Showing papers on "Protein–protein interaction published in 2010"
TL;DR: The affinity of the small-molecule inhibitor quercetin to its kinase PKA was determined in buffer and human serum, revealing a 400-fold reduced affinity in serum, which may allow to make more reliable conclusions on protein functionality, and may facilitate more efficient drug development.
Abstract: Protein interactions inside the human body are expected to differ from the situation in vitro. This is crucial when investigating protein functions or developing new drugs. In this study, we present a sample-efficient, free-solution method, termed microscale thermophoresis, that is capable of analysing interactions of proteins or small molecules in biological liquids such as blood serum or cell lysate. The technique is based on the thermophoresis of molecules, which provides information about molecule size, charge and hydration shell. We validated the method using immunologically relevant systems including human interferon gamma and the interaction of calmodulin with calcium. The affinity of the small-molecule inhibitor quercetin to its kinase PKA was determined in buffer and human serum, revealing a 400-fold reduced affinity in serum. This information about the influence of the biological matrix may allow to make more reliable conclusions on protein functionality, and may facilitate more efficient drug development.
915 citations
TL;DR: It is demonstrated that hydrogen bonding and optimized hydrophobic interactions both stabilize the ligands at the target site, and help alter binding affinity and drug efficacy.
Abstract: Background Weak intermolecular interactions such as hydrogen bonding and hydrophobic interactions are key players in stabilizing energetically-favored ligands, in an open conformational environment of protein structures. However, it is still poorly understood how the binding parameters associated with these interactions facilitate a drug-lead to recognize a specific target and improve drugs efficacy. To understand this, comprehensive analysis of hydrophobic interactions, hydrogen bonding and binding affinity have been analyzed at the interface of c-Src and c-Abl kinases and 4-amino substituted 1H-pyrazolo [3, 4-d] pyrimidine compounds. Methodology In-silico docking studies were performed, using Discovery Studio software modules LigandFit, CDOCKER and ZDOCK, to investigate the role of ligand binding affinity at the hydrophobic pocket of c-Src and c-Abl kinase. Hydrophobic and hydrogen bonding interactions of docked molecules were compared using LigPlot program. Furthermore, 3D-QSAR and MFA calculations were scrutinized to quantify the role of weak interactions in binding affinity and drug efficacy. Conclusions The in-silico method has enabled us to reveal that a multi-targeted small molecule binds with low affinity to its respective targets. But its binding affinity can be altered by integrating the conformationally favored functional groups at the active site of the ligand-target interface. Docking studies of 4-amino-substituted molecules at the bioactive cascade of the c-Src and c-Abl have concluded that 3D structural folding at the protein-ligand groove is also a hallmark for molecular recognition of multi-targeted compounds and for predicting their biological activity. The results presented here demonstrate that hydrogen bonding and optimized hydrophobic interactions both stabilize the ligands at the target site, and help alter binding affinity and drug efficacy.
336 citations
TL;DR: It is suggested that CD69 induces an S1P1 conformation that shares some properties of the ligand-bound state, thereby facilitating S1F1 internalization and degradation, and identifying an integral membrane interaction between CD69 and S1p1.
288 citations
TL;DR: LaLauren et al. as discussed by the authors characterized the interaction between synthetic Aβ42 oligomers and the recombinant human prion protein (PrP) using two biophysical techniques: site-directed spin labeling and surface plasmon resonance.
257 citations
TL;DR: A physical and functional interaction between Keap1 and SQSTM1 is demonstrated and an additional layer of regulation in the Keap 1-Nrf2 pathway is revealed.
225 citations
TL;DR: Developing techniques are helping researchers to build the protein interaction networks that underlie all cell functions.
Abstract: Developing techniques are helping researchers to build the protein interaction networks that underlie all cell functions.
222 citations
TL;DR: A mass spectrometry assay is developed for the large-scale identification of in vivo protein-hydrophobic small metabolite interactions in yeast and compounds that bind ergosterol biosynthetic proteins and protein kinases are analyzed.
208 citations
TL;DR: Two major molecular mechanisms underlie SOX-partner factor interactions: cooperative DNA binding; and protein interactions dependent upon DNA binding which elicit a large transactivation potential.
178 citations
175 citations
TL;DR: The findings suggest that the co-chaperone Bag3 might prevent the accumulation of denatured proteins by regulating sHsp activity and by targeting their substrate proteins for degradation.
Abstract: The molecular chaperone HspB8 [Hsp (heat-shock protein) B8] is member of the B-group of Hsps. These proteins bind to unfolded or misfolded proteins and protect them from aggregation. HspB8 has been reported to form a stable molecular complex with the chaperone cohort protein Bag3 (Bcl-2-associated athanogene 3). In the present study we identify the binding regions in HspB8 and Bag3 crucial for their interaction. We present evidence that HspB8 binds to Bag3 through the hydrophobic groove formed by its strands β4 and β8, a region previously known to be responsible for the formation and stability of higher-order oligomers of many sHsps (small Hsps). Moreover, we demonstrate that two conserved IPV (Ile-Pro-Val) motifs in Bag3 mediate its binding to HspB8 and that deletion of these motifs suppresses HspB8 chaperone activity towards mutant Htt43Q (huntingtin exon 1 fragment with 43 CAG repeats). In addition, we show that Bag3 can bind to the molecular chaperone HspB6. The interaction between HspB6 and Bag3 requires the same regions that are involved in the HspB8–Bag3 association and HspB6–Bag3 promotes clearance of aggregated Htt43Q. Our findings suggest that the co-chaperone Bag3 might prevent the accumulation of denatured proteins by regulating sHsp activity and by targeting their substrate proteins for degradation. Interestingly, a mutation in one of Bag3 IPV motifs has recently been associated with the development of severe dominant childhood muscular dystrophy, suggesting a possible important physiological role for HspB–Bag3 complexes in this disease.
172 citations
TL;DR: Results give strong support to the conjecture that the C-terminal region of the protein is not directly involved in the mechanism of aggregation and suggest that binding of NbSyn2 could be a useful probe for the identification of α-synuclein aggregation in vitro and possibly in vivo.
TL;DR: It is shown that the conformational change that allows Bax to insert into the outer mitochondrial membrane is the rate-limiting step in the multistep process of Bax activation, which resolves the paradox of similar structures but opposite functions of Bcl-XL and Bax.
Abstract: The dysregulation of apoptosis is a key step in developing tumours, and mediates resistance to cancer therapy. Many different signals for cell death converge on permeabilization of the outer mitochondrial membrane, which is controlled by the Bcl-2 family of proteins. The importance of this step is becoming increasingly relevant as the first generation of small molecules that inhibit the interaction of Bcl-2 family proteins enters clinical trials as anticancer agents. The Bcl-2 family can be divided into three classes: BH3-only proteins that are activated by various forms of cellular stress, Bax and Bak proteins that mediate mitochondrial membrane permeabilization, and inhibitory proteins such as Bcl-2 and Bcl-XL. The recently proposed embedded together model emphasizes the fact that many of the regulatory interactions between different classes of Bcl-2 family members occur at intracellular membranes, and binding to membranes causes conformational changes in the proteins that dictate functions in a dynamic manner. Within this context, recent results indicate that Bcl-XL functions as a dominant-negative Bax, a concept that resolves the paradox of similar structures but opposite functions of Bcl-XL and Bax. We have also shown that the conformational change that allows Bax to insert into the outer mitochondrial membrane is the rate-limiting step in the multistep process of Bax activation. Nevertheless, investigating the structure of activated Bax or Bak as monomers and as components of the oligomeric structures that mediate membrane permeabilization is the focus of ongoing research (and controversy) at many laboratories worldwide.
TL;DR: The combined analysis of SDPs in interfaces and ligand-binding sites provides a more complete picture of the organization of protein families, constituting the necessary framework for a large scale analysis of the evolution of protein function.
Abstract: The divergence accumulated during the evolution of protein families translates into their internal organization as subfamilies, and it is directly reflected in the characteristic patterns of differentially conserved residues. These specifically conserved positions in protein subfamilies are known as “specificity determining positions” (SDPs). Previous studies have limited their analysis to the study of the relationship between these positions and ligand-binding specificity, demonstrating significant yet limited predictive capacity. We have systematically extended this observation to include the role of differential protein interactions in the segregation of protein subfamilies and explored in detail the structural distribution of SDPs at protein interfaces. Our results show the extensive influence of protein interactions in the evolution of protein families and the widespread association of SDPs with protein interfaces. The combined analysis of SDPs in interfaces and ligand-binding sites provides a more complete picture of the organization of protein families, constituting the necessary framework for a large scale analysis of the evolution of protein function.
TL;DR: This review describes the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of theTAP method.
TL;DR: Full characterization of protein interaction networks of protein complexes and their dynamics in response to various cellular cues will provide essential information for us to understand how protein complexes work together in cells to maintain cell viability and normal homeostasis.
TL;DR: Crystal structures of ternary complexes revealed that the molecules bind to different sites in the interface of the 14–3–3 protein and PMA2, thus explaining the different binding kinetics.
Abstract: Two structurally unrelated small molecules that stabilize the interaction of a 14–3–3 protein with the proton pump PMA2 have been identified. The compounds are selective among different 14–3–3 protein–protein interactions and are active in vivo. Crystal structures of ternary complexes revealed that the molecules bind to different sites in the interface of the 14–3–3 protein and PMA2 (see picture), thus explaining the different binding kinetics.
TL;DR: The crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob is determined, providing an accurate framework for mapping all known ligand binding sites onto the surface ofCD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system.
Abstract: The human membrane cofactor protein (MCP, CD46) is a central component of the innate immune system. CD46 protects autologous cells from complement attack by binding to complement proteins C3b and C4b and serving as a cofactor for their cleavage. Recent data show that CD46 also plays a role in mediating acquired immune responses, and in triggering autophagy. In addition to these physiologic functions, a significant number of pathogens, including select adenoviruses, measles virus, human herpes virus 6 (HHV-6), Streptococci, and Neisseria, use CD46 as a cell attachment receptor. We have determined the crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob. Extracellular CD46 comprises four short consensus repeats (SCR1-SCR4) that form an elongated structure resembling a hockey stick, with a long shaft and a short blade. Domains SCR1, SCR2 and SCR3 are arranged in a nearly linear fashion. Unexpectedly, however, the structure reveals a profound bend between domains SCR3 and SCR4, which has implications for the interactions with ligands as well as the orientation of the protein at the cell surface. This bend can be attributed to an insertion of five hydrophobic residues in a SCR3 surface loop. Residues in this loop have been implicated in interactions with complement, indicating that the bend participates in binding to C3b and C4b. The structure provides an accurate framework for mapping all known ligand binding sites onto the surface of CD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system.
TL;DR: The finding suggests that direct protein interaction between Complexes I/II and STAT3 cannot be required for optimal ATP production, nor can it dramatically modulate oxidative phosphorylation in vivo, andSTAT3 is likely altering mitochondrial function via transcriptional regulation or indirect signaling pathways that warrant further investigation.
TL;DR: A physical association of RTE1 and ETR1 is demonstrated using in vivo and in vitro methods to indicate that a high affinity association of N terminus-based relationship between RTE 1 and E TR1 is important in the regulation of ETR 1.
TL;DR: The SOX protein interactions with partner co-factors, the ability ofSOX proteins to modulate Wnt signalling and the effect of post-translational modification of SOX proteins on their function are reviewed.
TL;DR: The first structure of the DUF283 domain is reported from the Arabidopsis thaliana DCL4, which resembles the structural similarity to the double-stranded RNA-binding domain and is suggested to have a potential functional role in target selection in small RNA processing.
Abstract: Dicer or Dicer-like (DCL) protein is a catalytic component involved in microRNA (miRNA) or small interference RNA (siRNA) processing pathway, whose fragment structures have been partially solved. However, the structure and function of the unique DUF283 domain within dicer is largely unknown. Here we report the first structure of the DUF283 domain from the Arabidopsis thaliana DCL4. The DUF283 domain adopts an alpha-beta-beta-beta-alpha topology and resembles the structural similarity to the double-stranded RNA-binding domain. Notably, the N-terminal alpha helix of DUF283 runs cross over the C-terminal alpha helix orthogonally, therefore, N- and C-termini of DUF283 are in close proximity. Biochemical analysis shows that the DUF283 domain of DCL4 displays weak dsRNA binding affinity and specifically binds to double-stranded RNA-binding domain 1 (dsRBD1) of Arabidopsis DRB4, whereas the DUF283 domain of DCL1 specifically binds to dsRBD2 of Arabidopsis HYL1. These data suggest a potential functional role of the Arabidopsis DUF283 domain in target selection in small RNA processing.
TL;DR: Calculations of the potential of mean force between proteins and protein clusters show that the addition of cholesterol dramatically reduces repulsive lipid-mediated interactions between proteins (protein clusters) with positive mismatch, but does not affect attractive interaction between proteins with negative mismatch.
TL;DR: The authors describe the development of the first high-throughput screening assay for the discovery of inhibitors of methyl-lysine binding proteins that will be used to initiate a full-scale discovery effort for this broad target class, malignant brain tumor domain-containing proteins.
Abstract: The histone code comprises many posttranslational modifications that occur mainly in histone tail peptides. The identity and location of these marks are read by a variety of histone-binding proteins that are emerging as important regulators of cellular differentiation and development and are increasingly being implicated in numerous disease states. The authors describe the development of the first high-throughput screening assay for the discovery of inhibitors of methyl-lysine binding proteins that will be used to initiate a full-scale discovery effort for this broad target class. They focus on the development of an AlphaScreen-based assay for malignant brain tumor (MBT) domain-containing proteins, which bind to the lower methylation states of lysine residues present in histone tail peptides. This assay takes advantage of the avidity of the AlphaScreen beads to clear the hurdle to assay development presented by the low micromolar binding constants of the histone binding proteins for their cognate peptides. The assay is applicable to other families of methyl-lysine binding proteins, and it has the potential to be used in screening efforts toward the discovery of novel small molecules with utility as research tools for cellular reprogramming and ultimately drug discovery.
TL;DR: A model is provided in which WDR20 serves as a stimulatory subunit for preserving and regulating the activity of the subset of the UAF1·USP complexes, which contradicts the previous report that USP12 and USP46 do not regulate the FA pathway.
TL;DR: Get3, Get4, and Get5 in Saccharomyces cerevisiae participate in the insertion of tail-anchored proteins into the endoplasmic reticulum membrane, and formed a tight complex, suggesting that they constitute subunits of a larger complex.
TL;DR: In silico search for TPR proteins in Arabidopsis and rice comprising of at least one three-motif TPR domain with conserved amino acid residues required for Hsp90/Hsp70 binding successfully identified in a genome-wide context CC-TPR proteins with potential to interact with HSp90/ Hsp70, and suggest that the Hsp 90/HSP70 system relies on TPR co-chaperones more than it was previously realized.
Abstract: The essential eukaryotic molecular chaperone Hsp90 operates with the help of different co-chaperones, which regulate its ATPase activity and serve as adaptors to recruit client proteins and other molecular chaperones, such as Hsp70, to the Hsp90 complex Several Hsp90 and Hsp70 co-chaperones contain the tetratricopeptide repeat (TPR) domain, which interacts with the highly conserved EEVD motif at the C-terminal ends of Hsp90 and Hsp70 The acidic side chains in EEVD interact with a subset of basic residues in the TPR binding pocket called a ‘carboxylate clamp’ Since the carboxylate clamp residues are conserved in the TPR domains of known Hsp90/Hsp70 co-chaperones, we carried out an in silico search for TPR proteins in Arabidopsis and rice comprising of at least one three-motif TPR domain with conserved amino acid residues required for Hsp90/Hsp70 binding This approach identified in Arabidopsis a total of 36 carboxylate clamp (CC)-TPR proteins, including 24 novel proteins, with potential to interact with Hsp90/Hsp70 The newly identified CC-TPR proteins in Arabidopsis and rice contain additional protein domains such as ankyrin, SET, octicosapeptide/Phox/Bem1p (Phox/PB1), DnaJ-like, thioredoxin, FBD and F-box, and protein kinase and U-box, indicating varied functions for these proteins To provide proof-of-concept of the newly identified CC-TPR proteins for interaction with Hsp90, we demonstrated interaction of AtTPR1 and AtTPR2 with AtHsp90 in yeast two-hybrid and in vitro pull down assays These findings indicate that the in silico approach used here successfully identified in a genome-wide context CC-TPR proteins with potential to interact with Hsp90/Hsp70, and further suggest that the Hsp90/Hsp70 system relies on TPR co-chaperones more than it was previously realized
TL;DR: Gel-filtration analysis of ethylene receptors solubilized from Arabidopsis membranes demonstrates that the receptors exist as components of high-molecular-mass protein complexes and suggests that different protein complexes may allow for tailoring of the ethylene signal to specific cellular environments and responses.
Abstract: Background
The gaseous plant hormone ethylene is perceived in Arabidopsis thaliana by a five-member receptor family composed of ETR1, ERS1, ETR2, ERS2, and EIN4.
Methodology/Principal Findings
Gel-filtration analysis of ethylene receptors solubilized from Arabidopsis membranes demonstrates that the receptors exist as components of high-molecular-mass protein complexes. The ERS1 protein complex exhibits an ethylene-induced change in size consistent with ligand-mediated nucleation of protein-protein interactions. Deletion analysis supports the participation of multiple domains from ETR1 in formation of the protein complex, and also demonstrates that targeting to and retention of ETR1 at the endoplasmic reticulum only requires the first 147 amino acids of the receptor. A role for disulfide bonds in stabilizing the ETR1 protein complex was demonstrated by use of reducing agents and mutation of Cys4 and Cys6 of ETR1. Expression and analysis of ETR1 in a transgenic yeast system demonstrates the importance of Cys4 and Cys6 of ETR1 in stabilizing the receptor for ethylene binding.
Conclusions/Significance
These data support the participation of ethylene receptors in obligate as well as ligand-dependent non-obligate protein interactions. These data also suggest that different protein complexes may allow for tailoring of the ethylene signal to specific cellular environments and responses.
TL;DR: Protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain–peptide interactions in high throughput using fluorescently labeled synthetic peptides are provided.
Abstract: Protein microarrays provide an efficient way to identify and quantify protein–protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain–peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (KDs) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein–ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein–protein interaction networks.
TL;DR: The results from the free energy decomposition indicate that the intermolecular van der Waals interaction and the nonpolar solvation term provide the driving force for binding process in the PA-PB1 complex.
Abstract: The emergence of the extremely aggressive influenza recently has highlighted the urgent need for new effective treatments. The influenza RNA-dependent RNA polymerase (RdRp) heterotrimer including PA, PB1 and PB2 has crucial roles in viral RNA replication and transcription. The highly conserved PB1 binding site on PA can be considered as a novel potential drug target site. The interaction between PB1 binding site and PA is crucial to many functions of the virus. In this study, to understand the detailed interaction profile and to characterize the binding hot spots in the interactions of the PA-PB1 complex, an 8 ns molecular dynamics simulation of the subunit PA-PB1 combined with MM-PBSA (molecular mechanics Poisson-Boltzmann surface area), MM-GBSA (molecular mechanics generalized Born surface area) computations and virtual alanine scanning were performed. The results from the free energy decomposition indicate that the intermolecular van der Waals interaction and the nonpolar solvation term provide the driving force for binding process. Through the pair interaction analysis and virtual alanine scanning, we identified the binding hot spots of PA and the basic binding motif of PB1. This information can provide some insights for the structure-based RNA-dependent RNA polymerase inhibitors design. The identified binding motif can be used as the starting point for the rational design of small molecules or peptide mimics. This study will also lead to new opportunities toward the development of new generation therapeutic agents exhibiting specificity and low resistance to influenza virus.
TL;DR: Main topics of this report are the localization of candidate membrane proteins in cholesterol-rich microdomains, the issue of specificity of cholesterol- protein interactions, and applications of the various cholesterol probes for these studies.
Abstract: Cholesterol is a major constituent of the plasma membrane in eukaryotic cells. It regulates the physical state of the phospholipid bilayer and is crucially involved in the formation of membrane microdomains. Cholesterol also affects the activity of several membrane proteins, and is the precursor for steroid hormones and bile acids. Here, methods are described that are used to explore the binding and/or interaction of proteins to cholesterol. For this purpose, a variety of cholesterol probes bearing radio-, spin-, photoaffinity- or fluorescent labels are currently available. Examples of proven cholesterol binding molecules are polyene compounds, cholesterol-dependent cytolysins, enzymes accepting cholesterol as substrate, and proteins with cholesterol binding motifs. Main topics of this report are the localization of candidate membrane proteins in cholesterol-rich microdomains, the issue of specificity of cholesterol– protein interactions, and applications of the various cholesterol probes for these studies.