Modeling EphB4-EphrinB2 protein–protein interaction using flexible docking of a short linear motif

Background

Many protein–protein interactions are mediated by a short linear motif. Usually, amino acid sequences of those motifs are known or can be predicted. It is much harder to experimentally characterize or predict their structure in the bound form. In this work, we test a possibility of using flexible docking of a short linear motif to predict the interaction interface of the EphB4-EphrinB2 complex (a system extensively studied for its significance in tumor progression).

Methods

In the modeling, we only use knowledge about the motif sequence and experimental structures of EphB4-EphrinB2 complex partners. The proposed protocol enables efficient modeling of significant conformational changes in the short linear motif fragment during molecular docking simulation. For the docking simulations, we use the CABS-dock method for docking fully flexible peptides to flexible protein receptors (available as a server at http://biocomp.chem.uw.edu.pl/CABSdock/). Based on the docking result, the protein–protein complex is reconstructed and refined.

Results

Using this novel protocol, we obtained an accurate EphB4-EphrinB2 interaction model.

Conclusions

The results show that the CABS-dock method may be useful as the primary docking tool in specific protein–protein docking cases similar to EphB4-EphrinB2 complex—that is, where a short linear motif fragment can be identified.

     

Functional dissection of an intrinsically disordered protein: Understanding the roles of different domains of Knr4 protein in protein�Cprotein interactions

Knr4, recently characterized as an intrinsically disordered Saccharomyces cerevisiae protein, participates in cell wall formation and cell cycle regulation. It is constituted of a functional central globular core flanked by a poorly structured N‐terminal and large natively unfolded C‐terminal domains. Up to now, about 30 different proteins have been reported to physically interact with Knr4. Here, we used an in vivo two‐hybrid system approach and an in vitro surface plasmon resonance (BIAcore) technique to compare the interaction level of different Knr4 deletion variants with given protein partners. We demonstrate the indispensability of the N‐terminal domain of Knr4 for the interactions. On the other hand, presence of the unstructured C‐terminal domain has a negative effect on the interaction strength. In protein interactions networks, the most highly connected proteins or “hubs” are significantly enriched in unstructured regions, and among them the transient hub proteins contain the largest and most highly flexible regions. The results presented here of our analysis of Knr4 protein suggest that these large disordered regions are not always involved in promoting the protein–protein interactions of hub proteins, but in some cases, might rather inhibit them. We propose that this type of regions could prevent unspecific protein interactions, or ensure the correct timing of occurrence of transient interactions, which may be of crucial importance for different signaling and regulation processes.     

Adrenergic regulation of HCN4 channel requires protein association with β2-adrenergic receptor

β(1)- and β(2)-adrenergic receptors utilize different signaling mechanisms to control cardiac function. Recent studies demonstrated that β(2)-adrenergic receptors (β(2)ARs) colocalize with some ion channels that are critical for proper cardiac function. Here, we demonstrate that β(2)ARs form protein complexes with the pacemaker HCN4 channel, as well as with other subtypes of HCN channels. The adrenergic receptor-binding site was identified at a proximal region of the N-terminal tail of the HCN4 channel. A synthetic peptide derived from the β(2)AR-binding domain of the HCN4 channel disrupted interaction between HCN4 and β(2)AR. In addition, treatment with this peptide prevented adrenergic augmentation of pacemaker currents and spontaneous contraction rates but did not affect adrenergic regulation of voltage-gated calcium currents. These results suggest that the ion channel-receptor complex is a critical mechanism in ion channel regulation.     

Assessment and significance of protein–protein interactions during development of protein biopharmaceuticals

Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein–protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein–protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibody–antigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.     

Nuclear localization of Sindbis virus nonstructural protein nsP2

In early infection, approximately 10% of nonstructural protein nsP2 of Sindbis virus was transported into the nuclei of virus-infected BHK-21 cells. Nuclear asP2 was dominantly associated with nuclear matrix. During the course of infection, increasing amounts of nsP2 accumulated in the nuclear fraction. A prominent accumulation of nuclear nsP2 occurred early in infection, from 1 h to 3 h postinfection. Meanwhile. a weak NTPase activity was found to be associated with the immunocomplexed nsP2. Nuclear localization of nsP2 and its possible role were diseussed in relation to the inhibition of host macromolecular synthesis.     

Identification of pseudo ‘phosphothreonyl-specific’ protein phosphatase T with a fraction of polycation-stimulated protein phosphatase 2A

Protein phosphatase T from rat liver, so termed due to its activity toward [³²P-Thr]casein and its marked preference for the phosphopeptide Arg-Arg-Ala-Thr(P)-Val-Ala over its phosphoseryl derivative (Donella Deana, A., Marchiori, F., Meggio, F. and Pinna, L.A. (1982) J. Biol. Chem. 257, 8565–8568), is shown here to belong to the family of type 2A protein phosphatase according to Cohen's nomenclature (Ingebritsen, T.S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255–261). In particular, protein phosphatase T is endowed with phosphorylase phosphatase activity that is stimulated by protamine, histone H1 and heparin, it is inhibited by spermine, it does not bind to heparin-Sepharose and it readily dephosphorylates the phosphopeptide Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser reproducing the phosphorylation site of the α-subunit of phosphorylase kinase. The M_r of protein phosphatase T determined by gel filtration under non-denaturating conditions is about 150 kDa and its activity ratio toward histone H1 phosphorylated by protein kinase C versus histone H1 phosphorylated by cAMP-dependent protein kinase is unusually high. Some properties of protein phosphatase T, such as its weak binding to DEAE-cellulose and its high stimulation by protamine as compared to a relatively poor stimulation by histone H1, suggest that it may be similar to subtype 2A_o of protein phosphatase 2A.     

Discovery of a small-molecule inhibitor and cellular probe of Keap1–Nrf2 protein–protein interaction

A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1–Nrf2 protein–protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure–activity relationships support its use as a lead for our ongoing optimization     

Accuracy modulating mutations of the ribosomal protein S4-S5 interface do not necessarily destabilize the rps4-rps5 protein–protein interaction

During the process of translation, an aminoacyl tRNA is selected in the A site of the decoding center of the small subunit based on the correct codon–anticodon base pairing. Though selection is usually accurate, mutations in the ribosomal RNA and proteins and the presence of some antibiotics like streptomycin alter translational accuracy. Recent crystallographic structures of the ribosome suggest that cognate tRNAs induce a “closed conformation” of the small subunit that stabilizes the codon–anticodon interactions at the A site. During formation of the closed conformation, the protein interface between rpS4 and rpS5 is broken while new contacts form with rpS12. Mutations in rpS12 confer streptomycin resistance or dependence and show a hyperaccurate phenotype. Mutations reversing streptomycin dependence affect rpS4 and rpS5. The canonical rpS4 and rpS5 streptomycin independent mutations increase translational errors and were called ribosomal ambiguity mutations (ram). The mutations in these proteins are proposed to affect formation of the closed complex by breaking the rpS4-rpS5 interface, which reduces the cost of domain closure and thus increases translational errors. We used a yeast two-hybrid system to study the interactions between the small subunit ribosomal proteins rpS4 and rpS5 and to test the effect of ram mutations on the stability of the interface. We found no correlation between ram phenotype and disruption of the interface.     

PCS-based structure determination of protein–protein complexes

A simple and fast nuclear magnetic resonance method for docking proteins using pseudo-contact shift (PCS) and ¹H^N/¹⁵N chemical shift perturbation is presented. PCS is induced by a paramagnetic lanthanide ion that is attached to a target protein using a lanthanide binding peptide tag anchored at two points. PCS provides long-range (~40 Å) distance and angular restraints between the lanthanide ion and the observed nuclei, while the ¹H^N/¹⁵N chemical shift perturbation data provide loose contact-surface information. The usefulness of this method was demonstrated through the structure determination of the p62 PB1-PB1 complex, which forms a front-to-back 20 kDa homo-oligomer. As p62 PB1 does not intrinsically bind metal ions, the lanthanide binding peptide tag was attached to one subunit of the dimer at two anchoring points. Each monomer was treated as a rigid body and was docked based on the backbone PCS and backbone chemical shift perturbation data. Unlike NOE-based structural determination, this method only requires resonance assignments of the backbone ¹H^N/¹⁵N signals and the PCS data obtained from several sets of two-dimensional ¹⁵N-heteronuclear single quantum coherence spectra, thus facilitating rapid structure determination of the protein–protein complex.     

Inhibition of protein–protein interaction of HER2–EGFR and HER2–HER3 by a rationally designed peptidomimetic

Protein–protein interactions (PPI) play a crucial role in many biological processes and modulation of PPI using small molecules to target hot spots has therapeutic value. As a model system we will use PPI of human epidermal growth factor receptors (EGFRs). Among the four EGFRs, EGFR–HER2 and HER2–HER3 are well known in cancer. We have designed a small molecule that is targeted to modulate HER2-mediated signaling. Our approach is novel because the small molecule designed disrupts dimerization not only of EGFR–HER2, but also of HER2–HER3. In the present study we have shown, using surface plasmon resonance analysis, that a peptidomimetic, compound 5, binds specifically to HER2 protein extracellular domain and disrupts the dimerization of EGFRs. To evaluate the effect of compound 5 on HER2 signaling in vitro, Western blot and PathHunter assays were used. Results indicated that compound 5 inhibits the phosphorylation of HER2 kinase domain and inhibits the heterodimerization in a dose-dependent manner. Molecular modeling methods were used to model the PPI of HER2–HER3 heterodimer.     

Monoubiquitination promotes calpain cleavage of the protein phosphatase 2A (PP2A) regulatory subunit α4, altering PP2A stability and microtubule-associated protein phosphorylation

Multiple neurodegenerative disorders are linked to aberrant phosphorylation of microtubule-associated proteins (MAPs). Protein phosphatase 2A (PP2A) is the major MAP phosphatase; however, little is known about its regulation at microtubules. α4 binds the PP2A catalytic subunit (PP2Ac) and the microtubule-associated E3 ubiquitin ligase MID1, and through unknown mechanisms can both reduce and enhance PP2Ac stability. We show MID1-dependent monoubiquitination of α4 triggers calpain-mediated cleavage and switches α4's activity from protective to destructive, resulting in increased Tau phosphorylation. This regulatory mechanism appears important in MAP-dependent pathologies as levels of cleaved α4 are decreased in Opitz syndrome and increased in Alzheimer disease, disorders characterized by MAP hypophosphorylation and hyperphosphorylation, respectively. These findings indicate that regulated inter-domain cleavage controls the dual functions of α4, and dysregulation of α4 cleavage may contribute to Opitz syndrome and Alzheimer disease.     

Pyruvate kinase M2 in chronic inflammations: a potpourri of crucial protein–protein interactions

Cell Biology and Toxicology - Chronic inflammation (CI) is a primary contributing factor involved in multiple diseases like cancer, stroke, diabetes, Alzheimer’s disease, allergy, asthma,...     

Selective blockade of trypanosomatid protein synthesis by a recombinant antibody anti-Trypanosoma cruzi P2β protein

The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope.     

Two-hybrid-based analysis of protein–protein interactions of the yeast multidrug resistance protein, Pdr5p

The ATP-binding cassette (ABC) transporters are a large family of proteins responsible for the translocation of a variety of compounds across the membranes of both prokaryotes and eukaryotes. The inter-protein and intra-protein interactions in these traffic ATPases are still only poorly understood. In the present study we describe, for the first time, an extensive yeast two-hybrid (Y2H)-based analysis of the interactions of the cytoplasmic loops of the yeast pleiotropic drug resistance (Pdr) protein, Pdr5p, an ABC transporter of Saccharomyces cerevisiae. Four of the major cytosolic loops that have been predicted for this protein [including the two nucleotide-binding domain (NBD)-containing loops and the cytosolic C-terminal region] were subjected to an extensive inter-domain interaction study in addition to being used as baits to identify potential interacting proteins within the cell using the Y2H system. Results of these studies have revealed that the first cytosolic loop (CL1) – containing the first NBD domain – and also the C-terminal region of Pdr5p interact with several candidate proteins. The possibility of an interaction between the CL1 loops of two neighboring Pdr5p molecules was also indicated, which could possibly have implications for dimerization of this protein. Electronic Publication     

The PP4R1 subunit of protein phosphatase PP4 targets TRAF2 and TRAF6 to mediate inhibition of NF-κB activation

TRAFs constitute a family of proteins that have been implicated in signal transduction by immunomodulatory cellular receptors and viral proteins. TRAF2 and TRAF6 have an E3-ubiquitin ligase activity, which is dependent on the integrity of their RING finger domain and it has been associated with their ability to activate the NF-κB and AP1 signaling pathways. A yeast two-hybrid screen with TRAF2 as bait, identified the regulatory subunit PP4R1 of protein phosphatase PP4 as a TRAF2-interacting protein. The interaction of TRAF2 with PP4R1 depended on the integrity of the RING finger domain of TRAF2. PP4R1 could interact also with the TRAF2-related factor TRAF6 in a RING domain-dependent manner. Exogenous expression of PP4R1 inhibited NF-κB activation by TRAF2, TRAF6, TNF and the Epstein–Barr virus oncoprotein LMP1. In addition, expression of PP4R1 downregulated IL8 induction by LMP1, whereas downregulation of PP4R1 by RNA interference enhanced the induction of IL8 by LMP1 and TNF. PP4R1 could mediate the dephosphorylation of TRAF2 Ser11, which has been previously implicated in TRAF2-mediated activation of NF-κB. Finally, PP4R1 could inhibit TRAF6 polyubiquitination, suggesting an interference with the E3 ubiquitin ligase activity of TRAF6. Taken together, our data identify a novel mechanism of NF-κB pathway inhibition which is mediated by PP4R1-dependent targeting of specific TRAF molecules.     

Identification of a disruptor of the MDM2-p53 protein–protein interaction facilitated by high-throughput in silico docking

NSC 333003 has been identified from the NCI Diversity Set as an inhibitor of the MDM2-p53 protein–protein interaction by in silico docking (virtual screening). Its potency and chemical characteristics render it well suited for lead optimization studies that can result in more potent analogs with improved drug-like properties. Its synthesis was achieved using an acid catalyzed condensation reaction from commercially available benzothiazole hydrazine and pyridyl phenyl ketone in refluxing methanol. Stereochemical implications for this compound are described.