Extended Data Fig. 7: Arabidopsis (At) PhyA has a compromised ATP-binding pocket and lacks autophosphorylation activity in vitro despite structural similarity to transmitter histidine kinases.

a, Orthogonal views of the HKRD CA region from At PhyA superposed with the same region from At PhyB (PDB ID code 7RZW²²) and the prokaryotic Walk transmitter HK from Lactobacillis plantarum (Lp) (PDB ID code 4U7O³²). b and c, Models showing the predicted position of ADP (red) in the At PhyA CA domain based on the binding pocket described in Lp WalK³². Residues expected to participate in binding are indicated in (b). ADP clashes with multiple residues in the pocket of this predicted PhyA-ATP model, indicating that conformational shifts in At PhyA induced by ATP or photoactivation would be necessary for binding. Sites with substantial clash are circled. d, Amino acid sequence alignment of the possible ATP-binding pocket of At PhyA and At PhyB with comparable CA domains from bona fide histidine kinases from Bacillus subtilis (Bs YFI), Thermotoga maritima (Tm HK853), L. plantarum (Lp Walk) and Streptococcus mutans (Sm Vick), and with those from bacterial Phys (BphPs) with HK/phosphatase activities from Pseudomonas syringae (Ps BphP)¹⁸ and Deinococcus radiodurans (Dr BphP)¹⁶. Identical and similar amino acids are colored in black and grey boxes, respectively. The signature N, G1/D, F and G2 boxes and ATP lid for histidine kinases are indicated³². Arrowheads locate key residues within the ATP-binding pocket that are critical for catalysis³². e-g, At PhyA is a poor kinase as compared to Ps BphP based on autophosphorylation assays. Equimolar amounts of recombinant biliproteins were incubated for 1 min to 2 hr at ambient temperature (~24 °C) with 150 μM ATP supplemented with 10 μCi of [γ-³²P]-ATP, quenched with SDS-PAGE sample buffer, and measured for ³²P incorporation by autoradiography of SDS-PAGE gels. e, Time course for autophosphorylation of Ps BphP as Pfr. f, Comparisons of autophosphorylation activities of At PhyB as Pr and Pfr with those of Ps BphP after 2 hr incubations. Arrowheads locate Ps BphP. The phosphorimager scans are representative of three independent experiments. g, Images of the SDS-PAGE gels stained for protein with Coomassie blue or for the bound bilin by zinc-induced fluorescence.