Protein-protein Interfaces Research Articles

Channeling of cAMP in PDE-PKA Complexes Promotes Signal Adaptation

Spatiotemporal control of the cAMP signaling pathway is governed by both hormonal stimulation of cAMP generation by adenylyl cyclases (activation phase) and cAMP hydrolysis by phosphodiesterases (PDEs) (termination phase). The termination phase is initiated by PDEs actively targeting the protein kinase A (PKA) R-subunit through formation of a PDE-PKAR-cyclic adenosine monophosphate (cAMP) complex (the termination complex). Our results using PDE8 as a model PDE, reveal that PDEs mediate active hydrolysis of cAMP bound to its receptor RIα by enhancing the enzymatic activity. This accelerated cAMP turnover occurs via formation of a stable PDE8-RIα complex, where the protein-protein interface forms peripheral contacts and the central ligand cements this ternary interaction. The basis for enhanced catalysis is active translocation of cAMP from its binding site on RIα to the hydrolysis site on PDE8 through direct “channeling.” Our results reveal cAMP channeling in the PDE8-RIα complex and a molecular description of how this channel facilitates processive hydrolysis of unbound cAMP. Thus, unbound cAMP maintains the PDE8-RIα complex while being hydrolyzed, revealing an undiscovered mode for amplification of PKA activity by cAMP-mediated sequestration of the R-subunit by PDEs. This novel regulatory mode explains the paradox of cAMP signal amplification by accelerated PDE-mediated cAMP turnover. This highlights how target effector proteins of small-molecule ligands can promote enzyme-mediated ligand hydrolysis by scaffolding effects. Enhanced activity of the PDE8-RIα complex facilitates robust desensitization, allowing the cell to respond to dynamic levels of cAMP rather than steady-state levels. The PDE8-RIα complex represents a new class of PDE-based complexes for specific drug discovery targeting the cAMP signaling pathway.

ProteinsPlus: a web portal for structure analysis of macromolecules.

With currently more than 126 000 publicly available structures and an increasing growth rate, the Protein Data Bank constitutes a rich data source for structure-driven research in fields like drug discovery, crop science and biotechnology in general. Typical workflows in these areas involve manifold computational tools for the analysis and prediction of molecular functions. Here, we present the ProteinsPlus web server that offers a unified easy-to-use interface to a broad range of tools for the early phase of structure-based molecular modeling. This includes solutions for commonly required pre-processing tasks like structure quality assessment (EDIA), hydrogen placement (Protoss) and the search for alternative conformations (SIENA). Beyond that, it also addresses frequent problems as the generation of 2D-interaction diagrams (PoseView), protein–protein interface classification (HyPPI) as well as automatic pocket detection and druggablity assessment (DoGSiteScorer). The unified ProteinsPlus interface covering all featured approaches provides various facilities for intuitive input and result visualization, case-specific parameterization and download options for further processing. Moreover, its generalized workflow allows the user a quick familiarization with the different tools. ProteinsPlus also stores the calculated results temporarily for future request and thus facilitates convenient result communication and re-access. The server is freely available at http://proteins.plus.

Predicting the Initial Steps of Salt-Stable Cowpea Chlorotic Mottle Virus Capsid Assembly with Atomistic Force Fields.

Computational prediction of native protein-protein interfaces still remains a challenging task. In virus capsids, each protein unit is in contact with copies of itself through several interfaces. The relative strengths of the different contacts affect the dynamics of the assembly, especially if the process is hierarchical. We investigate the dimerization of the salt-stable cowpea chlorotic mottle virus (CCMV) capsid protein using a combination of different computational tools. The best predictions of dimer configurations provided by blind docking with ZDOCK are rescored using geometry optimization with the Amber and Rosetta force fields. We also evaluate the relative stabilities of the three main interfaces present in the icosahedral capsid using locally restricted docking with Rosetta. Both the rescoring and locally restricted docking results report a particularly stable protein-protein interface, which is the most likely intermediate during the first stage of the hierarchical capsid assembly. The blind docking results rescored with both Amber and Rosetta yield docking funnels, i.e., three or more near-native structures among the top five predictions. The results support experimental observations on in vitro assembly of CCMV capsids. The cross-validation of the results suggests that energy-landscape-based methods with biomolecular force fields have the potential to improve existing docking procedures.

Inhibition of HIV-1 Maturation via Small-Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein.

The human immunodeficiency virus type 1 (HIV-1) capsid protein is an attractive therapeutic target, owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsids to HIV-1 infectivity. To date, small-molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here, we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, Boehringer-Ingelheim compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant cross-links in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle.IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here, we show that one such compound, compound 1, interferes with assembly of the conical viral capsid during virion maturation and results in perturbations at a specific protein-protein interface in the capsid lattice. We also identify and characterize a mutation in the capsid protein that confers resistance to the inhibitor. This study reveals a novel mechanism by which a capsid-targeting small molecule can inhibit HIV-1 replication.

Characterizing the Hot Spots Involved in RON-MSPβ Complex Formation Using In Silico Alanine Scanning Mutagenesis and Molecular Dynamics Simulation.

Purpose: Implication of protein-protein interactions (PPIs) in development of many diseases such as cancer makes them attractive for therapeutic intervention and rational drug design. RON (Recepteur d’Origine Nantais) tyrosine kinase receptor has gained considerable attention as promising target in cancer therapy. The activation of RON via its ligand, macrophage stimulation protein (MSP) is the most common mechanism of activation for this receptor. The aim of the current study was to perform in silico alanine scanning mutagenesis and to calculate binding energy for prediction of hot spots in protein-protein interface between RON and MSPβ chain (MSPβ). Methods: In this work the residues at the interface of RON-MSPβ complex were mutated to alanine and then molecular dynamics simulation was used to calculate binding free energy. Results: The results revealed that Gln193, Arg220, Glu287, Pro288, Glu289, and His424 residues from RON and Arg521, His528, Ser565, Glu658, and Arg683 from MSPβ may play important roles in protein-protein interaction between RON and MSP. Conclusion: Identification of these RON hot spots is important in designing anti-RON drugs when the aim is to disrupt RON-MSP interaction. In the same way, the acquired information regarding the critical amino acids of MSPβ can be used in the process of rational drug design for developing MSP antagonizing agents, the development of novel MSP mimicking peptides where inhibition of RON activation is required, and the design of experimental site directed mutagenesis studies.

Protein-Protein Interaction Interface Database Development based on Protein Network

Protein-Protein Interaction Interface Database Development based on Protein Network

Abstract A24: Translation control of tumor metastasis via FSTL1 binding with SPP1/OPN to modulated cytoskeleton remodeling

Abstract Follistatin-like protein 1 (FSTL1) plays a critical role in lung organogenesis. However, the prognostic significance and functional consequence of FSTL1 downregulation remains unclear in lung cancer. Here we show that the reduced levels of FSTL1 are widely detected in tumors compared to normal tissues and reflect a poor clinical outcome in non-small cell lung cancer, especially lung adenocarcinoma. Moreover, we found that FSTL1 expression negatively correlates with the metastatic potential of lung cancer cells in vitro and in vivo. Whereas the antibody-based neutralization of extracellular FSTL1 enhances the cellular migration/invasion ability, the inclusion of recombinant FSTL1 protein diminishes the metastatic capacity of lung cancer cells in vitro and in vivo. Notably, the administration of FSTL1 was capable of effectively preventing the metastatic progression of lung cancer cells in an orthotropic animal model. The in vitro protein-protein interaction (PPI) assay demonstrated that FSTL1 directly binds to the pro-form of secreted phosphoprotein 1 (SPP1), also named osteopontin (OPN), and thereby restrains the proteolytic activation of SPP1/OPN, which leads to the inactivation of integrin/CD44-associated signaling cascades and actin cytoskeleton. Runx2 and osterix transcription factors are required for the expression of SPP1/OPN. Thus, FSTL1 compromised the SPP1/OPN related translation control activity through PPI mechanism to reverse the phenotype in lung cancer. The signature of combining low-level FSTL1 with high-level SPP1 predicts a worse prognosis in lung cancer patients. This study is the first to document the PPI between FSTL1 and SPP1/OPN and our findings provide a novel and effective strategy to combat SPP1/OPN-driven metastatic cancers with FSTL1 downregulation. Citation Format: Jean Chiou, Yuan-Feng Lin, Michael Hsiao. Translation control of tumor metastasis via FSTL1 binding with SPP1/OPN to modulated cytoskeleton remodeling. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A24.

Ligand-induced allostery in the interaction of the Pseudomonas aeruginosa heme binding protein with heme oxygenase

A heme-dependent conformational rearrangement of the C-terminal domain of heme binding protein (PhuS) is required for interaction with the iron-regulated heme oxygenase (HemO). Herein, we further investigate the underlying mechanism of this conformational rearrangement and its implications for heme transfer via site-directed mutagenesis, resonance Raman (RR), hydrogen-deuterium exchange MS (HDX-MS) methods, and molecular dynamics (MD). HDX-MS revealed that the apo-PhuS C-terminal α6/α7/α8-helices are largely unstructured, whereas the apo-PhuS H212R variant showed an increase in structure within these regions. The increased rate of heme association with apo-PhuS H212R compared with the WT and lack of a detectable five-coordinate high-spin (5cHS) heme intermediate are consistent with a more folded and less dynamic C-terminal domain. HDX-MS and MD of holo-PhuS indicate an overall reduction in molecular flexibility throughout the protein, with significant structural rearrangement and protection of the heme binding pocket. We observed slow cooperative unfolding/folding events within the C-terminal helices of holo-PhuS and the N-terminal α1/α2-helices that are dampened or eliminated in the holo-PhuS H212R variant. Chemical cross-linking and MALDI-TOF MS mapped these same regions to the PhuS:HemO protein-protein interface. We previously proposed that the protein-protein interaction induces conformational rearrangement, promoting a ligand switch from His-209 to His-212 and triggering heme release to HemO. The reduced conformational freedom of holo-PhuS H212R combined with the increase in entropy and decrease in heme transfer on interaction with HemO further support this model. This study provides significant insight into the role of protein dynamics in heme binding and release in bacterial heme transport proteins.

Systematic substitutions at BLIP position 50 result in changes in binding specificity for class A \u03b2-lactamases

BackgroundThe production of β-lactamases by bacteria is the most common mechanism of resistance to the widely prescribed β-lactam antibiotics. β-lactamase inhibitory protein (BLIP) competitively inhibits class A β-lactamases via two binding loops that occlude the active site. It has been shown that BLIP Tyr50 is a specificity determinant in that substitutions at this position result in large differential changes in the relative affinity of BLIP for class A β-lactamases.ResultsIn this study, the effect of systematic substitutions at BLIP position 50 on binding to class A β-lactamases was examined to further explore the role of BLIP Tyr50 in modulating specificity. The results indicate the sequence requirements at position 50 are widely different depending on the target β-lactamase. Stringent sequence requirements were observed at Tyr50 for binding Bacillus anthracis Bla1 while moderate requirements for binding TEM-1 and relaxed requirements for binding KPC-2 β-lactamase were seen. These findings cannot be easily rationalized based on the β-lactamase residues in direct contact with BLIP Tyr50 since they are identical for Bla1 and KPC-2 suggesting that differences in the BLIP-β-lactamase interface outside the local environment of Tyr50 influence the effect of substitutions.ConclusionsResults from this study and previous studies suggest that substitutions at BLIP Tyr50 may induce changes at the interface outside its local environment and point to the complexity of predicting the impact of substitutions at a protein-protein interaction interface.

A Luciferase-fragment Complementation Assay to Detect Lipid Droplet-associated Protein-Protein Interactions

A critical challenge for all organisms is to carefully control the amount of lipids they store. An important node for this regulation is the protein coat present at the surface of lipid droplets (LDs), the intracellular organelles dedicated to lipid storage. Only limited aspects of this regulation are understood so far. For the probably best characterized case, the regulation of lipolysis in mammals, some of the major protein players have been identified, and it has been established that this process crucially depends on an orchestrated set of protein-protein interactions. Proteomic analysis has revealed that LDs are associated with dozens, if not hundreds, of different proteins, most of them poorly characterized, with even fewer data regarding which of them might physically interact. To comprehensively understand the mechanism of lipid storage regulation, it will likely be essential to define the interactome of LD-associated proteins.Previous studies of such interactions were hampered by technical limitations. Therefore, we have developed a split-luciferase based protein-protein interaction assay and test for interactions among 47 proteins from Drosophila and from mouse. We confirmed previously described interactions and identified many new ones. In 1561 complementation tests, we assayed for interactions among 487 protein pairs of which 92 (19%) resulted in a successful luciferase complementation. These results suggest that a prominent fraction of the LD-associated proteome participates in protein-protein interactions.In targeted experiments, we analyzed the two proteins Jabba and CG9186 in greater detail. Jabba mediates the sequestration of histones to LDs. We successfully applied our split luciferase complementation assay to learn more about this function as we were e.g. able to map the interaction between Jabba and histones. For CG9186, expression levels affect the positioning of LDs. Here, we reveal the ubiquitination of CG9186, and link this posttranslational modification to LD cluster induction.

Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer.

Collagen prolyl 4-hydroxylase (C-P4H), an α2β2 heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The β-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 Å. The dimerization domains from the two α-subunits form a protein-protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/β-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/β-subunits locate at the far ends of the βααβ complex. The PDI/β-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the α-subunits has an important role in achieving the correct PSB-CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.

Protein Folding upon Binding Revealed by Molecular Dynamics Simulation

Macromolecules frequently associate in living cells and tissues, where specific binding interactions based on dynamic conformation regulate molecular localization and activity. Despite the rapidly expanding experimental characterization of the structural proteome, the combinatorial explosion of its interactome provides a strong motivation for the development of new tools to predict macromolecular binding modes. Molecular simulation is one such promising approach, where advances in hardware, software, and parameterization are driving increasingly accurate models of protein folding. Here, we use temperature replica-exchange simulations to study folding at a protein-protein interface and reveal the disorder-to-order transition that is necessary for binding between a G protein-coupled receptor and either a G protein or a regulatory kinase. Specifically, we recapitulate a known G protein binding mode and predict the experimentally uncharacterized binding mode of G protein coupled receptor kinase 2. The macromolecular signaling assembly that we conformationally define has important implications for kinase function and provides new insight into biased signaling from these receptors.

Protein-Protein Complex Structure Prediction using the Solution Theory in the Energy Representation

Proteins conduct their functions through interactions with other molecules. Thus, accurate prediction of protein complex is a key to understand functional mechanisms. We have developed a method to evaluate binding free energy differences of complex models generated by docking prediction using the all-atom molecular dynamics simulation and solution theory in the energy representation. By evaluating binding free energy difference with the method, we previously show that “near-native” models similar to crystal structure are successfully selected as the lowest energy structures in two protein-protein complexes, bovine trypsin with CMTI-1 squash inhibitor and RNase SA with barstar [Takemura, J. Chem. Phys. 2012]. The method requires relatively short MD simulations (2 ns) and can calculate binding free energy difference among several hundreds of complex models. As a protein-protein complex structure prediction method, we combined the evaluation method with CyClus [Omori, Proteins, 2013] that performs fast clustering/reranking using a cylindrical approximation of interface and improves the results of the rigid-body docking.The initial complex models were prepared using the protein-protein rigid body docking program, ZDOCK. The generated complex models were then clustered and reranked using CyClus. Free energy analysis for top 100 or 300 complex models returned by CyClus were conducted using conformational energies, solute entropies, and solvation free energies calculated with the solution theory in the energy representation. Our analysis improved the results of CyClus and the complex models similar to the native structure have the lowest binding free energies, suggesting that this procedure is effective in protein-protein complex structure predictions. We further improved the procedure by placing interface waters into protein-protein interface before free energy evaluations.

Using Unnatural Amino Acids to Probe the Interaction between Tarantula Toxins and Voltage Sensing Domains in KV Channels

Peptide toxins folded into an inhibitor cystine knot (ICK) are found in the venoms of a variety of organisms (spider, scorpion, snakes, etc.), and many of these target the voltage-sensing domains in voltage-activated ion channels to modulate their gating properties. Our previous studies suggest that tarantula toxins partition into the lipid membrane and bind to the S3b-S4 paddle motif within the voltage-sensing domains to allosterically inhibit Kv channels. Alanine scanning mutagenesis studies have identified candidate residues in both toxin and channel that may contribute to forming the protein-protein interface when in complex. To definitively identify protein interaction surfaces between toxin and Kv channel, we employed unnatural amino acids and Strain - Promoted Azide Alkyne Click (SPAAC) chemistry to carry out site-specific crosslinking. Azidohomoalanine (AHA), an analog of methionine, was incorporated in Kv channels expressed in Xenopus laevis oocytes through endogenous methionyl t-RNA synthetase by supplementing excess of AHA in the medium. Simultaneously, guangxitoxin-1E (GxTx-1E) was engineered with spinster cysteine residues to incorporate an azide-reactive dibenzocyclooctyne (DBCO) group using cysteine-maleimide conjugation method. Currently, we are assaying for toxin-channel crosslinks in a functional manner by analyzing the recovery of macroscopic ionic currents and kinetics of toxin dissociation using two-electrode voltage clamp (TEVC) technique.

Refinement of Protein Docking with Atom-Atom Contact Potentials, Backbone Flexibility and Side-Chain Repacking

Protein-protein docking is a challenging task for proteins with large conformational changes upon binding (unbound/bound interface RMSD g (ii) a neighbor-dependent Ramachandran probability distributions for the backbone loops torsional degrees of freedom; and (iii) our library of protein-protein interface side-chain rotamers. The procedure was benchmarked on the Dockground (http://dockground.compbio.ku.edu) X-ray unbound set 4.0, which contains 396 co-crystallized protein-protein complexes and corresponding unbound structures for both proteins. The procedure was evaluated by CAPRI assessment criteria for its ability to refine docking predictions with ligand RMSD < 10 A, classified as incorrect, to the acceptable or better quality category, using only the atom-atom contact potentials and the rotational/translational degrees of freedom.

Borrelia burgdorferi BBI39 Paralogs, Targets of Protective Immunity, Reduce Pathogen Persistence Either in Hosts or in the Vector.

Borrelia burgdorferi genome harbors several paralogous gene families (pgf) that can encode immunogenic proteins of unknown function. Protein-protein interaction assays using a transmission-blocking vaccine candidate, BBA52, as bait identified an interacting partner in spirochetes-a member of pgf 54, annotated as BBI39. We show that BBI39 is a surface-exposed membrane antigen that is immunogenic during spirochete infection, despite the gene being primarily transcribed in the vector with a transient expression in the host only at tick-bite sites. Immunization of rodents with BBI39, or a diverse paralog, BBI36, or their combination impaired pathogen acquisition by the vector, transmission from ticks to hosts, or induction of disease. High-titer BBI39 immunoglobulin G antibodies, which have borreliacidal properties, could be generated through routine subcutaneous or oral immunization, further highlighting use of BBI39 proteins as novel Lyme disease vaccines that can target pathogens in the host or in ticks.

Evolution of Chaperonin Gene Duplication in Stigonematalean Cyanobacteria (Subsection V).

Chaperonins promote protein folding and are known to play a role in the maintenance of cellular stability under stress conditions. The group I bacterial chaperonin complex comprises GroEL, that forms a barrel-like oligomer, and GroES that forms the lid. In most eubacteria the GroES/GroEL chaperonin is encoded by a single-copy bicistronic operon, whereas in cyanobacteria up to three groES/groEL paralogs have been documented. Here we study the evolution and functional diversification of chaperonin paralogs in the heterocystous, multi-seriate filament forming cyanobacterium Chlorogloeopsis fritschii PCC 6912. The genome of C. fritschii encodes two groES/groEL operons (groESL1, groESL1.2) and a monocistronic groEL gene (groEL2). A phylogenetic reconstruction reveals that the groEL2 duplication is as ancient as cyanobacteria, whereas the groESL1.2 duplication occurred at the ancestor of heterocystous cyanobacteria. A comparison of the groEL paralogs transcription levels under different growth conditions shows that they have adapted distinct transcriptional regulation. Our results reveal that groEL1 and groEL1.2 are upregulated during diazotrophic conditions and the localization of their promoter activity points towards a role in heterocyst differentiation. Furthermore, protein–protein interaction assays suggest that paralogs encoded in the two operons assemble into hybrid complexes. The monocistronic encoded GroEL2 is not forming oligomers nor does it interact with the co-chaperonins. Interaction between GroES1.2 and GroEL1.2 could not be documented, suggesting that the groESL1.2 operon does not encode a functional chaperonin complex. Functional complementation experiments in Escherichia coli show that only GroES1/GroEL1 and GroES1/GroEL1.2 can substitute the native operon. In summary, the evolutionary consequences of chaperonin duplication in cyanobacteria include the retention of groESL1 as a housekeeping gene, subfunctionalization of groESL1.2 and neofunctionalization of the monocistronic groEL2 paralog.

BTB-BACK Domain Protein POB1 Suppresses Immune Cell Death by Targeting Ubiquitin E3 ligase PUB17 for Degradation.

Hypersensitive response programmed cell death (HR-PCD) is a critical feature in plant immunity required for pathogen restriction and prevention of disease development. The precise control of this process is paramount to cell survival and an effective immune response. The discovery of new components that function to suppress HR-PCD will be instrumental in understanding the regulation of this fundamental mechanism. Here we report the identification and characterisation of a BTB domain E3 ligase protein, POB1, that functions to suppress HR-PCD triggered by evolutionarily diverse pathogens. Nicotiana benthamiana and tobacco plants with reduced POB1 activity show accelerated HR-PCD whilst those with increased POB1 levels show attenuated HR-PCD. We demonstrate that POB1 dimerization and nuclear localization are vital for its function in HR-PCD suppression. Using protein-protein interaction assays, we identify the Plant U-Box E3 ligase PUB17, a well established positive regulator of plant innate immunity, as a target for POB1-mediated proteasomal degradation. Using confocal imaging and in planta immunoprecipitation assays we show that POB1 interacts with PUB17 in the nucleus and stimulates its degradation. Mutated versions of POB1 that show reduced interaction with PUB17 fail to suppress HR-PCD, indicating that POB1-mediated degradation of PUB17 U-box E3 ligase is an important step for negative regulation of specific immune pathways in plants. Our data reveals a new mechanism for BTB domain proteins in suppressing HR-PCD in plant innate immune responses.

Ultrasensitive Firefly Luminescent Intermediate-Based Protein-Protein Interaction Assay (FlimPIA) Based on the Functional Complementation of Mutant Firefly Luciferases.

We recently developed a protein-protein interaction assay, FlimPIA (Firefly luminescent intermediate-based Protein-protein Interaction Assay) based on the catalytic mechanism of firefly luciferase (Fluc) that can be divided into two half-reactions: the adenylation step and the oxidative luminescent steps. We engineered two mutant Fluc enzymes named "Donor" and "Acceptor" where the oxidative luminescent activity of the Donor is almost eliminated and the adenylation activity of the Acceptor is suppressed. When the Donor and the Acceptor are each fused to one of two interacting partners, and put together to interact, the Donor and the Acceptor come sufficiently close such that the Acceptor can react with the luciferyl-adenylate intermediate (LH2-AMP) produced by the Donor, and thus emit luminescence.FlimPIA can be used in vitro and in cultured cells. Owing to recent improvements, it has several advantages in terms of signal/background ratio, detectable size of interacting partner, and sensitivity over conventional protein-protein interaction assays based on Förster/fluorescence resonance energy transfer and protein-fragment complementation performed in vitro. Here, we describe a protocol to make use of the latest version of FlimPIA which shows even lower background and higher signal than previously described ones.

Detection of Membrane Protein Interactions by Cell-based Tango Assays.

The Tango assay is a protein-protein interaction assay, in which a transcription factor (rTA) is fused to a membrane-bound protein via a linker that contains a cleavage site for TEV protease, whereas a soluble interaction partner is fused to TEV protease (Barnea et al., 2008). Association between the two interaction partners leads to an efficient cleavage of the transcription factor, allowing it to translocate to the nucleus and activate a luciferase reporter gene as measurement of the interactions. In this modified assay, we fused one copy of the membrane-spanning amyloid precursor protein (APP) C99 region to TEV site-rTA (C99-TEV site-rTA) and a second copy to TEV protease (C99-TEV) to analyze intramembrane C99-C99 interaction in live cells.