Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

North Carolina State University

Does not respond to nucleotides 1 (DORN1) has recently been identified as the first membrane-integral plant ATP receptor, which is required for ATP-induced calcium response, mitogen-activated protein kinase activation and defense responses in Arabidopsis thaliana. In order to understand DORN1-mediated ATP sensing and signal transduction, crystallization and preliminary X-ray studies were conducted on the extracellular domain of DORN1 (atDORN1-ECD) and that of an orthologous protein, Camelina sativa lectin receptor kinase I.9 (csLecRK-I.9-ECD or csI.9-ECD). A variety of deglycosylation strategies were employed to optimize the glycosylated recombinant atDORN1-ECD for crystallization. In addition, the glycosylated csI.9-ECD protein was crystallized at 291 K. X-ray diffraction data were collected at 4.6 Å resolution from a single crystal. The crystal belonged to space group C222 or C222, with unit-cell parameters a = 94.7, b = 191.5, c = 302.8 Å. These preliminary studies have laid the foundation for structural determination of the DORN1 and I.9 receptor proteins, which will lead to a better understanding of the perception and function of extracellular ATP in plants.

X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa.

Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In Arabidopsis, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65 Ǻ and 2.75 Ǻ respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our in vitro biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs).

Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions.

During reproduction in flowering plants, the male gametophyte delivers an immotile male gamete to the female gametophyte in the pistil by formation of pollen tubes. In Arabidopsis thaliana, two synergid cells situated on either side of the egg cell produce cysteine-rich chemoattractant peptide LURE that guides the pollen tube to the female gametophyte for sexual reproduction. Recently, in Arabidopsis thaliana, Pollen Receptor Kinase 3 (PRK3), along with PRK1, PRK6, and PRK8, have been predicted to be the receptors responsible for sensing LURE. These receptors belong to the Leucine Rich Repeat Receptor Like Kinases (LRR-RLKs), the largest family of receptor kinases found in Arabidopsis thaliana. How PRKs regulate the growth and development of the pollen tube remains elusive. In order to better understand the PRK-mediated signaling mechanism in pollen tube growth and guidance, we have determined the crystal structure of the extracellular domain (ecd) of PRK3 at 2.5 Å, which resembles the SERK family of plant co-receptors. The structure of ecdPRK3 is composed of a conserved surface that coincides with the conserved receptor-binding surface of the SERK family of co-receptors. Our structural analyses of PRK3 have provided a template for future functional studies of the PRK family of LRR-RLK receptors in the regulation of pollen tube development.

The Extracellular Domain of Pollen Receptor Kinase 3 is structurally similar to the SERK family of co-receptors.

Activation of IκB kinase β (IKKβ) is a central event in the NF-κB-mediated canonical pro-inflammatory pathway. Numerous studies have reported that oligomerization-mediated trans autophosphorylation of IKKβ is indispensable for its phosphorylation, leading to its activation and IKKβ-mediated phosphorylation of substrates such as IκB proteins. Moreover, IKKβ's interaction with the NF-κB essential modifier (NEMO) is necessary for IKKβ activation. Interestingly, some viruses encode virulence factors that target IKKβ to inhibit NF-κB-mediated antiviral immune responses. One of these factors is the vaccinia viral protein B14, which directly interacts with and inhibits IKKβ. Here we mapped the interaction interface on the B14 and IKKβ proteins. We observed that B14 binds to the junction of the kinase domain (KD) and scaffold and dimerization domain (SDD) of IKKβ. Molecular docking analyses identified key interface residues in both IKKβ and B14 that were further confirmed by mutational studies to promote binding of the two proteins. During trans autophosphorylation of protein kinases in the IKK complex, the activation segments of neighboring kinases need to transiently interact with each other's active sites, and we found that the B14-IKKβ interaction sterically hinders direct contact between the kinase domains of IKKβ in the IKK complex, containing IKKβ, IKKα, and NEMO in human cells. We conclude that binding of B14 to IKKβ prevents IKKβ trans autophosphorylation and activation, thereby inhibiting NF-κB signaling. Our study provides critical structural and mechanistic information for the design of potential therapeutic agents to target IKKβ activation for the management of inflammatory disorders.

Sayan Chakraborty

Department of Molecular and Structural Biochemistry,
College of Agriculture and Life Sciences,
Department of Molecular and Structural Biochemistry, College of Agriculture and Life Sciences

North Carolina State University

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

Sayan Chakraborty

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular and Structural Biochemistry,College of Agriculture and Life Sciences,Department of Molecular and Structural Biochemistry, College of Agriculture and Life Sciences

North Carolina State University

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

Department of Molecular and Structural Biochemistry,
College of Agriculture and Life Sciences,
Department of Molecular and Structural Biochemistry, College of Agriculture and Life Sciences