Protein–protein interactions (PPIs) govern all aspects of cell function and, as such, are a major target for research and therapeutic intervention. A major rate-limiting step in PPI research is the expression and purification of full-length proteins. The use of peptides to study PPIs significantly facilitates the structural and biophysical characterization of PPIs as well as the effort to develop drugs to control PPIs. Here we describe examples for the use of peptides to study PPI and some of the important experimental methods that are used in the field. Peptides have proved to be excellent tools to study PPIs and have been contributing both for understanding mechanisms of PPIs as well as for drug design for PPI modulation.

The Wnt pathway tumor-suppressor protein Axin coordinates the formation of a critical multiprotein destruction complex that serves to downregulate β-catenin protein levels, thereby preventing target gene activation. Given the lack of structural information on some of the major functional parts of Axin, it remains unresolved how the recruitment and positioning of Wnt pathway kinases, such as glycogen synthase kinase 3β, are coordinated to bring about β-catenin phosphorylation. Using various biochemical and biophysical methods, we demonstrate here that the central region of Axin that is implicated in binding glycogen synthase kinase 3β and β-catenin is natively unfolded. Our results support a model in which the unfolded nature of these critical scaffolding regions in Axin facilitates dynamic interactions with a kinase and its substrate, which in turn act upon each other.

Screening of arrays and libraries of compounds is well-established as a high-throughput method for detecting and analyzing interactions in both biological and chemical systems. Arrays and libraries can be composed from various types of molecules, ranging from small organic compounds to DNA, proteins and peptides. The applications of libraries for detecting and characterizing biological interactions are wide and diverse, including for example epitope mapping, carbohydrate arrays, enzyme binding and protein–protein interactions. Here, we will focus on the use of peptide arrays to study protein–protein interactions. Characterization of protein–protein interactions is crucial for understanding cell functionality. Using peptides, it is possible to map the precise binding sites in such complexes. Peptide array libraries usually contain partly overlapping peptides derived from the sequence of one protein from the complex of interest. The peptides are attached to a solid support using various techniques such as SPOT-synthesis and photolithography. Then, the array is incubated with the partner protein from the complex of interest. Finally, the detection of the protein-bound peptides is carried out by using immunodetection assays. Peptide array screening is semi-quantitative, and quantitative studies with selected peptides in solution are required to validate and complement the screening results. These studies can improve our fundamental understanding of cellular processes by characterizing amino acid patterns of protein–protein interactions, which may even develop into prediction algorithms. The binding peptides can then serve as a basis for the design of drugs that inhibit or activate the target protein–protein interactions. In the current review, we will introduce the recent work on this subject performed in our and in other laboratories. We will discuss the applications, advantages and disadvantages of using peptide arrays as a tool to study protein–protein interactions.

The human immunodeficiency virus type-1 (HIV-1) Vif protein neutralizes the cellular defense mechanism against the virus. The C-terminal domain of Vif (CTD, residues 141–192) mediates many of its interactions. Full-length Vif is difficult to purify in large amounts, hence the only available structure of Vif is of residues 140–155 within the ElonginBC complex. Other structural information, derived from modeling and indirect experiments, indicates that the Vif CTD may be unstructured. Here, we chemically synthesized the Vif CTD using pseudo-proline-building blocks, studied its solution structure in the unbound state using biophysical techniques and found that it is unstructured under physiological conditions. The circular dichroism (CD) spectrum of Vif CTD showed a pattern of random coil with residual helical structure. The ¹⁵N-HSQC nuclear magnetic resonance (NMR) spectrum was characteristic of natively unfolded peptides. Vif CTD eluted from an analytical gel filtration column earlier than expected, indicating an extended conformation. Disorder predictions found the CTD to be unstructured, in agreement with our experimental results. CD experiments showed that Vif CTD underwent a conformational change upon interacting with membrane-mimicking DPC micelles, but not upon binding to a peptide derived from its binding region in ElonginC. Our results provide direct evidence for the unfolded structure of the free Vif CTD and indicate that it may gain structure upon binding its natural ligands.

Phosphopeptides have been used to study phosphorylation and dephosphorylation, which are key events in protein expression. Backbone cyclization has been shown to increase the stability and selectivity of peptides. Backbone cyclic peptides with conformational diversity have produced bioactive peptides with improved pharmaceutical properties, metabolic stability, and enhanced intestinal permeability. We demonstrate a successful methodology for incorporating phospho-amino acids into backbone cyclic peptides. The nuclear factor-kappa B (NF-κB) is a latent mammalian protein prototype of dimeric transcription factors that exists in all cell types and plays a pivotal role in a huge number of genes, such as those responsible for chronic and acute inflammatory diseases. To inhibit NF-κB, backbone cyclic phosphopeptides were designed and synthesized based on the conserved sequence of the Inhibitor kappa B (IκB). The peptides were screened for inhibiting IκB ubiquitylation. The best compound showed 90% inhibition at a concentration of 3 μM, and its solution structure showed similarity to a related β-catenin protein. This general methodology can be use for synthesizing cyclic phosphorylated, as well as backbone cyclic phosphorylated peptides for various biological targets.

We used computational methods to study the interaction between two key proteins in apoptosis regulation: the transcription factor NF-kappa-B (NFkappaB) and the proapoptotic protein ASPP2. The C-terminus of ASPP2 contains ankyrin repeats and SH3 domains (ASPP2(ANK-SH3)) that mediate interactions with numerous apoptosis-related proteins, including the p65 subunit of NFkappaB (NFkappaB(p65)). Using peptide-based methods, we have recently identified the interaction sites between NFkappaB(p65) and ASPP2(ANK-SH3) (Rotem et al., J Biol Chem 283, 18990-18999). Here we conducted a computational study of protein docking and molecular dynamics to obtain a structural model of the complex between the full length proteins and propose a mechanism for the interaction. We found that ASPP2(ANK-SH3) binds two sites in NFkappaB(p65), at residues 236-253 and 293-313 that contain the nuclear localization signal (NLS). These sites also mediate the binding of NFkappaB to its natural inhibitor IkappaB, which also contains ankyrin repeats. Alignment of the ankyrin repeats of ASPP2(ANK-SH3) and IkappaB revealed that both proteins share highly similar interfaces at their binding sites to NFkappaB. Protein docking of ASPP2(ANK-SH3) and NFkappaB(p65), as well as molecular dynamics simulations of the proteins, provided structural models of the complex that are energetically similar to the NFkappaB-IkappaB determined structure. Our results show that ASPP2(ANK-SH3) binds NFkappaB(p65) in a similar manner to its natural inhibitor IkappaB, suggesting a possible novel role for ASPP2 as an NFkappaB inhibitor.

Background

The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN), catalyzes the integration of viral DNA into the host cell genome. IN catalyzes the first step of the integration process, namely the 3′-end processing in which IN removes a pGT dinucleotide from the 3′ end of each viral long terminal repeat (LTR). Following nuclear import of the viral preintegration complex, the host chromosomal DNA becomes accessible to the viral cDNA and the second step of the integration process, namely the strand-transfer step takes place. This ordered sequence of events, centered on integration, is mandatory for HIV replication.

Methodology/Principal Findings

Using an integrase peptide library, we selected two peptides, designated INr-1 and INr-2, which interact with the Rev protein and probably mediate the Rev-integrase interaction. Using an in-vitro assay system, we show that INr-1 and INr-2 are able to abrogate the inhibitory effects exerted by Rev and Rev-derived peptides on integrase activity. Both INr-1 and INr-2 were found to be cell-permeable and nontoxic, allowing a study of their effect in HIV-1-infected cultured cells. Interestingly, both INr peptides stimulated virus infectivity as estimated by production of the viral P24 protein, as well as by determination of the appearance of newly formed virus particles. Furthermore, kinetics studies revealed that the cell-permeable INr peptides enhance the integration process, as was indeed confirmed by direct determination of viral DNA integration by real-time PCR.

Conclusions/Significance

The results of the present study raise the possibility that in HIV-infected cells, the Rev protein may be involved in the integration of proviral DNA by controlling/regulating the activity of the integrase. Release from such inhibition leads to stimulation of IN activity and multiple viral DNA integration events.

Following fusion of the human immunodeficiency virus type-1 (HIV-1) with host cells' membrane and reverse transcription of the viral RNA, the resulted cDNA is integrated into the host genome by the viral integrase enzyme (IN). Quantitative estimations have revealed that only 1–2 copies are integrated per infected cell, although many copies of the viral RNA are reverse-transcribed. The molecular mechanism that restricts the integration degree has not, so far, been elucidated. Following integration, expressed partially spliced and unspliced transcripts are exported from the nuclei by the viral Rev protein. Here, we show that in virally infected cells, the Rev interacts with the IN forming a Rev–IN complex and consequently limits the number of integration events. Disruption of the Rev–IN complex by selected IN-derived peptides or infection by a Rev-deficient virus stimulate integration resulting in large numbers of integration event/cell. Conversely, infection of Rev-expression cells blocks integration and inhibits virus production. Increased integration appears to correlate with increased cell death of infected cultures. Our results thus demonstrate a new regulatory function of Rev and probably establish a link between Rev restriction of HIV-1 integration and protection of HIV-1-infected cells from premature cell death.

The HIV-1 integrase enzyme (IN) catalyzes integration of viral DNA into the host genome. We previously developed peptides that inhibit IN in vitro and HIV-1 replication in cells. Here we present the design, synthesis and evaluation of several derivatives of one of these inhibitory peptides, the 20-mer IN1. The peptide corresponding to the N-terminal half of IN1 (IN1 1–10) was easier to synthesize and much more soluble than the 20-mer IN1. IN1 1–10 bound IN with improved affinity and inhibited IN activity as well as HIV replication and integration in infected cells. While IN1 bound the IN tetramer, its shorter derivatives bound dimeric IN. Mapping the peptide binding sites in IN provided a model that explains this difference. We conclude that IN1 1–10 is an improved lead compound for further development of IN inhibitors.

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein is an emerging target for the development of anti-HIV drugs. We recently described a new approach for inhibiting IN by “shiftides”—peptides that inhibit the protein by shifting its oligomerization equilibrium from the active dimer to the inactive tetramer. In this study, we used the yeast two-hybrid system with the HIV-1 IN as a bait and a combinatorial peptide aptamer library as a prey to select peptides of 20 amino acids that specifically bind IN. Five non-homologous peptides, designated as IN-1 to IN-5, were selected. ELISA studies confirmed that IN binds the free peptides. All the five peptides interact with IN with comparable affinity (K_d≈10 μM), as was revealed by fluorescence anisotropy studies. Only one peptide, IN-1, inhibited the enzymatic activity of IN in vitro and the HIV-1 replication in cultured cells. In correlation, fluorescence anisotropy binding experiments revealed that of the five peptides, only the inhibitory IN-1 inhibited the DNA binding of IN. Analytical gel filtration experiments revealed that only the IN-1 and not the four other peptides shifted the oligomerization equilibrium of IN towards the tetramer. Thus, the results show a distinct correlation between the ability of the selected peptides to inhibit IN activity and that to shift its oligomerization equilibrium.

We have characterized the molecular basis of the interaction between ASPP2 and Bcl-2, which are key proteins in the apoptotic pathway. The C-terminal ankyrin repeats and SH3 domain of ASPP2 (ASPP2_Ank-SH3) mediate its interactions with the antiapoptotic protein Bcl-2. We used biophysical and computational methods to identify the interaction sites of Bcl-2 and its homologues with ASPP2. Using peptide array screening, we found that ASPP2_Ank-SH3 binds two homologous sites in all three Bcl proteins tested: (i) the conserved BH4 motif, and (ii) a binding site for proapoptotic regulators. Quantitative binding studies revealed that binding of ASPP2_Ank-SH3 to the Bcl-2 family members is selective at two levels: (i) interaction with Bcl-2-derived peptides is the tightest compared to peptides from the other family members, and (ii) within Bcl-2, binding of ASPP2_Ank-SH3 to the BH4 domain is tightest. Sequence alignment of the ASPP2-binding peptides combined with binding studies of mutated peptides revealed that two nonconserved positions where only Bcl-2 contains positively charged residues account for its tighter binding. The experimental binding results served as a basis for docking analysis, by which we modeled the complexes of ASPP2_Ank-SH3 with the full-length Bcl proteins. Using peptide arrays and quantitative binding studies, we found that Bcl-2 binds three loops in ASPP2_Ank-SH3 with similar affinity, in agreement with our predicted model. Based on our results, we propose a mechanism in which ASPP2 induces apoptosis by inhibiting functional sites of the antiapoptotic Bcl-2 proteins.

The HIV-1 Integrase protein (IN) mediates the integration of the viral cDNA into the host genome. IN is an emerging target for anti-HIV drug design, and the first IN-inhibitor was recently approved by the FDA. We have developed a new approach for inhibiting IN by “shiftides”: peptides derived from its cellular binding protein LEDGF/p75 that inhibit IN by shifting its oligomerization equilibrium from the active dimer to an inactive tetramer. In addition, we described two peptides derived from the HIV-1 Rev protein that interact with IN and inhibit its activity in vitro and in cells. In the current study, we show that the Rev-derived peptides also act as shiftides. Analytical gel filtration and cross-linking experiments showed that IN was dimeric when bound to the viral DNA, but tetrameric in the presence of the Rev-derived peptides. Fluorescence anisotropy studies revealed that the Rev-derived peptides inhibited the DNA binding of IN. The Rev-derived peptides inhibited IN catalytic activity in vitro in a concentration-dependent manner. Inhibition was much more significant when the peptides were added to free IN before it bound the viral DNA than when the peptides were added to a preformed IN-DNA complex. This confirms that the inhibition is due to the ability of the peptides to shift the oligomerization equilibrium of the free IN toward a tetramer that binds much weaker to the viral DNA. We conclude that protein–protein interactions of IN may serve as a general valuable source for shiftide design.

ASPP2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response. The C terminus of ASPP2 contains ankyrin (Ank) repeats and a SH3 domain, which mediate its interactions with numerous partner proteins such as p53, NFκB, and Bcl-2. It also contains a proline-rich domain (ASPP2 Pro), whose structure and function are unclear. Here we used biophysical and biochemical methods to study the structure and the interactions of ASPP2 Pro, to gain insight into its biological role. We show, using biophysical and computational methods, that the ASPP2 Pro domain is natively unfolded. We found that the ASPP2 Pro domain interacts with the ASPP2 Ank-SH3 domains, and mapped the interaction sites in both domains. Using a combination of peptide array screening, biophysical and biochemical techniques, we found that ASPP2 Ank-SH3, but not ASPP2 Pro, mediates interactions of ASPP2 with peptides derived from its partner proteins. ASPP2 Pro-Ank-SH3 bound a peptide derived from its partner protein NFκB weaker than ASPP2 Ank-SH3 bound this peptide. This suggested that the presence of the proline-rich domain inhibited the interactions mediated by the Ank-SH3 domains. Furthermore, a peptide from ASPP2 Pro competed with a peptide derived from NFκB on binding to ASPP2 Ank-SH3. Based on our results, we propose a model in which the interaction between the ASPP2 domains regulates the intermolecular interactions of ASPP2 with its partner proteins.

We present a new approach for the conversion of active sequences of proteins and peptides into small molecules. A library of macrocyclic disulfide molecules was made, in which the active pharmacophores of the parent peptide are preserved while the size of the macromolecular scaffold on which the pharmacophores are arranged is varied. This enables a systematic search for macromolecules in which the pharmacophores are in an appropriate conformation for biological activity. We developed two procedures for the synthesis of such libraries from building blocks that include commercial amino acids and functionalized aldehydes. Chemical synthesis using the “tea-bag” method gave a library with higher diversity, but low yields, compared to the manual synthesis of the library, in which the compounds were synthesized in individual vessels and the yield and purity improved dramatically. As a proof of concept, we synthesized a 34-member library derived from the sequence of the activation loop of insulin-like growth factor-1 receptor. Selected compounds were screened, and one was found to be biologically active in the low micromolar range. The concept presented here may prove particularly useful in cases where the pharmacophores are known but need to be systematically screened for a spatial arrangement that will enable biological activity.

Peptides are valuable tools for studying protein–protein interactions, especially in cases of isolated protein domains and natively unfolded proteins. Here, we used peptides to quantitatively characterize the interaction between the natively unfolded HIV-1 Tat protein and the tetramerization domain of the cellular tumor suppressor protein p53. We used peptide mapping, fluorescence anisotropy, and NMR spectroscopy to perform a detailed structural and biophysical characterization of the interaction between the two proteins and elucidate its molecular mechanism, which have so far been studied using cell-based methods. We show that the p53 tetramerization domain, p53(326–355), binds directly to residues 1–35 and 47–57 in Tat. We have characterized the interaction between p53(326–355) and Tat(47–57) in detail. The p53 residues that are mainly involved in binding to Tat(47–57) are E343 and E349, which bind to the positively charged arginine-rich motif of Tat by a partly electrostatic mechanism. All oligomerization states of p53(326–355) bind Tat(47–57) without inhibiting p53 tetramerization, since the residues in p53(326–355) that bind Tat(47–57) face away from the tetramerization interface. We conclude that p53 is able to bind Tat as a transcriptionally active tetramer.

The LTR retrotransposon of Schizosacharomyces pombe, Tf1, has several distinctive properties that can be related to the unique properties of its reverse transcriptase (RT). Consequently, we expressed, purified and studied the recombinant Tf1 RT. This monomeric protein possesses all activities typical to RTs: DNA and RNA-dependent DNA polymerase as well as an inherent ribonuclease H. The DNA polymerase activity shows preference to Mn(+)(2) or Mg(+)(2), depending on the substrate used, whereas the ribonuclease H strongly prefers Mn(+)(2). The most outstanding feature of Tf1 RT is its capacity to add non-templated nucleotides to the 3'-ends of the nascent DNA. This is mainly apparent in the presence of Mn(+)(2), as is the noticeable low fidelity of DNA synthesis. In all, Tf1 RT has a marked infidelity in synthesizing DNA at template ends, a phenomenon that can explain, as discussed herein, some of the features of Tf1 replication in the host cells.

Proteins are involved in various equilibria that play a major role in their activity or regulation. The design of molecules that shift such equilibria is of great therapeutic potential. This fact was demonstrated in the cases of allosteric inhibitors, which shift the equilibrium between active and inactive (R and T) states, and chemical chaperones, which shift folding equilibrium of proteins. Here, we expand these concepts and propose the shifting of oligomerization equilibrium of proteins as a general methodology for drug design. We present a strategy for inhibiting proteins by “shiftides”: ligands that specifically bind to an inactive oligomeric state of a disease-related protein and modulate its activity by shifting the oligomerization equilibrium of the protein toward it. We demonstrate the feasibility of our approach for the inhibition of the HIV-1 integrase (IN) protein by using peptides derived from its cellular-binding protein, LEDGF/p75, which specifically inhibit IN activity by a noncompetitive mechanism. The peptides inhibit the DNA-binding of IN by shifting the IN oligomerization equilibrium from the active dimer toward the inactive tetramer, which is unable to catalyze the first integration step of 3′ end processing. The LEDGF/p75-derived peptides inhibit the enzymatic activity of IN in vitro and consequently block HIV-1 replication in cells because of the lack of integration. These peptides are promising anti-HIV lead compounds that modulate oligomerization of IN via a previously uncharacterized mechanism, which bears advantages over the conventional interface dimerization inhibitors.

ASPP (apoptosis-stimulating protein of p53) 2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response. Here, we provide an overview of the structure and protein–protein interactions of ASPP2. The C-terminus of ASPP2 contains Ank (ankyrin) repeats and an SH3 domain (Src homology 3 domain). The Ank–SH3 domains mediate interactions between ASPP2 and numerous proteins involved in apoptosis such as p53 and Bcl-2. The proline-rich domain of ASPP2 is unfolded in its native state, but was not shown to mediate intermolecular interactions. Instead, it makes an intramolecular domain–domain interaction with the Ank–SH3 C-terminal domains of ASPP2. This intramolecular interaction between the unstructured proline-rich domain and the structured Ank–SH3 domains in ASPP2, which is possible due to the unfolded nature of the proline-rich domain, is proposed to have an important role in regulating the intermolecular interactions of ASPP2 with its partner proteins.

MDC1 (NFBD1), a mediator of the cellular response to DNA damage, plays an important role in checkpoint activation and DNA repair. Here we identified a cross-talk between the DNA damage response and cell cycle regulation. We discovered that MDC1 binds the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that controls the cell cycle. The interaction is direct and is mediated by the tandem BRCA1 C-terminal domains of MDC1 and the C terminus of the Cdc27 (APC3) subunit of the APC/C. It requires the phosphorylation of Cdc27 and is enhanced after induction of DNA damage. We show that the tandem BRCA1 C-terminal domains of MDC1, known to directly bind the phosphorylated form of histone H2AX (γ-H2AX), also bind the APC/C by the same mechanism, as phosphopeptides that correspond to the C termini of γ-H2AX and Cdc27 competed with each other for the binding to MDC1. Our results reveal a link between the cellular response to DNA damage and cell cycle regulation, suggesting that MDC1, known to have a role in checkpoint regulation, executes part of this role by binding the APC/C.

The p53 tumor suppressor is a nucleocytoplasmic shuttling protein that is found predominantly in the nucleus of cells. In addition to mutation, abnormal p53 cellular localization is one of the mechanisms that inactivate p53 function. To further understand features of p53 that contribute to the regulation of its trafficking within the cell, we analysed the subnuclear localization of wild-type and mutant p53 in human cells that were either permeabilized with detergent or treated with the proteasome inhibitor MG132. We, here, show that either endogenously expressed or exogenously added p53 protein localizes to the nucleolus in detergent-permeabilized cells in a concentration- and ATP hydrolysis-dependent manner. Two discrete regions within the carboxyl terminus of p53 are essential for nucleolar localization in permeabilized cells. Similarly, localization of p53 to the nucleolus after proteasome inhibition in unpermeabilized cells requires sequences within the carboxyl terminus of p53. Interestingly, genotoxic stress markedly decreases the association of p53 with the nucleolus, and phosphorylation of p53 at S392, a site that is modified by such stress, partially impairs its nucleolar localization. The possible significance of these findings is discussed.