1. Volume 6, Issue 3, Pages 959-970 (May 2013)
Structure–Function Analysis of Arabidopsis thaliana Histidine Kinase AHK5 Bound to Its Cognate Phosphotransfer Protein AHP1 
Johannes Bauer, Kerstin Reiss, Manikandan Veerabagu, Michael Heunemann, Klaus Harter, Thilo Stehle 
Molecular Plant 
Volume 6, Issue 3, Pages (May 2013)
DOI: /mp/sss126
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 Schematic Overview of His–Asp Phosphorelay Systems.
(A) Overview of phosphorelay signaling in two-component systems (TCS). Signal perception induces ATP-binding to the catalytic domain (CA), followed by phosphorylation of a conserved histidine residue in the dimerization and phosphotransfer domain (DHP) of the kinase. Finally, the phosphoryl group (P) is relayed onto a conserved aspartic acid residue in the receiver domain of the cognate response regulator (RR).
(B) Overview of phosphorelay signaling in multi-step phosphorelay systems (MSP). MSP signaling involves a hybrid histidine kinase containing a fused receiver domain and an additional AHP protein.
Molecular Plant 2013 6, DOI: ( /mp/sss126)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
Structure of the AHK5RD–AHP1 Complex.AHP1 and AHK5RD are shown in all figures as ribbon tracings and colored green and blue, respectively.
(A, B) Overall structure. The conserved Asp and His residues are drawn as red sticks. In (A), AHP1 and AHK5RD are additionally shown in surface representation.
(C) Binding interface of AHP1 (left) and AHK5RD (right). Contacting residues are shown in stick representation. Residues involved in hydrogen bonds are highlighted orange, with oxygens in red and nitrogens in blue. Hydrophobic patch residues are highlighted blue. The histidine and aspartic residues that donate and accept the phosphoryl group are shown as yellow sticks.
(D) Mg2+-coordination in the phosphorylation site. The Mg2+-ion (yellow) and water molecules (red) are shown as spheres. The octahedral coordination geometry of Mg2+ is indicated by black broken lines; further hydrogen bonds of Mg2+-coordinating water molecules are indicated by orange broken lines. The phosphoryl-transferring histidine and aspartic acid residues are shown as sticks, with oxygens in red and nitrogens in blue. All other Mg2+-contacting residues are drawn in stick representation with carbons colored light blue. F903 and T884 of AHK5RD are involved in ‘F-T’-coupling and are shown as blue sticks; the predicted reorientation upon phosphorylation is indicated by black arrows.
Molecular Plant 2013 6, DOI: ( /mp/sss126)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

4. Figure 3 Interaction Studies of AHK5RD–AHP Complexes.
(A–F) Surface plasmon resonance analyses. Double-referenced sensorgrams of the AHK5RD-interaction with AHP1 (A), AHP2 (B), and AHP3 (C) are shown. Red boxes show the range used for calculation of averaged equilibrium response values. (D–F) Averaged equilibrium response values plotted against the injected concentrations of AHP1 (D), AHP2 (E), and AHP3 (F).
(G, H) BiFC analysis of the AHK5RD-interaction with AHP1-6 in planta. (G) Confocal images of tobacco leaf cells expressing both the YFP-C::AHK5RD fusion protein and respective AHP::YFP–N fusion proteins. The fluorescence images (left column), corresponding bright field images (middle column), and respective overlays (right column) are shown. (H) Western blot analysis of protein extracts from tobacco leaves used for BiFC analysis. Immunodetection was carried out by α-myc antibody for AHP::YFP–N fusion proteins and by α-HA antibody for YFP::C–AHK5RD. N, protein extract from non-transformed tobacco leaves; M, protein size standard.
Molecular Plant 2013 6, DOI: ( /mp/sss126)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

5. Figure 4 Interface Architectures of AHP1, YPD1, and CheA3HP.
(A) Conservation of interface residues in AHP proteins based on the AHK5RD–AHP1 complex structure. AHP1 is shown in surface representation with non-interface residues colored white. The phosphorylation site is shown in yellow. Functionally conserved hydrogen bonds in AHP1–6 are colored orange; not functionally conserved hydrogen bonds are colored purple. All hydrophobic residues in the interface are conserved and colored blue. All other AHP interface residues within a radius of 5 Å to AHK5RD are shown in green and they are all conserved in terms of their surface complementarity with respect to the interface.
(B–D) Comparison of interface architectures of AHP1 (B), YPD1 (C), and CheA3HP (D) based on the AHK5RD–AHP1 complex structure, the phosphorylated SLN1RD–YPD1 complex structure, and the phosphorylated CheY6–CheA3HP (CheY6–CheA3HP•P) complex structure, respectively. All three proteins are shown in surface representation, with non-interface residues colored white. The phosphorylation site is shown in yellow. Amino acids that contribute hydrogen bonds are colored orange; residues contributing hydrophobic interactions with their respective complex partner are colored blue.
Molecular Plant 2013 6, DOI: ( /mp/sss126)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Superposition of AHK5RD–AHP1 Complex with Structurally Similar Complexes.
(A) Superposition of the AHK5RD–AHP1 complex (blue) with the phosphorylated SLN1RD–YPD1 complex (green), the apo SLN1RD–YPD1 complex (yellow), and the phosphorylated CheY6–CheA3HP complex (red) by superposing the receiver domains only. The phosphoryl-transferring histidine and aspartic acid residues are highlighted in stick representation.
(B) Comparison of the AHK5RD–AHP1 complex (blue) with the phosphorylated SLN1RD–YPD1 complex (green) and the phosphorylated CheY6–CheA3HP complex (red). The superpositions are based on the receiver domains only and, for reasons of clarity, only AHK5RD–AHP1 is shown as a complete complex, with phosphoryl-transferring histidine and aspartic acid residues highlighted in stick representation. The extra helix of AHP1 and YPD1 is indicated.
Molecular Plant 2013 6, DOI: ( /mp/sss126)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions