Structural Study Of The Arabidopsis Receptor-like Kinase GHR1and Structural Basis For An ATPase Subunit Of An ECF Transporter From Thermoanaerobacter Tengcongensis

In plants, the largest group of membrane receptors is the receptor-like kinases (RLKs) that play important roles in diverse biological processes, particularly plant development and plant immune response. The sequenced Arabidopsis genome contains more than600RLKs, representing nearly2.5%of the annotated protein-coding genes in Arabidopsis. RLKs are a family of transmembrane proteins that feature an N-terminal extracellular domain linked to a C-terminal intracellular kinase domain via a middle transmembrane domain. Arabidopsis GUARD CELL HYDROGEN PEROXIDE-RESISTANT1(GHRl), recently identified as a member of the leucine-rich repeat receptor kinase family (LRR-RKs), is a critical early component in abscisic acid (ABA) signaling. Here, we present strategies for expression and purification of the extracellular domain of GHR1(GHR1-ECD) and the kinase domain of GHR1(GHR1-KD), and large-scale production was obtained for structural study. GHR1is a receptor serine/threonine kinase whose extracellular domain is predicted to contain22LRRs. We engineered an N-terminal secretory signal peptide and a C-terminal poly-His tag of GHR1-ECD for secretory expression and affinity purification. Using the Bac-to-Bac(?) Baculovirus Expression System, large-scale production of GHR1-ECD was purified, and crystals were successfully obtained. GHRl-KD is a serine/threonine kinase, in contrast to most other protein kinases which efficiently utilize ATP as a phosphoryl donor, we report the GHRl-KD structure that substitutes a GTP molecule for ATP. The residue Thr780of GHRl-KD is critical for binding GTP, and this site is a conserved Ala in other ATP-utilizing kinases. The structure of GHRl-KD provides new understanding about protein kinases in plants.Cellular survival requires excretion of waste products and uptake of essential nutrients across the cell membrane. An intact ABC transporter consists of two variable hydrophobic transmembrane domains (TMDs) and two conserved hydrophilic ABC-ATPases(A components), are vital participants in this process, which use the chemical energy of ATP hydrolysis to translocate various substrates, ranging from small ions to large macromolecules. Energy-coupling factor (ECF) transporters are a new group of ABC transporters in prokaryotes that contain an ECF module and employ membrane proteins to bind substrates. CbiMNQO protein complex is a typical ECF transporter and is a widespread group of microbial transporters for cobalt. CbiO from the CbiMNQO protein complex corresponds to an A component. Here we report the crystal structure of CbiO at a resolution of2.3A. CbiO contains an N-terminal ATPase domain and C-terminal helical domain. Structural and biochemical data show that CbiO forms a homodimer, interactions mainly via conserved hydrophobic amino acids between the two C-terminal domains result in formation of a four-helix bundle. Structural comparison shows that Arg82in CbiO important for the interaction within an ECF module of other ECF transporters adopts a strikingly different rotamer. The data provide information on understanding of the structural organization and interaction of the CbiMNQO system.