Abstract
Nitrate transport activity of the LtnT permease of the cyanobacterium Synechococcus elongatus is activated when LtnA, a response regulator without an effector domain, is phosphorylated by LtnB, a hybrid histidine kinase. We identified a protein (LtnC) that is required for activation of LtnT. LtnC consists of an N-terminal histidine-containing phosphoacceptor (HisKA) domain, a receiver domain, and a unique C-terminal domain found in some cyanobacterial proteins. Because LtnC lacks an ATP-binding kinase domain of a histidine kinase, it is incapable of autophosphorylation, but LtnC is phosphorylated by LtnA. The histidine residue in the HisKA domain but not the aspartate residue in the receiver domain is essential for phosphorylation of LtnC and activation of LtnT. LtnC phosphorylation leads to oligomerization of the protein. Fusion of the C-terminal domain of LtnC to glutathione S-transferase, which forms oligomers, also activates LtnT, suggesting that oligomerization of the LtnC C-terminal domain causes LtnT activation. These results indicate that the C-terminal domain of LtnC acts as an effector domain that directs the output of the signal from the phosphorelay system. The two-step (His-Asp-His) phosphorelay system, composed of the LtnB, LtnA, and LtnC proteins, is distinct from the known phosphorelay systems, namely, the typical two-component system (His-Asp) and the multistep phosphorelay system (His-Asp-His-Asp), because the HisKA domain of LtnC is the terminal phosphoacceptor that determines the signal output. LtnC is a new class of signal transducer in His-Asp phosphorelay systems that contains a HisKA domain and an effector domain.
Highlights
The REC domain of a response regulator is the terminal acceptor of the phosphoryl group, and the phosphorylated response regulators act as molecular switches to certain cellular events by regulating the activity of their C-terminal effector domains
To overexpress glutathione S-transferase (GST) and its fusion with the C-terminal region of LtnC, the pSE1GST plasmid and its derivative carrying the 3Ј region of ltnC were transformed into E. coli JM105, expression of the encoded proteins was induced by 50 M isopropyl-thio--Dgalactopyranoside, and the proteins were purified on glutathione-Sepharose resin
Downstream of the ltnA and -B genes involved in activation of the permease encoded by ltnT are two open reading frames (ORFs) syc2279_d and syc2280_d, which are oriented in the same direction as ltnAB and which overlap with the upstream genes by 8 and 77 bases, respectively (Fig. 1). syc2279_d encodes a protein of 411 amino acids, which is similar to hybrid histidine kinase and carries a HisKA domain in the N-terminal region and a REC domain in the central region, 37870 JOURNAL OF BIOLOGICAL CHEMISTRY
Summary
Pseudorevertant of NA3, nonsense mutation in the ltnB gene NA3R ltnA::CmR NA3R ltnB::CmR NA3R ltnC::CmR NA3R ltnD::CmR. KmR, Synechococcus shuttle expression vector pSE1 derivative encoding LtnA and LtnB lacking N-terminal region and both REC domains pSE1 derivative encoding LtnC pSE1 derivative encoding LtnC(H16E) pSE1 derivative encoding LtnC(D223N) pSE1 derivative encoding LtnC lacking HisKA and REC domains pSE1 derivative encoding LtnD pSE1 derivative encoding LtnD lacking a CAP_ED domain pSE1 derivative encoding glutathione S-transferase pSE1GST derivative encoding the C-terminus region of LtnC pSE1GST derivative encoding the C-terminus region of LtnD. AmpR, E. coli His-tagged expression vector pQE30 derivative encoding LtnA pQE31 derivative encoding LtnB lacking the N-terminal region pQE31 derivative encoding LtnB lacking the N-terminal region and both REC domains pQE30 derivative encoding LtnC pQE30 derivative encoding LtnC(H16E) pQE30 derivative encoding LtnC(D223N)
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