Split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts

We developed a split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts. In this assay, the N- and C-terminal fragments of Renilla reniforms luciferase are translationally fused to bait and prey proteins, respectively. When the proteins interact, split luciferase becomes activated and emits luminescence that can be measured by a microplate luminometer. Split luciferase activity was measured by first transforming protoplasts with a DNA vector in a 96-well plate. DNA vector expressing both bait and prey genes was constructed through two independent in vitro DNA recombinant reactions, Gateway and Cre-loxP. As proof of concept, we detected the protein-protein interactions between the nuclear histones 2A and 2B, as well as between membrane proteins SYP (syntaxin of plant) 51 and SYP61, in Arabidopsis protoplasts.     

Non‐interacting surface solvation and dynamics in protein–protein interactions

Protein–protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure‐based drug design methods, as well as design of de novo binders with preferred interaction properties. At a structural and molecular level, interface and rim regions are not enough to fully account for the energetics of protein–protein binding, even for simple lock‐and‐key rigid binders. As we have recently shown, properties of the global surface might also play a role in protein–protein interactions. Here, we report on molecular dynamics simulations performed to understand solvent effects on protein–protein surfaces. We compare properties of the interface, rim, and non‐interacting surface regions for five different complexes and their free components. Interface and rim residues become, as expected, less mobile upon complexation. However, non‐interacting surface appears more flexible in the complex. Fluctuations of polar residues are always lower compared with charged ones, independent of the protein state. Further, stable water molecules are often observed around polar residues, in contrast to charged ones. Our analysis reveals that (a) upon complexation, the non‐interacting surface can have a direct entropic compensation for the lower interface and rim entropy and (b) the mobility of the first hydration layer, which is linked to the stability of the protein–protein complex, is influenced by the local chemical properties of the surface. These findings corroborate previous hypotheses on the role of the hydration layer in shielding protein–protein complexes from unintended protein–protein interactions. Proteins 2015; 83:445–458. © 2014 Wiley Periodicals, Inc.     

A fluorescent reporter for mapping cellular protein‐protein interactions in time and space

We introduce a fluorescent reporter for monitoring protein–protein interactions in living cells. The method is based on the Split‐Ubiquitin method and uses the ratio of two auto‐fluorescent reporter proteins as signal for interaction (SPLIFF). The mating of two haploid yeast cells initiates the analysis and the interactions are followed online by two‐channel time‐lapse microscopy of the diploid cells during their first cell cycle. Using this approach we could with high spatio‐temporal resolution visualize the differences between the interactions of the microtubule binding protein Stu2p with two of its binding partners, monitor the transient association of a Ran‐GTPase with its receptors at the nuclear pore, and distinguish between protein interactions at the polar cortical domain at different phases of polar growth. These examples further demonstrate that protein–protein interactions identified from large‐scale screens can be effectively followed up by high‐resolution single‐cell analysis.     

Dissecting protein‐protein interactions in proteasome assembly: Implication to its self‐assembly

The 26S proteasome is a multi‐catalytic ATP‐dependent protease complex that recognizes and cleaves damaged or misfolded proteins to maintain cellular homeostasis. The 26S subunit consists of 20S core and 19S regulatory particles. 20S core particle consists of a stack of heptameric alpha and beta subunits. To elucidate the structure‐function relationship, we have dissected protein‐protein interfaces of 20S core particle and analyzed structural and physiochemical properties of intra‐alpha, intra‐beta, inter‐beta, and alpha‐beta interfaces. Furthermore, we have studied the evolutionary conservation of 20S core particle. We find the size of intra‐alpha interfaces is significantly larger and is more hydrophobic compared with other interfaces. Inter‐beta interfaces are well packed, more polar, and have higher salt‐bridge density than other interfaces. In proteasome assembly, residues in beta subunits are better conserved than alpha subunits, while multi‐interface residues are the most conserved. Among all the residues at the interfaces of both alpha and beta subunits, Gly is highly conserved. The largest size of intra‐alpha interfaces complies with the hypothesis that large interfaces form first during the 20S assembly. The tight packing of inter‐beta interfaces makes the core particle impenetrable from outer wall of the cylinder. Comparing the three domains, eukaryotes have large and well‐packed interfaces followed by archaea and bacteria. Our findings provide a structural basis of assembly of 20S core particle in all the three domains of life.     

LuTHy: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ?L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems.     

Split‐Cre recombinase effectively monitors protein–protein interactions in living bacteria

The ability of Cre recombinase to excise genetic material has been used extensively for genome engineering in prokaryotic and eukaryotic cells. Recently, split‐Cre fragments have been described that advance control of recombinase activity in mammalian cells. However, whether these fragments can be utilized for monitoring protein‐protein interactions has not been reported. In this work, we developed a protein‐fragment complementation assay (PCA) based on split‐Cre for monitoring and engineering pairwise protein interactions in living Escherichia coli cells. This required creation of a dual‐fluorescent reporter plasmid that permits visualization of reconstituted Cre recombinase activity by switching from red to green in the presence of an interacting protein pair. The resulting split‐Cre PCA faithfully links cell fluorescence with differences in binding affinity, thereby allowing the facile isolation of high‐affinity binders based on phenotype. Given the resolution of its activity and sensitivity to interactions, our system may prove a viable option for poorly expressed or weakly interacting protein pairs that evade detection in other PCA formats. Based on these findings, we anticipate that our split‐Cre PCA will become a highly complementary and useful new addition to the protein‐protein interaction toolbox.     

Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions

A reversible green fluorogenic protein‐fragment complementation assay was developed based on the crystal structure of UnaG, a recently discovered fluorescent protein. In living mammalian cells, the nonfluorescent fragments complemented and rapidly became fluorescent upon rapamycin‐induced FKBP and Frb protein interaction, and lost fluorescence when the protein interaction was inhibited. This reversible fluorogenic reporter, named uPPI [UnaG‐based protein‐protein interaction (PPI) reporter], uses bilirubin (BR) as the chromophore and requires no exogenous cofactor. BR is an endogenous molecule in mammalian cells and is not fluorescent by itself. uPPI may have many potential applications in visualizing spatiotemporal dynamics of PPIs.     

Optimization of protein–protein docking for predicting Fc–protein interactions

The antibody crystallizable fragment (Fc) is recognized by effector proteins as part of the immune system. Pathogens produce proteins that bind Fc in order to subvert or evade the immune response. The structural characterization of the determinants of Fc–protein association is essential to improve our understanding of the immune system at the molecular level and to develop new therapeutic agents. Furthermore, Fc‐binding peptides and proteins are frequently used to purify therapeutic antibodies. Although several structures of Fc–protein complexes are available, numerous others have not yet been determined. Protein–protein docking could be used to investigate Fc–protein complexes; however, improved approaches are necessary to efficiently model such cases. In this study, a docking‐based structural bioinformatics approach is developed for predicting the structures of Fc–protein complexes. Based on the available set of X‐ray structures of Fc–protein complexes, three regions of the Fc, loosely corresponding to three turns within the structure, were defined as containing the essential features for protein recognition and used as restraints to filter the initial docking search. Rescoring the filtered poses with an optimal scoring strategy provided a success rate of approximately 80% of the test cases examined within the top ranked 20 poses, compared to approximately 20% by the initial unrestrained docking. The developed docking protocol provides a significant improvement over the initial unrestrained docking and will be valuable for predicting the structures of currently undetermined Fc–protein complexes, as well as in the design of peptides and proteins that target Fc.     

Investigation of the pH‐dependence of dye‐doped protein–protein interactions

Proteins can dramatically change their conformation under environmental conditions such as temperature and pH. In this context, Glycoprotein's conformational determination is challenging. This is due to the variety of domains which contain rich chemical characters existing within this complex. Here we demonstrate a new, straightforward and efficient technique that uses the pH‐dependent properties of dyes‐doped Pig Gastric Mucin (PGM) for predicting and controlling protein–protein interaction and conformation. We utilize the PGM as natural host matrix which is capable of dynamically changing its conformational shape and adsorbing hydrophobic and hydrophilic dyes under different pH conditions and investigate and control the fluorescent properties of these composites in solution. It is shown at various pH conditions, a large variety of light emission from these complexes such as red, green and white is obtained. This phenomenon is explained by pH‐dependent protein folding and protein–protein interactions that induce different emission spectra which are mediated and controlled by means of dye–dye interactions and surrounding environment. This process is used to form the technologically challenging white light‐emitting liquid or solid coating for LED devices.     

UPA, a universal protein array system for quantitative detection of protein–protein, protein–DNA, protein–RNA and protein–ligand interactions

Protein–protein interactions have been widely used to study gene expression pathways and may be considered as a new approach to drug discovery. Here I report the development of a universal protein array (UPA) system that provides a sensitive, quantitative, multi-purpose, effective and easy technology to determine not only specific protein–protein interactions, but also specific interactions of proteins with DNA, RNA, ligands and other small chemicals. (i) Since purified proteins are used, the results can be easily interpreted. (ii) UPA can be used multiple times for different targets, making it economically affordable for most laboratories, hospitals and biotechnology companies. (iii) Unlike DNA chips or DNA microarrays, no additional instrumentation is required. (iv) Since the UPA uses active proteins (without denaturation and renaturation), it is more sensitive compared with most existing methods. (v) Because the UPA can analyze hundreds (even thousands on a protein microarray) of proteins in a single experiment, it is a very effective method to screen proteins as drug targets in cancer and other human diseases.     

Cold spots are universal in protein–protein interactions

Proteins interact with each other through binding interfaces that differ greatly in size and physico‐chemical properties. Within the binding interface, a few residues called hot spots contribute the majority of the binding free energy and are hence irreplaceable. In contrast, cold spots are occupied by suboptimal amino acids, providing possibility for affinity enhancement through mutations. In this study, we identify cold spots due to cavities and unfavorable charge interactions in multiple protein–protein interactions (PPIs). For our cold spot analysis, we first use a small affinity database of PPIs with known structures and affinities and then expand our search to nearly 4000 homo‐ and heterodimers in the Protein Data Bank (PDB). We observe that cold spots due to cavities are present in nearly all PPIs unrelated to their binding affinity, while unfavorable charge interactions are relatively rare. We also find that most cold spots are located in the periphery of the binding interface, with high‐affinity complexes showing fewer centrally located colds spots than low‐affinity complexes. A larger number of cold spots is also found in non‐cognate interactions compared to their cognate counterparts. Furthermore, our analysis reveals that cold spots are more frequent in homo‐dimeric complexes compared to hetero‐complexes, likely due to symmetry constraints imposed on sequences of homodimers. Finally, we find that glycines, glutamates, and arginines are the most frequent amino acids appearing at cold spot positions. Our analysis emphasizes the importance of cold spot positions to protein evolution and facilitates protein engineering studies directed at enhancing binding affinity and specificity in a wide range of applications.     

Mass spectrometry-based footprinting of protein–protein interactions

We present a high-resolution mass spectrometric (MS) footprinting method enabling identification of contact amino acids in protein–protein complexes. The method is based on comparing surface topologies of a free protein versus its complex with the binding partner using differential accessibility of small chemical group selective modifying reagents. Subsequent MS analysis reveals the individual amino acids selectively shielded from modification in the protein–protein complex. The current report focuses on probing interactions between full-length HIV-1 integrase and its principal cellular partner lens epithelium-derived growth factor. This method has a generic application and is particularly attractive for studying large protein–protein interactions that are less amenable for crystallographic or NMR analysis.     

A discriminative approach for identifying domain–domain interactions from protein–protein interactions

Protein domains are functional and structural units of proteins. Therefore, identification of domain–domain interactions (DDIs) can provide insight into the biological functions of proteins. In this article, we propose a novel discriminative approach for predicting DDIs based on both protein–protein interactions (PPIs) and the derived information of non‐PPIs. We make a threefold contribution to the work in this area. First, we take into account non‐PPIs explicitly and treat the domain combinations that can discriminate PPIs from non‐PPIs as putative DDIs. Second, DDI identification is formalized as a feature selection problem, in which it tries to find out a minimum set of informative features (i.e., putative DDIs) that discriminate PPIs from non‐PPIs, which is plausible in biology and is able to predict DDIs in a systematic and accurate manner. Third, multidomain combinations including two‐domain combinations are taken into account in the proposed method, where multidomain cooperations may help proteins to interact with each other. Numerical results on several DDI prediction benchmark data sets show that the proposed discriminative method performs comparably well with other top algorithms with respect to overall performance, and outperforms other methods in terms of precision. The PPI data sets used for prediction of DDIs and prediction results can be found at http://csb.shu.edu.cn/dipd . Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Peptidic modulators of protein‐protein interactions: Progress and challenges in computational design

With the decline in productivity of drug‐development efforts, novel approaches to rational drug design are being introduced and developed. Naturally occurring and synthetic peptides are emerging as novel promising compounds that can specifically and efficiently modulate signaling pathways in vitro and in vivo. We describe sequence‐based approaches that use peptides to mimic proteins in order to inhibit the interaction of the mimicked protein with its partners. We then discuss a structure‐based approach, in which protein‐peptide complex structures are used to rationally design and optimize peptidic inhibitors. We survey flexible peptide docking techniques and discuss current challenges and future directions in the rational design of peptidic inhibitors. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 505–513, 2009. This article was originally published online as an accepted preprint. The “Published Online”date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com