The inhibitor of κB kinase ß (IKKß) phosphorylates IκBα twice in a single binding event through a sequential mechanism.

Phosphorylation of Inhibitor of κB (IκB) proteins by IκB Kinase ß (IKKß) leads to IκB degradation and subsequent activation of nuclear factor κB transcription factors. Of particular interest is the IKKß-catalyzed phosphorylation of IκBα residues Ser32 and Ser36 within a conserved destruction box motif. To investigate the catalytic mechanism of IKKß, we performed pre-steady-state kinetic analysis of the phosphorylation of IκBα protein substrates catalyzed by constitutively active, human IKKß. Phosphorylation of full-length IκBα catalyzed by IKKß was characterized by a fast exponential phase followed by a slower linear phase. The maximum observed rate (kp) of IKKß-catalyzed phosphorylation of IκBα was 0.32 s-1 and the binding affinity of ATP for the IKKß•IκBα complex (Kd) was 12 µM. Substitution of either Ser32 or Ser36 with Ala, Asp, or Cys reduced the amplitude of the exponential phase by approximately 2-fold. Thus, the exponential phase was attributed to phosphorylation of IκBα at Ser32 and Ser36, whereas the slower linear phase was attributed to phosphorylation of other residues. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic amino acid substitution mutant was nearly twice that of WT IκBα and 4-fold faster than any of the other IκBα amino acid substitution mutants, suggesting that phosphorylation of Ser32 increases the phosphorylation rate of Ser36. These conclusions were supported by parallel experiments using GST-IκBα(1-54) fusion protein substrates bearing the first 54 residues of IκBα. Our data suggest a model wherein, IKKß phosphorylates IκBα at Ser32 followed by Ser36 within a single binding event.

Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA.

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.

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

Activation of IκB kinase ß (IKKß) is a central event in the NF-κB-mediated canonical pro-inflammatory pathway. Numerous studies have reported that oligomerization-mediated trans autophosphorylation of IKKß is indispensable for its phosphorylation, leading to its activation and IKKß-mediated phosphorylation of substrates such as IκB proteins. Moreover, IKKß's interaction with the NF-κB essential modifier (NEMO) is necessary for IKKß activation. Interestingly, some viruses encode virulence factors that target IKKß to inhibit NF-κB-mediated antiviral immune responses. One of these factors is the vaccinia viral protein B14, which directly interacts with and inhibits IKKß. Here we mapped the interaction interface on the B14 and IKKß proteins. We observed that B14 binds to the junction of the kinase domain (KD) and scaffold and dimerization domain (SDD) of IKKß. Molecular docking analyses identified key interface residues in both IKKß 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-IKKß interaction sterically hinders direct contact between the kinase domains of IKKß in the IKK complex, containing IKKß, IKKα, and NEMO in human cells. We conclude that binding of B14 to IKKß prevents IKKß 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 IKKß activation for the management of inflammatory disorders.

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

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 Arabidopsis 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.

Crystal structure of inhibitor of κB kinase ß.

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 IKKß 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 IKKß dimerization, but dimerization per se is not important for maintaining IKKß activity and instead is required for IKKß activation. Other IKK family members, IKKα, TBK1 and IKK-i, may have a similar trimodular architecture and function.

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

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.

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 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.

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

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.

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

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).

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

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 IKKß, 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 (Dmax=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.

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

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 Šresolution from a single crystal. The crystal belonged to space group C222 or C2221, 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.

Cloning of DNA Fragments Possessing Promoter Function from Kluyveromyces cicerisporus.

Using three yeast promoter-probe plasmids pSK-kan401, pSK-kan1105, pSK-kan1238 with different reading frames, eight DNA fragments possessing high efficient promoter function have been cloned from K. cicerisporus. Their 3'-DNA sequences have been analyzed. The relative strength of their promoter function was studied by comparing their ability to promote the expression of the reporter gene APHI with that of glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) promoter in K. lactis. The results showed that the insertion fragments in pSK-kan401-41 and pSK-kan1105-51 have stronger promoter function and they are also functional in K. cicerisporus Y179.

Cloning of GAP Gene from K. fragilis.

The K. fragilis CBS 397 gene library was screened with a GAP probe, which was designed according to the homology with S. cerevisiae, K. lactis, K. marxianus GAP gene. One positive clone pG1 containing GAP1 gene was isolated and confirmed by Southern hybridization. The GAP1 gene was partially sequenced. By using a fragment of the clone as a probe, another positive clone pG2 was acquired and also confirmed by Southern hybridization. The GAP2 gene from pG2 was completely sequenced. The upstream sequences of both genes were shown to have promoter activity. The fragment of pG1 could hybridize with three chromosomes of K. fragilis.

[Comparative clinical observation of the short-term bone fusion effect of composite biosynthetic bone and ilium auto-grafting].

OBJECTIVE: To evaluate the short-term bone fusion after implantation of composite biosynthetic bone made of coralline hydroxyapatite composite (CHC) as a substitute for autologous bone grafting. METHODS: A clinical sequential trial was designed and the biosynthetic bone and the patient's ilium were respectively used in the treatment of femoral nonunions and interbody fusion. X-ray examinations were performed 10 weeks after the operations and the bone fusion was graded according to Lane-Sandhu's method. RESULTS: The biosynthetic bone was capable of bone fusion as observed in this experiment. Sequential analysis found that the upper bound was reached when the X-ray examination scores of 15th pairs were transcribed in the chart with 2 pairs of cases dismissed for having the same grade. CONCLUSION: The bone fusion effect of CHC is better than auto-grafting judging from the present observation at 10 weeks after operation.

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

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.

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

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.

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

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.