Publications by authors named "Guozhou Xu"

13 Publications

  • Page 1 of 1

Expression, purification, and phylogenetic analysis of MDIS1-INTERACTING RECEPTOR-LIKE KINASE1 (MIK1).

Protein J 2020 10 26;39(5):461-471. Epub 2020 Oct 26.

Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, USA.

An abundance of protein structures has been solved in the last six decades that are paramount in defining the function of such proteins. For unsolved protein structures, however, predictions based on sequence and phylogenetic similarity can be useful for identifying key domains of interaction. Here, we describe expression and purification of a recombinant plant LRR-RLK ectodomain MIK1 using a modified baculovirus-mediated expression system with subsequent N-linked glycosylation analysis using LC-MS/MS and computational sequence-based analyses. Though highly ubiquitous, glycosylation site specificity and the degree of glycosylation influenced by genetic and exogenous factors are still largely unknown. Our experimental analysis of N-glycans on MIK1 identified clusters of glycosylation that may explicate the regions involved in MIK1 ectodomain binding. Whether these glycans are necessary for function is yet to be determined. Phylogenetic comparison using multiple sequence alignment between MIK1 and other LRR-RLKs, namely TDR in Arabidopsis thaliana, revealed conserved structural motifs that are known to play functional roles in ligand and receptor binding.
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http://dx.doi.org/10.1007/s10930-020-09926-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706394PMC
October 2020

Plant Leucine-Rich Repeat Receptor Kinase (LRR-RK): Structure, Ligand Perception, and Activation Mechanism.

Molecules 2019 Aug 25;24(17). Epub 2019 Aug 25.

Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA.

In recent years, secreted peptides have been recognized as essential mediators of intercellular communication which governs plant growth, development, environmental interactions, and other mediated biological responses, such as stem cell homeostasis, cell proliferation, wound healing, hormone sensation, immune defense, and symbiosis, among others. Many of the known secreted peptide ligand receptors belong to the leucine-rich repeat receptor kinase (LRR-RK) family of membrane integral receptors, which contain more than 200 members within making it the largest family of plant receptor kinases (RKs). Genetic and biochemical studies have provided valuable data regarding peptide ligands and LRR-RKs, however, visualization of ligand/LRR-RK complex structures at the atomic level is vital to understand the functions of LRR-RKs and their mediated biological processes. The structures of many plant LRR-RK receptors in complex with corresponding ligands have been solved by X-ray crystallography, revealing new mechanisms of ligand-induced receptor kinase activation. In this review, we briefly elaborate the peptide ligands, and aim to detail the structures and mechanisms of LRR-RK activation as induced by secreted peptide ligands within plants.
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http://dx.doi.org/10.3390/molecules24173081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6749341PMC
August 2019

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

J Vis Exp 2018 08 20(138). Epub 2018 Aug 20.

Department of Molecular and Structural Biochemistry, College of Agriculture and Life Sciences, North Carolina State University;

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.
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http://dx.doi.org/10.3791/58283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128212PMC
August 2018

Mechanism of vaccinia viral protein B14-mediated inhibition of IκB kinase β activation.

J Biol Chem 2018 06 10;293(26):10344-10352. Epub 2018 May 10.

From the Department of Molecular and Structural Biochemistry, College of Agriculture and Life Sciences, North Carolina State University, Raleigh, North Carolina 27695

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.
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http://dx.doi.org/10.1074/jbc.RA118.002817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028965PMC
June 2018

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

Sci Rep 2018 02 12;8(1):2796. Epub 2018 Feb 12.

Department of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, Raleigh, 27695, USA.

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.
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http://dx.doi.org/10.1038/s41598-018-21218-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809528PMC
February 2018

Evidence for M1-Linked Polyubiquitin-Mediated Conformational Change in NEMO.

J Mol Biol 2017 12 27;429(24):3793-3800. Epub 2017 Oct 27.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, and Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, United States. Electronic address:

The NF-κB essential modulator (NEMO) is the scaffolding subunit of the inhibitor of κB kinase (IKK) holocomplex and is required for the activation of the catalytic IKK subunits, IKKα and IKKβ, during the canonical inflammatory response. Although structures of shorter constructs of NEMO have been solved, efforts to elucidate the full-length structure of NEMO have proved difficult due to its apparent high conformational plasticity. To better characterize the gross dimensions of full-length NEMO, we employed in-line size exclusion chromatography-small-angle X-ray scattering. We show that NEMO adopts a more compact conformation (D=320Å) than predicted for a fully extended coiled-coil structure (>500Å). In addition, we map a region of NEMO (residues 112-150) in its coiled-coil 1 domain that impedes the binding of linear (M1-linked) di-ubiquitin to its coiled-coil 2-leucine zipper ubiquitin binding domain. This ubiquitin binding inhibition can be overcome by a longer chain of linear, but not K63-linked polyubiquitin. Collectively, these observations suggest that NEMO may be auto-inhibited in the resting state by intramolecular interactions and that during signaling, NEMO may be allosterically activated by binding to long M1-linked polyubiquitin chains.
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http://dx.doi.org/10.1016/j.jmb.2017.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705538PMC
December 2017

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

PLoS One 2017 6;12(4):e0175317. Epub 2017 Apr 6.

Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina, United States of America.

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).
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175317PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5383425PMC
September 2017

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

Acta Crystallogr F Struct Biol Commun 2016 10 22;72(Pt 10):782-787. Epub 2016 Sep 22.

Department of Molecular and Structural Biochemistry, North Carolina State University, 26 Polk Hall, Raleigh, NC 27695, USA.

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.
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http://dx.doi.org/10.1107/S2053230X16014278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053164PMC
October 2016

Structure of the FANCI-FANCD2 complex: insights into the Fanconi anemia DNA repair pathway.

Science 2011 Jul;333(6040):312-6

Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the ~300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each protein has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.
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http://dx.doi.org/10.1126/science.1205805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3310437PMC
July 2011

Crystal structure of inhibitor of κB kinase β.

Nature 2011 Apr 20;472(7343):325-30. Epub 2011 Mar 20.

Department of Biochemistry, Weill Cornell Medical College, New York, New York 10021, USA.

Inhibitor of κB (IκB) kinase (IKK) phosphorylates IκB proteins, leading to their degradation and the liberation of nuclear factor κB for gene transcription. Here we report the crystal structure of IKKβ in complex with an inhibitor, at a resolution of 3.6 Å. The structure reveals a trimodular architecture comprising the kinase domain, a ubiquitin-like domain (ULD) and an elongated, α-helical scaffold/dimerization domain (SDD). Unexpectedly, the predicted leucine zipper and helix-loop-helix motifs do not form these structures but are part of the SDD. The ULD and SDD mediate a critical interaction with IκBα that restricts substrate specificity, and the ULD is also required for catalytic activity. The SDD mediates IKKβ dimerization, but dimerization per se is not important for maintaining IKKβ activity and instead is required for IKKβ activation. Other IKK family members, IKKα, TBK1 and IKK-i, may have a similar trimodular architecture and function.
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http://dx.doi.org/10.1038/nature09853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3081413PMC
April 2011

Structure of a beta-TrCP1-Skp1-beta-catenin complex: destruction motif binding and lysine specificity of the SCF(beta-TrCP1) ubiquitin ligase.

Mol Cell 2003 Jun;11(6):1445-56

Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

The SCF ubiquitin ligases catalyze protein ubiquitination in diverse cellular processes. SCFs bind substrates through the interchangeable F box protein subunit, with the >70 human F box proteins allowing the recognition of a wide range of substrates. The F box protein beta-TrCP1 recognizes the doubly phosphorylated DpSGphiXpS destruction motif, present in beta-catenin and IkappaB, and directs the SCF(beta-TrCP1) to ubiquitinate these proteins at specific lysines. The 3.0 A structure of a beta-TrCP1-Skp1-beta-catenin complex reveals the basis of substrate recognition by the beta-TrCP1 WD40 domain. The structure, together with the previous SCF(Skp2) structure, leads to the model of SCF catalyzing ubiquitination by increasing the effective concentration of the substrate lysine at the E2 active site. The model's prediction that the lysine-destruction motif spacing is a determinant of ubiquitination efficiency is confirmed by measuring ubiquitination rates of mutant beta-catenin peptides, solidifying the model and also providing a mechanistic basis for lysine selection.
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http://dx.doi.org/10.1016/s1097-2765(03)00234-xDOI Listing
June 2003

Mutational analyses of the p35-caspase interaction. A bowstring kinetic model of caspase inhibition by p35.

J Biol Chem 2003 Feb 27;278(7):5455-61. Epub 2002 Nov 27.

Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA.

Apoptosis is a highly regulated multistep process for programmed cellular destruction. It is centered on the activation of a group of intracellular cysteine proteases known as caspases. The baculoviral p35 protein effectively blocks apoptosis through its broad spectrum caspase inhibition. It harbors a caspase recognition sequence within a highly protruding reactive site loop (RSL), which gets cleaved by a target caspase before the formation of a tight complex. The crystal structure of the post-cleavage complex between p35 and caspase-8 shows that p35 forms a thioester bond with the active site cysteine of the caspase. The covalent bond is prevented from hydrolysis by the N terminus of p35, which repositions into the active site of the caspase to eliminate solvent accessibility of the catalytic residues. Here, we report mutational analyses of the pre-cleavage and post-cleavage p35/caspase interactions using surface plasmon resonance biosensor measurements, pull-down assays and kinetic inhibition experiments. The experiments identify important structural elements for caspase inhibition by p35, including the strict requirement for a Cys at the N terminus of p35 and the rigidity of the RSL. A bowstring kinetic model for p35 function is derived in which the tension generated in the bowstring system during the pre-cleavage interaction is crucial for the fast post-cleavage conformational changes required for inhibition.
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http://dx.doi.org/10.1074/jbc.M211607200DOI Listing
February 2003