Receiver domain of sensor histidine kinase CKI1

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Receiver domain of CKI1 from Arabidopsis, 3mmn
Receiver domain of CKI1 from Arabidopsis, 3mmn














Receiver domain of sensor histidine kinase CKI1 (CKI1RD) catalyses the transphosphorylation reaction during hormonal and abiotic signalling in plants. Membrane-bound histidine kinase Cytokinin-independet 1 (CKI1) is a member of the Multistep phosphorelay (MSP) signalling pathway in Arabidopsis. CKI1 was found to be constitutively active activator of a cytokinin-like response. Intracellularly located C-terminal CKI1RD is responsible for the recognition of CKI1 downstream signalling partners from family of Arabidopsis histidine-containing phosphotransfer proteins (AHP) and triggers the cytokinin-like signal transmission. Divalent magnesium ion bound in the active site of CKI1RD is essential for the transphosphorylation reaction. Crystal structure of CKI1RD was determined as magnesium-free and magnesium-bound form. Magnesium binding induces the rearrangement of residues around the active site of CKI1RD, as was determined by both X-ray crystallography and NMR spectroscopy.

Biological Function

CKI1 acts as constitutively active histidine-kinaze in MSP signaling in Arabidopsis.
CKI1 acts as constitutively active histidine-kinaze in MSP signaling in Arabidopsis.

CKI1 as member of Multistep phosphorelay signaling in Arabidopsis
The sensor histidine kinase CKI1 was identified as an activator of a cytokinin-like response when overexpressed in hypocotyl explants [1] and it was shown to be essential for the female gametophyte development[2][3]. Cytokinin response in Arabidopsis involves shoot and root growth regulation, leaf senesce, circadian rhythms and more[4]. No cytokinin binding to CKI1 has been detected, and in contrast to the genuine cytokinin receptors of Arabidopsis, CKI1 was found to be constitutively active in bacteria and yeast or Arabidopsis protoplasts[5][6]. Cytokinin signalling in plants is triggered by MSP[7] , which was adopted by plants from bacterial Two-component system[8]. The signalling molecule is bound to the sensory histidin-kinase and then is transferred via AHPs (AHP1-AHP5) to nuclear response regulators (ARRs). ARRs act as transcription factors or interact with other effector proteins[9] to perform specific cellular response to initial environmental stimuli. In contrast to ancestral Two-component signalling in bacteria, protein interactions in plant MSP are supposed to be rather nonspecific. The current MSP interaction maps from Arabidopsis are based on yeast two-hybrid system[10][11][12] and show the AHPs as highly promiscuous, able to interact with all cytokinin receptors, number of other histidine-kinases and number of ARRs. Recently was shown that CKI1RD is responsible for a specific interaction with individual subset of second messengers from AHP family (AHP2, AHP3, AHP5)[13].

CKI1RD structure and effects of magnesium binding in the active site

The crystal structure of CKI1RD shows the conformational conservation of RDs belonging to CheY-like protein superfamily [14][15]. CKI1RD is folded in a (α/β)5 manner with central β-sheet formed from parallel beta-strands (β2-β1-β3-β4-β5) surrounded on both sides by two (α1 and α5) and three (α2, α3, α4) α-helices. Secondary structure elements are connected by five loops L1-L5 on the face side of the protein. The active site with phosphoacceptor D1050 is located at the C-termini of the central β3-strand in a pocket delineated loops L1, L3 and L5. A highly conserved triad of carboxyl oxygens, formed by D1050 together with D992 and D993 and carbonyl oxygen of Q1052 give the active site an acidic character. This architecture of the active site is well conserved among CheY-like superfamily and corresponds to the phosphotransfer function of the receiver domains.

Structure and sequence conservation among known receiver domains. Left: Superimposition of 35 known crystal structures of receiver domains. Right: Representation of highly conserved residues among receiver domains (CKI1RD numbering)
Structure and sequence conservation among known receiver domains. Left: Superimposition of 35 known crystal structures of receiver domains. Right: Representation of highly conserved residues among receiver domains (CKI1RD numbering)

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The octahedral coordination geometry of magnesium ion in this crystal is not complete. Magnesium ion is four-coordinated with carboxyl oxygens of D993 and D1050, carbonyl oxygen of Q1052 and with one water molecule that forms a hydrogen bridge to the carboxyl oxygen of D992.
Magnesium binding mediates slight structural changes of the active site. Upon magnesium binding, the side chain of the D1050 rotates by 90° toward the divalent cation. The connection via salt bridge between D1050 and K1105 induces the rotation of K1105, whereas the salt bridge remains established. More, the NMR analysis shows magnesium binding to stabilize conformational flexibility of the loop L3 in the solution[16].

(a) Magnesium-free and (b)magnesium-bound forms of the CKI1RD active site. Magnesium ion (magenta) is approximately octahedrally coordinated. (c) 2Fo-Fc omit map of CKI1RD magnesium bound contoured at 1σ.  Conservation of the structure and sequence among known receiver domains. The magnesium ion and all atoms in radius of 5Å were omitted from phasing.
(a) Magnesium-free and (b)magnesium-bound forms of the CKI1RD active site. Magnesium ion (magenta) is approximately octahedrally coordinated. (c) 2Fo-Fc omit map of CKI1RD magnesium bound contoured at 1σ. Conservation of the structure and sequence among known receiver domains. The magnesium ion and all atoms in radius of 5Å were omitted from phasing.

Magnesium-bound form of CKI1RD, (PDB entry 3mmn)

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3D Structures of CKI1RD and similar receiver domains

3mm4 - CKI1RD in metal free form
3mmn - CKI1RD in magnesium bound form

3dcf - ETR1RD, receiver domain of plant hormone ethylene receptor from Arabidopsis
2chy - CheY, chemotaxis response regulatory protein from Salmonella enterica
3dgf - response regulatory signalling protein from Thermotoga maritina

References

  1. Kakimoto T. CKI1, a histidine kinase homolog implicated in cytokinin signal transduction. Science. 1996 Nov 8;274(5289):982-5. PMID:8875940
  2. Pischke MS, Jones LG, Otsuga D, Fernandez DE, Drews GN, Sussman MR. An Arabidopsis histidine kinase is essential for megagametogenesis. Proc Natl Acad Sci U S A. 2002 Nov 26;99(24):15800-5. Epub 2002 Nov 8. PMID:12426401 doi:10.1073/pnas.232580499
  3. Hejatko J, Pernisova M, Eneva T, Palme K, Brzobohaty B. The putative sensor histidine kinase CKI1 is involved in female gametophyte development in Arabidopsis. Mol Genet Genomics. 2003 Jul;269(4):443-53. Epub 2003 May 28. PMID:12774227 doi:10.1007/s00438-003-0858-7
  4. Brenner WG, Ramireddy E, Heyl A, Schmulling T. Gene regulation by cytokinin in Arabidopsis. Front Plant Sci. 2012;3:8. Epub 2012 Jan 31. PMID:22639635 doi:10.3389/fpls.2012.00008
  5. Yamada H, Suzuki T, Terada K, Takei K, Ishikawa K, Miwa K, Yamashino T, Mizuno T. The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane. Plant Cell Physiol. 2001 Sep;42(9):1017-23. PMID:11577198
  6. Hwang I, Sheen J. Two-component circuitry in Arabidopsis cytokinin signal transduction. Nature. 2001 Sep 27;413(6854):383-9. PMID:11574878 doi:10.1038/35096500
  7. Urao T, Yamaguchi-Shinozaki K, Shinozaki K. Two-component systems in plant signal transduction. Trends Plant Sci. 2000 Feb;5(2):67-74. PMID:10664616
  8. Mount SM, Chang C. Evidence for a plastid origin of plant ethylene receptor genes. Plant Physiol. 2002 Sep;130(1):10-4. PMID:12226482 doi:10.1104/pp.005397
  9. Heyl A, Schmulling T. Cytokinin signal perception and transduction. Curr Opin Plant Biol. 2003 Oct;6(5):480-8. PMID:12972049
  10. Urao T, Miyata S, Yamaguchi-Shinozaki K, Shinozaki K. Possible His to Asp phosphorelay signaling in an Arabidopsis two-component system. FEBS Lett. 2000 Aug 4;478(3):227-32. PMID:10930573
  11. Dortay H, Mehnert N, Burkle L, Schmulling T, Heyl A. Analysis of protein interactions within the cytokinin-signaling pathway of Arabidopsis thaliana. FEBS J. 2006 Oct;273(20):4631-44. Epub 2006 Sep 11. PMID:16965536 doi:10.1111/j.1742-4658.2006.05467.x
  12. Dortay H, Gruhn N, Pfeifer A, Schwerdtner M, Schmulling T, Heyl A. Toward an interaction map of the two-component signaling pathway of Arabidopsis thaliana. J Proteome Res. 2008 Sep;7(9):3649-60. Epub 2008 Jul 22. PMID:18642946 doi:10.1021/pr0703831
  13. Pekarova B, Klumpler T, Triskova O, Horak J, Jansen S, Dopitova R, Papouskova V, Nejedla E, Sklenar V, Marek J, Zidek L, Hejatko J, Janda L. Structure and binding specificity of the receiver domain of sensor histidine kinase CKI1 from Arabidopsis thaliana. Plant J. 2011 May 13. doi: 10.1111/j.1365-313X.2011.04637.x. PMID:21569135 doi:10.1111/j.1365-313X.2011.04637.x
  14. Stock AM, Martinez-Hackert E, Rasmussen BF, West AH, Stock JB, Ringe D, Petsko GA. Structure of the Mg(2+)-bound form of CheY and mechanism of phosphoryl transfer in bacterial chemotaxis. Biochemistry. 1993 Dec 14;32(49):13375-80. PMID:8257674
  15. Wilson D, Pethica R, Zhou Y, Talbot C, Vogel C, Madera M, Chothia C, Gough J. SUPERFAMILY--sophisticated comparative genomics, data mining, visualization and phylogeny. Nucleic Acids Res. 2009 Jan;37(Database issue):D380-6. Epub 2008 Nov 26. PMID:19036790 doi:10.1093/nar/gkn762
  16. Pekarova B, Klumpler T, Triskova O, Horak J, Jansen S, Dopitova R, Papouskova V, Nejedla E, Sklenar V, Marek J, Zidek L, Hejatko J, Janda L. Structure and binding specificity of the receiver domain of sensor histidine kinase CKI1 from Arabidopsis thaliana. Plant J. 2011 May 13. doi: 10.1111/j.1365-313X.2011.04637.x. PMID:21569135 doi:10.1111/j.1365-313X.2011.04637.x

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Tomas Klumpler, Michal Harel, Alexander Berchansky

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