mg2+ binding and beryllofluoridation alter the conformational equilibrium of the β3–α3 loop close to the phosphorylation site. Mutations that perturbed the conformational behavior of the β3–α3 loop while keeping the active-site aspartate intact resulted in suppression of CKI1 function. Mechanistically, homology modeling indicated that the β3–α3 loop directly interacts with the ATP-binding site of the CKI1 histidine kinase domain. The functional relevance of the conformational dynamics observed in the β3–α3 loop of CKI1RD was supported by a comparison with another A. thaliana histidine kinase, ETR1. In contrast to the highly dynamic β3–α3 loop of CKI1RD, the corresponding loop of the ETR1 receiver domain (ETR1RD) exhibited little conformational exchange and adopted a different orientation in crystals. Biochemical data indicated that ETR1RD is involved in phosphorylation-independent signaling, implying a direct link between conformational behavior and the ability of eukaryotic receiver domains to participate in MSP.

The influence of Mg2+ coordination on 13C and 15N chemical shifts in CKI1RD protein domain from experiment and molecular dynamics/density functional theory calculations

Sequence dependence of 13C and 15N chemical shifts in the receiver domain of CKI1 protein from Arabidopsis thaliana, CKI1RD, and its complexed form, CKI1RD•Mg2+, was studied by means of MD/DFT calculations. MD simulations of a 20–ns production run length were performed. Nine explicitly hydrated structures of increasing complexity were explored, up to a 40‐amino‐acid structure. The size of the model necessary depended on the type of nucleus, the type of amino acid and its sequence neighbors, other spatially close amino acids, and the orientation of amino acid NH groups and their surface/interior position. Using models covering a 10 and a 15 Å environment of Mg2+, a semi‐quantitative agreement has been obtained between experiment and theory for the V67−I73 sequence. The influence of Mg2+ binding was described better by the 15 Å as compared to the 10 Å model. Thirteen chemical shifts were analyzed in terms of the effect of Mg2+ insertion and geometry preparation. The effect of geometry was significant and opposite in sign to the effect of Mg2+ binding. The strongest individual effects were found for 15N of D70, S74, and V68, where the electrostatics dominated; for 13Cβ of D69 and 15N of K76, where the influences were equal, and for 13Cα of F72 and 13Cβ of K76, where the geometry adjustment dominated. A partial correlation between dominant geometry influence and torsion angle shifts upon the coordination has been observed. Proteins 2016; 84:686–699.

Conformation and dynamics of intrinsically disordered microtubule associated protein 2c (MAP2c)

Microtubule associated protein 2c (MAP2c) regulates structure and dynamics (polymerization and degradation) of microtubules in developing neurons and other cells. MAP2c is a 49 kDa intrinsically disordered protein (IDP) consisting of several structural and functional regions. The N-terminal part contains two important regions: An N-terminal region with a high content of negatively charged amino acids and a proline-rich region. The former segment contains a binding site for the regulatory subunit of cAMP-dependent protein kinase (PKA) and proposed binding site for steroids, while the latter one contains several phosphorylation sites. The second important part of MAP2c is a highlyconserved C-terminal domain that binds to microtubules. In order to investigate relation between highly dynamic structural features of MAP2c and its functions, we studied dynamics of MAP2c using nuclear magnetic resonance (NMR) relaxation and performed quantitative conformational analysis of NMR chemical shifts, small angle X-ray scattering, and paramagnetic relaxation enhancement. Pools of possible chain conformations were generated by the program Flexible-Meccano, which builds consecutively a polypeptide chain. The ensemble of structures reproducing the experimental data was selected by the program ASTEROIDS which uses a genetic algorithm. We found out that the least flexible amino acids are involved in transient long-range contacts between the acidic N-terminal domain and the microtubule-binding domain and that more ordered regions correlate with the regions of known or proposed function. Our results thus indicate importance of the N-terminal regions for the specificity of regulatory roles of MAP2c and a close relation between biological functions and conformational behavior of this protein

Quantitative mapping of microtubule-associated protein 2c (MAP2c) phosphorylation and regulatory protein 14-3-3ζ-binding sites reveals key differences between MAP2c and its homolog Tau.

There was an error in Fig. 6. Panels A and B were inadvertently exchanged. The correct legend should read as follows. Microscale thermophoretic analysis of interaction between 5 M E52C/C348S MAP2c and 14-3-3 in 50 mM Tris buffer. A, phosphorylated MAP2c; B, unphosphorylated MAP2c. The plots show interactions in the 14-3-3 monomer concentration range from 36.6 nM to 1.2 mM. The mean values S.D. for each concentration point were calculated from triplicate measurements. The results are described correctly in the text, and the error does not affect the results or conclusions of the work. ADDITIONS AND CORRECTIONS