Identification and mapping of protein-protein interactions by a combination of cross-linking, cleavage, and proteomics

Protein-protein interactions are vital for almost all cellular functions, and many require the formation of multiprotein complexes. Identification of the macroscopic and microscopic protein interactions within these complexes is essential in understanding their mechanisms, both under physiologic as well as pathologic conditions. This review describes the current technology available to investigate interactions between proteins utilizing chemical cross-linking and site-directed cleavage reagents, outlining the necessary steps involved in identifying interacting proteins both in vitro and in vivo. Once interacting proteins are identified, more information about the architecture of the assemblies is necessary. Unique separation techniques coupled with cross-linking and mass spectrometry can now identify specific interaction sites and lead to the development of quaternary structural protein models. Furthermore, combination of these methods with proteomic approaches enables the identification and analysis of complex interactions in vivo. Finally, future directions in cross-linking methodologies are discussed.     

Mapping protein-protein interactions between MutL and MutH by cross-linking

Strand discrimination in Escherichia coli DNA mismatch repair requires the activation of the endonuclease MutH by MutL. There is evidence that MutH binds to the N-terminal domain of MutL in an ATP-dependent manner; however, the interaction sites and the molecular mechanism of MutH activation have not yet been determined. We used a combination of site-directed mutagenesis and site-specific cross-linking to identify protein interaction sites between the proteins MutH and MutL. Unique cysteine residues were introduced in cysteine-free variants of MutH and MutL. The introduced cysteines were modified with the cross-linking reagent 4-maleimidobenzophenone. Photoactivation resulted in cross-links verified by mass spectrometry of some of the single cysteine variants to their respective Cys-free partner proteins. Moreover, we mapped the site of interaction by cross-linking different combinations of single cysteine MutH and MutL variants with thiol-specific homobifunctional cross-linkers of varying length. These results were used to model the MutH.MutL complex and to explain the ATP dependence of this interaction.     

Quantification of protein-protein interactions with chemical cross-linking and mass spectrometry

Chemical cross-linking in combination with mass spectrometry has largely been used to study protein structures and protein-protein interactions. Typically, it is used in a qualitative manner to identify cross-linked sites and provide a low-resolution topological map of the interacting regions of proteins. Here, we investigate the capability of chemical cross-linking to quantify protein-protein interactions using a model system of calmodulin and substrates melittin and mastoparan. Calmodulin is a well-characterized protein which has many substrates. Melittin and mastoparan are two such substrates which bind to calmodulin in 1:1 ratios in the presence of calcium. Both the calmodulin-melittin and calmodulin-mastoparan complexes have had chemical cross-linking strategies successfully applied in the past to investigate topological properties. We utilized an excess of immobilized calmodulin on agarose beads and formed complexes with varying quantities of mastoparan and melittin. Then, we applied disuccinimidyl suberate (DSS) chemical cross-linker, digested and detected cross-links through an LC-MS analytical method. We identified five interpeptide cross-links for calmodulin-melittin and three interpeptide cross-links for calmodulin-mastoparan. Using cross-linking sites of calmodulin-mastoparan, we demonstrated that mastoparan also binds in two orientations to calmodulin. We quantitatively demonstrated that both melittin and mastoparan preferentially bind to calmodulin in a parallel fashion, which is opposite to the preferred binding mode of the majority of known calmodulin binding peptides. We also demonstrated that the relative abundances of cross-linked peptide products quantitatively reflected the abundances of the calmodulin peptide complexes formed.     

Elucidation of protein-protein interactions using chemical cross-linking or label transfer techniques

Understanding the architectures of multiprotein complexes is a central problem in biology. Of the many chemical methods available, label transfer and cross-linking are becoming more popular. Recently, label transfer has been applied to very large protein complexes with great success, and new oxidative methods for protein cross-linking have been developed that are fast and highly efficient. Advances in these techniques should increase the understanding of biological structures and mechanisms.     

Identification of hot-spot residues in protein-protein interactions by computational docking

Background  

The study of protein-protein interactions is becoming increasingly important for biotechnological and therapeutic reasons. We can define two major areas therein: the structural prediction of protein-protein binding mode, and the identification of the relevant residues for the interaction (so called 'hot-spots'). These hot-spot residues have high interest since they are considered one of the possible ways of disrupting a protein-protein interaction. Unfortunately, large-scale experimental measurement of residue contribution to the binding energy, based on alanine-scanning experiments, is costly and thus data is fairly limited. Recent computational approaches for hot-spot prediction have been reported, but they usually require the structure of the complex.     

Identification of novel small molecules that inhibit protein-protein interactions between MAGE and KAP-1

The Class I MAGE proteins are normally expressed only in developing germ cells but are often aberrantly expressed in malignancies, particularly melanoma, making them good therapeutic targets. MAGE proteins promote tumor survival by binding to the RBCC region of KAP-1 and suppressing p53. Although, suppression of MAGE expression, by RNA interference, relieves p53 suppression and inhibits tumor growth, its therapeutic uses are limited by lack of methods for systemic delivery of small interfering RNA. To overcome this barrier, we sought to discover chemical compounds that inhibit binding between MAGE and KAP-1 proteins. Based on previously published effects of MAGE suppression, we developed a strategy for screening a small molecule library based on selective death of MAGE positive cells, activation of p53 and lack of caspase activity. We screened the Maybridge HitFinder library of compounds and eight compounds fulfilled these criteria. Seven of these compounds interfered with co-precipitation of MAGE and KAP-1, and three interfered with binding of MAGE and KAP-1 in a mammalian two hybrid assay. We now report identification of three potential compounds that interfere with MAGE/KAP-1 binding and can be developed as novel chemo-therapeutic agents for treatment of advanced melanoma and other cancers.     

Comprehensive and quantitative mapping of energy landscapes for protein-protein interactions by rapid combinatorial scanning

A novel, quantitative saturation (QS) scanning strategy was developed to obtain a comprehensive data base of the structural and functional effects of all possible mutations across a large protein-protein interface. The QS scan approach was applied to the high affinity site of human growth hormone (hGH) for binding to its receptor (hGHR). Although the published structure-function data base describing this system is probably the most extensive for any large protein-protein interface, it is nonetheless too sparse to accurately describe the nature of the energetics governing the interaction. Our comprehensive data base affords a complete view of the binding site and provides important new insights into the general principles underlying protein-protein interactions. The hGH binding interface is highly adaptable to mutations, but the nature of the tolerated mutations challenges generally accepted views about the evolutionary and biophysical pressures governing protein-protein interactions. Many substitutions that would be considered chemically conservative are not tolerated, while conversely, many non-conservative substitutions can be accommodated. Furthermore, conservation across species is a poor predictor of the chemical character of tolerated substitutions across the interface. Numerous deviations from generally accepted expectations indicate that mutational tolerance is highly context dependent and, furthermore, cannot be predicted by our current knowledge base. The type of data produced by the comprehensive QS scan can fill the gaps in the structure-function matrix. The compilation of analogous data bases from studies of other protein-protein interactions should greatly aid the development of computational methods for explaining and designing molecular recognition.     

Identification of functional modules from conserved ancestral protein-protein interactions

MOTIVATION: The increasing availability of large-scale protein-protein interaction (PPI) data has fueled the efforts to elucidate the building blocks and organization of cellular machinery. Previous studies have shown cross-species comparison to be an effective approach in uncovering functional modules in protein networks. This has in turn driven the research for new network alignment methods with a more solid grounding in network evolution models and better scalability, to allow multiple network comparison. RESULTS: We develop a new framework for protein network alignment, based on reconstruction of an ancestral PPI network. The reconstruction algorithm is built upon a proposed model of protein network evolution, which takes into account phylogenetic history of the proteins and the evolution of their interactions. The application of our methodology to the PPI networks of yeast, worm and fly reveals that the most probable conserved ancestral interactions are often related to known protein complexes. By projecting the conserved ancestral interactions back onto the input networks we are able to identify the corresponding conserved protein modules in the considered species. In contrast to most of the previous methods, our algorithm is able to compare many networks simultaneously. The performed experiments demonstrate the ability of our method to uncover many functional modules with high specificity. AVAILABILITY: Information for obtaining software and supplementary results are available at http://bioputer.mimuw.edu.pl/papers/cappi.     

Understanding protein-protein interactions by genetic suppression

Protein-protein interactions influence many cellular processes and it is increasingly being felt that even a weak and remote interplay between two subunits of a protein or between two proteins in a complex may govern the fate of a particular biochemical pathway. In a bacterial system where the complete genome sequence is available, it is an arduous task to assign function to a large number of proteins. It is possible that many of them are peripherally associated with a cellular event and it is very difficult to probe such interaction. However, mutations in the genes that encode such proteins (primary mutations) are useful in these studies. Isolation of a suppressor or a second-site mutation that restores the phenotype abolished by the primary mutation could be an elegant yet simple way to follow a set of interacting proteins. Such a reversion site need not necessarily be geometrically close to the primary mutation site.     

Structural interactions between retroviral Gag proteins examined by cysteine cross-linking.

We have examined structural interactions between Gag proteins within Moloney murine leukemia virus (M-MuLV) particles by making use of the cysteine-specific cross-linking agents iodine and bis-maleimido hexane. Virion-associated wild-type M-MuLV Pr65Gag proteins in immature particles were intermolecularly cross-linked at cysteines to form Pr65Gag oligomers, from dimers to pentamers or hexamers. Following a systematic approach of cysteine-to-serine mutagenesis, we have shown that cross-linking of Pr65Gag occurred at cysteines of the nucleocapsid (NC) Cys-His motif, suggesting that the Cys-His motifs within virus particles are packed in close proximity. The M-MuLV Pr65Gag protein did not cross-link to the human immunodeficiency virus Pr55Gag protein when the two molecules were coexpressed, indicating either that they did not coassemble or that heterologous Gag proteins were not in close enough proximity to be cross-linked. Using an assembly-competent, protease-minus, cysteine-minus Pr65Gag protein as a template, novel cysteine residues were generated in the M-MuLV capsid domain major homology region (MHR). Cross-linking of proteins containing MHR cysteines showed above-background levels of Gag-Gag dimers but also identified a novel cellular factor, present in virions, that cross-linked to MHR residues. Although the NC cysteine mutation was compatible with M-MuLV particle assembly, deletions of the NC domain were not tolerated. These results suggest that the Cys-His motif is held in close proximity within immature M-MuLV particles by interactions between CA domains and/or non-Cys-His motif domains of the NC.     

Prediction of protein-protein interactions between Ralstonia solanacearum and Arabidopsis thaliana

Ralstonia solanacearum is a devastating bacterial pathogen that has an unusually wide host range. R. solanacearum, together with Arabidopsis thaliana, has become a model system for studying the molecular basis of plant-pathogen interactions. Protein-protein interactions (PPIs) play a critical role in the infection process, and some PPIs can initiate a plant defense response. However, experimental investigations have rarely addressed such PPIs. Using two computational methods, the interolog and the domain-based methods, we predicted 3,074 potential PPIs between 119 R. solanacearum and 1,442 A. thaliana proteins. Interestingly, we found that the potential pathogen-targeted proteins are more important in the A. thaliana PPI network. To facilitate further studies, all predicted PPI data were compiled into a database server called PPIRA (http://protein.cau.edu.cn/ppira/). We hope that our work will provide new insights for future research addressing the pathogenesis of R. solanacearum.     

Identification of subunit-subunit interactions in bacteriophage P22 procapsids by chemical cross-linking and mass spectrometry

Viral capsids are dynamic structures which self-assemble and undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Chemical cross-linking analyzed by mass spectrometry was used to identify residue specific inter- and intra-subunit interactions in the P22 procapsids. All the intersubunit cross-links occurred between residues clustered in a loop region (residues 157-207) which was previously identified by mass spectrometry based on hydrogen/deuterium exchange and biochemical experiments. DSP and BS3 which have similar distance constraints (12 angstroms and 11.4 angstroms, respectively) cross-linked the same residues between two subunits in the procapsids (K183-K183), whereas DST, a shorter cross-linker, cross-linked lysine 175 in one subunit to lysine 183 in another subunit. The replacement of threonine with a cysteine at residue 182 immediately adjacent to the K183 cross-linking site resulted in slow spontaneous disulfide bond formation in the procapsids without perturbing capsid integrity, thus suggesting flexibility within the loop region and close proximity between neighboring loop regions. To build a detailed structure model, we have predicted the secondary structure elements of the P22 coat protein, and attempted to thread the prediction onto identified helical elements of cryoEM 3D reconstruction. In this model, the loop regions where chemical cross-linkings occurred correspond to the extra density (ED) regions which protrude upward from the outside of the capsids and face one another around the symmetry axes.     

Prediction of protein-protein interactions by docking methods

Recently, developments have been made in predicting the structure of docked complexes when the coordinates of the components are known. The process generally consists of a stage during which the components are combined rigidly and then a refinement stage. Several rapid new algorithms have been introduced in the rigid docking problem and promising refinement techniques have been developed, based on modified molecular mechanics force fields and empirical measures of desolvation, combined with minimisations that switch on the short-range interactions gradually. There has also been progress in developing a benchmark set of targets for docking and a blind trial, similar to the trials of protein structure prediction, has taken place.     

IDQD算法预测人类蛋白质相互作用

蛋白质是一切生命活动的物质基础,研究蛋白质的相互作用有助于理解生物过程的分子机制,阐明疾病的分子机理。本文依据蛋白质序列组分特征,应用基于多样性增量的二次判别分析方法,对人类的1 963对蛋白质相互作用进行了预测。自洽检验的各项预测指标均在79%以上,且交叉检验的总精度也大于60%,表明本算法可以用于蛋白质相互作用预测。     

Localization of protein-protein interactions between subunits of phytochrome.

We have used a novel assay based on protein fusions with lambda repressor to identify two small regions within phytochrome's carboxy-terminal domain that are capable of mediating dimerization. Using an in vivo assay, fusions between the DNA binding, amino-terminal domain of lambda repressor and fragments from oat PhyA phytochrome have been assayed for increased repressor activity, an indicator of dimerization. In this assay system, regions of oat phytochrome between amino acids V623-S673 and N1049-Q1129 have been shown to increase repressor activity. These short spans are highly conserved between proteins belonging to the phytochrome PhyA family. Embedded within these sequences are four segments that could potentially form amphipathic alpha helices. Two of the segments are well conserved between PhyA phytochrome and phytochromes encoded by the phyB and phyC genes, suggesting that heterodimers might form by way of subunit interaction at these sites.     

Diversity of protein-protein interactions

In this review, we discuss the structural and functional diversity of protein-protein interactions (PPIs) based primarily on protein families for which three-dimensional structural data are available. PPIs play diverse roles in biology and differ based on the composition, affinity and whether the association is permanent or transient. In vivo, the protomer's localization, concentration and local environment can affect the interaction between protomers and are vital to control the composition and oligomeric state of protein complexes. Since a change in quaternary state is often coupled with biological function or activity, transient PPIs are important biological regulators. Structural characteristics of different types of PPIs are discussed and related to their physiological function, specificity and evolution.     

Novel protein-protein interactions between Entamoeba histolyticad-phosphoglycerate dehydrogenase and phosphoserine aminotransferase

Physical interactions between d-phosphoglycerate dehydrogenase (EhPGDH) and phosphoserine aminotransferase (EhPSAT) from an enteric human parasite Entamoeba histolytica was observed by pull-down assay, gel filtration chromatography, chemical cross-linking, emission anisotropy, molecular docking and molecular dynamic simulations. The protein–protein complex had a 1:1 stochiometry with a dissociation constant of 3.453 × 10⁻⁷ M. Ionic interactions play a significant role in complex formation and stability. Analysis of the energy minimized average simulated model of the protein complex show that the nucleotide binding domain of EhPGDH specifically interacts with EhPSAT. Denaturation studies suggest that the nucleotide binding domain (Nbd) and substrate binding domain (Sbd) of EhPGDH are independent folding/unfolding units. Thus the Nbd-EhPGDH was separately cloned over-expressed and purified to homogeneity. Fluorescence anisotropy study show that the purified Nbd interacts with EhPSAT. Forward enzyme catalyzed reaction for the EhPGDH-PSAT complex showed efficient Km values for 3-phosphoglyceric acid as compared to only EhPGDH suggesting a possibility of substrate channelling in the protein complex.     

A tri-functional amino acid enables mapping of binding sites for posttranslational-modification-mediated protein-protein interactions

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HBc蛋白与NIRF蛋白细胞外相互作用鉴定

构建原核表达质粒pGST-HBc,真核表达质粒pFLAG-NIRF,表达并纯化GST-HBc蛋白,表达FLAG-NIRF蛋白,利用GST pull down技术鉴定HBc与NIRF蛋白的相互作用。利用PCR技术分别扩增HBc编码框和NIRF基因全长,并将其分别插入到原核表达载体pGEX-4T-1和真核表达载体p3*FLAG-CMV-10中,构建出pGST-HBc及pFLAG-NIRF。pFLAG-NIRF转染HEK293,培养48 h,收获前10 h加入MG132抑制蛋白酶体,收获细胞,经NP40裂解后离心收集上清pGST-HBc转化BL21(DE3)细菌,优化培养条件使GST-HBc在上清中大量表达,然后用GST4Bgel亲合纯化该蛋白,结合有GST-HBc蛋白与含FLAG-NIRF的细胞上清孵育后,离心收集GST 4Bgel,经洗脱液洗脱后蛋白电泳,western blotting分析。构建了pGST-HBc及pFLAG-NIRF,在上清中成功表达GST-HBc并纯化该蛋白,在细胞中成功表达FLAG-NIRF,GST pull down结果显示两者存在体外相互结合。HBc与NIRF能够在细胞外发生相互结合。