Determination of the sequence specificity of XIAP BIR domains by screening a combinatorial peptide library

Inhibitor of apoptosis (IAP) proteins regulate programmed cell death by inhibiting members of the caspase family of proteases. The X-chromosome-linked IAP (XIAP) contains three baculovirus IAP repeat (BIR) domains, which bind directly to the N-termini of target proteins including those of caspases-3, -7, and -9. In the present study, we defined the consensus sequences of the motifs that interact with the three BIR domains in an unbiased manner. A combinatorial peptide library containing four random residues at the N-terminus was constructed and screened using BIR domains as probes. We found that the BIR3 domain binds a highly specific motif containing an alanine or valine at the N-terminus (P1 position), an arginine or proline at the P3 position, and a hydrophobic residue (Phe, Ile, and Tyr) at the P4 position. The BIR2-binding motif is less stringent. Although it still requires an N-terminal alanine, it tolerates a wide variety of amino acids at P2-P4 positions. The BIR1 failed to bind to any peptides in the library. SPR analysis of individually synthesized peptides confirmed the library screening results. Database searches with the BIR2- and BIR3-binding consensus sequences revealed a large number of potential target proteins. The combinatorial library method should be readily applicable to other BIR domains or other types of protein modular domains.     

Defining SH2 domain and PTP specificity by screening combinatorial peptide libraries

Src homology 2 (SH2) domains mediate protein-protein interactions by recognizing short phosphotyrosyl (pY) peptide motifs in their partner proteins. Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of pY proteins, counteracting the protein tyrosine kinases. Both types of proteins exhibit primary sequence specificity, which plays at least a partial role in dictating their physiological interacting partners or substrates. A combinatorial peptide library method has been developed to systematically assess the sequence specificity of SH2 domains and PTPs. A "one-bead-one-compound" pY peptide library is synthesized on 90-microm TentaGel beads and screened against an SH2 domain or PTP of interest for binding or catalysis. The beads that carry the tightest binding sequences against the SH2 domain or the most efficient substrates of the PTP are selected by an enzyme-linked assay and individually sequenced by a partial Edman degradation/mass spectrometry technique. The combinatorial method has been applied to determine the sequence specificity of 8 SH2 domains from Src and Csk kinases, adaptor protein Grb2, and phosphatases SHP-1, SHP-2, and SHIP1 and a prototypical PTP, PTP1B.     

Affinity chromatographic screening of soluble combinatorial peptide libraries.

Affinity chromatography using immobilized S-protein was used for the screening of affinity peptide ligands from two soluble peptide libraries. Peptide library I consisted of octamers with glycine (G) at both termini of each peptide, i.e. GXXXXXXG. The six center positions were constructed using random sequences of six L-amino acids (Y, N, F, E, V, and L). Peptide library II also consisted of octamers but with glycine and valine (V) at both termini of each peptide (GVZZZZVG). The four variable center positions of peptide library II were random sequences of 18 L-amino acids. Peptides that were retained specifically on the immobilized S-protein column were eluted by 2% acetic acid. The peptides in the acid eluate were further separated using reversed-phase HPLC. Each separated peptide fraction was collected and the peptide sequences deconvoluted by mass spectrometry (MS/MS). The screenings of peptide libraries I and II resulted in 12 and 7 affinity peptides, respectively. Eight out of the twelve peptides from peptide library I contained the clear consensus sequence NFEV. Peptide library II resulted in affinity peptides with the sequences GVNFEVVG, GVNFTVVG and GVFFEL(I)VG. The advantages and limitations of affinity chromatography in peptide library screening are discussed.     

Quantitative assessment of peptide sequence diversity in M13 combinatorial peptide phage display libraries

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0+/-1.6% of the random dodecapeptides and 7.9+/-2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usage patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a beta-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.     

High‐energy water sites determine peptide binding affinity and specificity of PDZ domains

PDZ domains have well known binding preferences for distinct C‐terminal peptide motifs. For most PDZ domains, these motifs are of the form [S/T]‐W‐[I/L/V]. Although the preference for S/T has been explained by a specific hydrogen bond interaction with a histidine in the PDZ domain and the (I/L/V) is buried in a hydrophobic pocket, the mechanism for Trp specificity at the second to last position has thus far remained unknown. Here, we apply a method to compute the free energies of explicit water molecules and predict that potency gained by Trp binding is due to a favorable release of high‐energy water molecules into bulk. The affinities of a series of peptides for both wild‐type and mutant forms of the PDZ domain of Erbin correlate very well with the computed free energy of binding of displaced waters, suggesting a direct relationship between water displacement and peptide affinity. Finally, we show a correlation between the magnitude of the displaced water free energy and the degree of Trp‐sensitivity among subtypes of the HTRA PDZ family, indicating a water‐mediated mechanism for specificity of peptide binding.     

Reverse interactomics: decoding protein-protein interactions with combinatorial peptide libraries

Identification of binding partners is the crucial first step towards understanding the biological function of a protein. Many protein-protein interactions occur via modular domains that recognize short peptide motifs in their target proteins. Here we describe a chemical/bioinformatics approach for predicting the binding partners of modular domains. The optimal binding motif(s) of a protein domain is identified by screening a combinatorial peptide library. The resulting consensus sequence is used to search protein and genomic databases for potential binding proteins, which are subsequently confirmed (or disproved) by conventional protein binding assays (e.g. pull-down and co-immunoprecipitation).     

The emerging contribution of sequence context to the specificity of protein interactions mediated by PDZ domains

The canonical binding mode of PDZ domains to target motifs involves a small interface, unlikely to fully account for PDZ-target interaction specificities. Here, we review recent work on sequence context, defined as the regions surrounding not only the PDZ domains but also their target motifs. We also address the theoretical problem of defining the core of PDZ domains and the practical issue of designing PDZ constructs. Sequence context is found to introduce structural diversity, to impact the stability and solubility of constructs, and to deeply influence binding affinity and specificity, thereby increasing the difficulty of predicting PDZ-motif interactions. We expect that sequence context will have similar importance for other protein interactions mediated by globular domains binding to short linear motifs.     

Investigation of S-farnesyl transferase substrate specificity with combinatorial tetrapeptide libraries

Using biased tetrapeptide libraries made up of proteinogenic amino acids of the general formula Cys-O2-X3-X4, we searched for new substrates of partly purified rat brain S-farnesyl transferase (FTase). To achieve this task, an assay was developed in which the consumption of the co-substrate (farnesyl pyrophosphate) was measured. After three steps of deconvolution including each synthesis and enzymatic assay, the most efficient substrates found under these particular conditions were Cys-Lys-Gln-Gln (peptide I) and Cys-Lys-Gln-Met (peptide II). As a control, we used another tetrapeptide library (Cys-Val-O3-X4) in which the valine position was arbitrarily fixed, corresponding to Cys-Val-Ile-Met in the CAAX box of K-RasB, although this sublibrary was only marginally active compared with Cys-Lys-X3-X4 in the first round of deconvolution. The best substrate sublibrary was Cys-Val-Thr-X4, threonine being more favourable than the aliphatic amino acids (Val, Ile, Leu, Ala) in this position. Deconvolution finally led to Cys-Val-Thr-Gln, -Met, -Thr and -Ser as the most efficient substrates of FTase. Those tetrapeptides were not substrates of a partly purified geranylgeranyl transferase 1 (GGTase1). We also investigated the influence of the -1 position (at the N-terminus of cysteine) on the specificity of the enzyme, by using a series of pentapeptides constructed on the basis of the best tetrapeptide core (peptide 1). Among this family of analogues, only His-Cys-Lys-Gln-Gln did not behave as a substrate, whereas all the other pentapeptides were measurable substrates, with Gly-, Asn- and Thr-Cys-Lys-Gln-Gln displaying kinetic constants similar to that of Cys-Lys-Gln-Gln. The present work provides strong evidence that the best tetrapeptide substrates of FTase do not necessarily belong to the classical CAAX box, in which A's are lipophilic residues, but rather contain hydrophilic amino acids in the middle of their sequences. Among them, peptides I and II are potent FTase in vitro substrates that are not recognised by GGTase1 and might be new starting points for the design of FTase inhibitors.     

Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries.

Hsp70 chaperones assist protein folding by ATP-dependent association with linear peptide segments of a large variety of folding intermediates. The molecular basis for this ability to differentiate between native and non-native conformers was investigated for the DnaK homolog of Escherichia coli. We identified binding sites and the recognition motif in substrates by screening 4360 cellulose-bound peptides scanning the sequences of 37 biologically relevant proteins. DnaK binding sites in protein sequences occurred statistically every 36 residues. In the folded proteins these sites are mostly buried and in the majority found in beta-sheet elements. The binding motif consists of a hydrophobic core of four to five residues enriched particularly in Leu, but also in Ile, Val, Phe and Tyr, and two flanking regions enriched in basic residues. Acidic residues are excluded from the core and disfavored in flanking regions. The energetic contribution of all 20 amino acids for DnaK binding was determined. On the basis of these data an algorithm was established that predicts DnaK binding sites in protein sequences with high accuracy.     

Design, synthesis and screening of antisense peptide based combinatorial peptide libraries towards an aromatic region of SARS-CoV

A combination of high-performance affinity chromatography and antisense peptide based combinatorial peptide libraries was used to screen a potential inhibitor for SARS-CoV. An aromatic-amino acid-rich region within the transmembrane domain at the C terminal of spike (S) protein identified as a membrane-active region was chosen as the target sense peptide (SP) and immobilized as affinity ligand. Four antisense peptides were designed based on the degeneracy of genetic codes. One of them was screened as the lead peptide to construct the extended peptide libraries (EPL). The library screening was carried out at pH 5.5 so as to mimic the low-pH milieu required by virus fusion. After five cycles of screening, a dodecapeptide KKKKYRNIRRPG (DP) was identified to possess the highest binding affinity to the immobilized sense peptide. The dissociation constant of the complex between the DP and the SP was 5.64 x 10(-7) M in a physiological condition. The recognition between the DP and recombinant SARS S protein was demonstrated by ELISA assay to be in a saturable way. The competitive inhibition of the sense peptide in the competitive ELISA reveals the affinity binding between the DP and SARS S protein is specific and directed towards the target SP of the S protein. The results indicate this preferred polypeptide can be used as a lead compound of potent inhibitor of SARS-CoV. The mechanism study suggests the specific recognition between the DP and the target peptide was due to sequence-dependent and multi-modal affinity interaction.     

Integrating computational and mixture-based screening of combinatorial libraries

Mixture-based synthetic combinatorial library (MB-SCL) screening is a well-established experimental approach for rapidly retrieving structure–activity relationships (SAR) and identifying hits. Virtual screening is also a powerful approach that is increasingly being used in drug discovery programs and has a growing number of successful applications. However, limited efforts have been made to integrate both techniques. To this end, we combined experimental data from a MB-SCL of bicyclic guanidines screened against the κ-opioid receptor and molecular similarity methods. The activity data and similarity analyses were integrated in a biometric analysis–similarity map. Such a map allows the molecules to be categorized as actives, activity cliffs, low similarity to the reference compounds, or missed hits. A compound with IC₅₀ = 309 nM was found in the “missed hits” region, showing that active compounds can be retrieved from a MS-SCL via computational approaches. The strategy presented in this work is general and is envisioned as a general-purpose approach that can be applied to other MB-SCLs.     

Structural analysis of the N- and C-termini in a peptide with consensus sequence.

We present a structural analysis of a peptide, the sequence of which includes amino acids that show preferences for specific positions near the N- and C-termini in protein helices. This peptide has the sequence ac-YMSEDELKAAEAAFKRHGVP-amide, which includes a strong version of an N-terminal Harper-Rose capping box structure as well as a Gly located close to the C-terminus designed to elucidate its role in C-terminal capping. The sequence of five residues at the middle is inserted to separate effects at the two ends via a helix-stabilizing linker. Application of a simulated annealing procedure using interproton distance constraints derived from 1H NOESY experiments in water reveals the presence of a C-terminal structure in this model. The C-terminus forms a folded back structure in a significant fraction of structures generated by the annealing, in most of which Gly assumes an alpha L conformation. This structure occurs within a highly flexible region of the molecule and hence is occupied only a fraction of the time.     

Determination of the substrate specificity of tripeptidyl-peptidase I using combinatorial peptide libraries and development of improved fluorogenic substrates

Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant kcat/KM. In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1' and P2' residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.     

Exploring the chicken egg white proteome with combinatorial peptide ligand libraries

The use of two types of peptide ligand libraries (PLL), containing hexapeptides terminating either with a primary amine or modified with a terminal carboxyl group, allowed the discovery and identification of a large number of previously unreported egg white proteins. Whereas the most comprehensive list up to date ( Mann, K. , Proteomics 2007, 7, 3558- 3568 ) tabulated 78 unique gene products, our findings have almost doubled that value to 148 unique protein species. From the initial nontreated egg, it was possible to find 41 protein species; the difference (107 proteins) was generated as a result of the use of PLLs from which a similar number of species (112 and 109, respectively) was evidenced. Of those, 35 proteins were the specific catch of the amino-terminus PLL, while 33 were uniquely captured by the carboxy-terminus PLL. While a number of these low-abundance proteins might have a biological role in maintaining the integrity of the egg white and protecting the yolk, others might be derived from decaying epithelial cells lining the oviduct and/or represent remnants of products from the magnum and eggshell membrane components secreted by the isthmus, which might ultimately be incorporated, even if in trace amounts, into the egg white. The list of egg white components here reported is by far the most comprehensive at present and could serve as a starting point for isolation and functional characterization of proteins possibly having novel pharmaceutical and biomedical applications.     

Distinct ligand specificity of the Tiam1 and Tiam2 PDZ domains

Guanine nucleotide exchange factor proteins of the Tiam family are activators of the Rho GTPase Rac1 and critical for cell morphology, adhesion, migration, and polarity. These proteins are modular and contain a variety of interaction domains, including a single post-synaptic density-95/discs large/zonula occludens-1 (PDZ) domain. Previous studies suggest that the specificities of the Tiam1 and Tiam2 PDZ domains are distinct. Here, we sought to conclusively define these specificities and determine their molecular origin. Using a combinatorial peptide library, we identified a consensus binding sequence for each PDZ domain. Analysis of these consensus sequences and binding assays with peptides derived from native proteins indicated that these two PDZ domains have overlapping but distinct specificities. We also identified residues in two regions (S(0) and S(-2) pockets) of the Tiam1 PDZ domain that are important determinants of ligand specificity. Site-directed mutagenesis of four nonconserved residues in these two regions along with peptide binding analyses confirmed that these residues are crucial for ligand affinity and specificity. Furthermore, double mutant cycle analysis of each region revealed energetic couplings that were dependent on the ligand being investigated. Remarkably, a Tiam1 PDZ domain quadruple mutant had the same specificity as the Tiam2 PDZ domain. Finally, analysis of Tiam family PDZ domain sequences indicated that the PDZ domains segregate into four distinct families based on the residues studied here. Collectively, our data suggest that Tiam family proteins have highly evolved PDZ domain-ligand interfaces with distinct specificities and that they have disparate PDZ domain-dependent biological functions.     

PDZ domains and their binding partners: structure, specificity, and modification

PDZ domains are abundant protein interaction modules that often recognize short amino acid motifs at the C-termini of target proteins. They regulate multiple biological processes such as transport, ion channel signaling, and other signal transduction systems. This review discusses the structural characterization of PDZ domains and the use of recently emerging technologies such as proteomic arrays and peptide libraries to study the binding properties of PDZ-mediated interactions. Regulatory mechanisms responsible for PDZ-mediated interactions, such as phosphorylation in the PDZ ligands or PDZ domains, are also discussed. A better understanding of PDZ protein-protein interaction networks and regulatory mechanisms will improve our knowledge of many cellular and biological processes.     

Determination of the binding specificity of the SH2 domains of protein tyrosine phosphatase SHP-1 through the screening of a combinatorial phosphotyrosyl peptide library

A method for the rapid identification of high-affinity ligands to Src homology-2 (SH2) domains is reported. A phosphotyrosyl (pY) peptide library containing completely randomized residues at positions -2 to +3 relative to the pY was synthesized on TentaGel resin, with a unique peptide sequence on each resin bead (total 2.5 x 10(6) different sequences). The library was screened against the biotinylated N- and C-terminal SH2 domains of protein tyrosine phosphatase SHP-1, and the beads that carry high-affinity ligands of the SH2 domains were identified using an enzyme-linked assay involving a streptavidin-alkaline phosphatase conjugate. Peptide ladder sequencing of the selected beads using matrix-assisted laser desorption ionization mass spectrometry revealed consensus sequences for both SH2 domains. The N-terminal SH2 domain strongly selects for peptides with a leucine at the -2 position; at the C-terminal side of the pY residue, it can recognize two distinct classes of peptides with consensus sequences of LXpY(M/F)X(F/M) and LXpYAXL (X = any amino acid), respectively. The C-terminal SH2 domain exhibits almost exclusive selectivity for peptides of the consensus sequence, (V/I/L)XpYAX(L/V). Several representative sequences selected from the library were individually synthesized and tested for binding to the SH2 domains by surface plasmon resonance and for their ability to stimulate the catalytic activity of SHP-1. Both experiments have demonstrated that the selected peptides are capable of binding to the SH2 domains with dissociation constants (K(D)) in the low micromolar range.     

Capillary electrophoresis laser-induced fluorescence for screening combinatorial peptide libraries in assays of botulinum neurotoxin A

Botulinum neurotoxin serotype A (BoNT/A) is a proteolytic enzyme that induces muscle paralysis. It is a cause of food poisoning, a potential bioterrorist threat and, in low doses an emerging pharmaceutical product. No effective treatment is currently available for BoNT intoxication. Previously we developed a BoNT/A light chain enzyme assay using a peptide substrate based on the SNAP-25 protein target, with HPLC separation and UV detection of assay products, and applied the method to screen combinatorial peptide libraries for inhibitory activity to BoNT/A. We now report on development of a capillary electrophoresis laser-induced fluorescence (CE-LIF) method for measuring BoNT/A activity. The enzyme assay products were labeled with CBQCA dye followed by CE separation on a bare fused silica column in a HEPES-based buffer and LIF detection. All assay products were separated in CE within 8 min compared to incomplete separation of assay products within 1h by HPLC. The labeled products showed linear dependence of intensity versus concentration, and quantitative mole-fraction assignments. We used the CE-LIF method to screen combinatorial peptide libraries for potential modulating effects on BoNT/A peptidase activity. With some of the libraries, peptides co-migrated with assay products and interfered with quantitation. In such cases, interference was reduced by substituting sodium dodecyl sulfate (SDS) for Tween-20 in the running buffer. Separation in the capillaries then occurred by micellar electrokinetic chromatography (MEKC). The CE-LIF method is quick and lends itself to high-throughput or microfluidic formats.     

Synthesis and screening of a random dimeric peptide library using the one-bead-one-dimer combinatorial approach

Large combinatorial libraries of random peptides have been used for a variety of applications that include analysis of protein-protein interactions, epitope mapping, and drug targeting. The major obstacle in screening these libraries is the loss of specific but low affinity binding peptides during washing steps. Loss of these specific binders often results in isolation of peptides that bind nonspecifically to components used in the selection process. Previously, it has been demonstrated that dimerizing or multimerizing a peptide can remarkably improve its binding kinetics by 10- to 1000-fold due to an avidity effect. To take advantage of this observation, we constructed a random library of 12 amino acid dimeric peptides on polyethylene glycol acrylamide (PEGA) beads by modifying the 'one-bead-one-compound' approach. The chemical synthesis of 100,000 peptides as dimers can be problematic due to steric and aggregation effects and the presence of many peptide sequences that are difficult to synthesize. We have designed a method, described in detail here, to minimize the problems inherent in the synthesis of a dimeric library by modifying the existing 'split and pool' synthetic method. Using this approach the dimeric library was used to isolate a series of peptides that bound selectively to epithelial cancer cells. One peptide with the amino acid sequence QMARIPKRLARH bound as a dimer to prostate cancer cells spiked into the blood but did not bind to circulating hematopoeitic cells. The monomeric form of this peptide, however, did not bind well to the same LNCaP cell line. These data demonstrate that "hits" obtained from such a 'one-bead-one-dimer' library can be used directly for the final application or used as leads for construction of second generation libraries.     

A review of the utility of soluble peptide combinatorial libraries

This is paper reviews the preparation and use of soluble synthetic combinatorial libraries (SCLs) made up of millions of peptide and nonpeptide sequences for the identification of highly active individual compounds. First presented in 1991. SCLs have been prepared in a number of different lengths and formats, and are composed entirely of L -, D -, and unnatural amino acids. Also, existing peptide libraries have been chemically transformed to yield large diversities of nonpeptidic compounds. This review encompasses the published work from this laboratory using SCLs for the identification of antigenic sequences recognized by monoclonal antibodies, novel peptide agonists and antagonists to opioid receptors, new trypsin inhibitors, novel antibacterials, and compounds that inhibit melittin's hemolytic activity. SCLs offer a fundamental, practical advance in the study of interactions between peptide and nonpeptide sequences and their biochemical or pharmacological targets. © 1994 John Wiley & Sons, Inc.