Exploring Protein-Peptide Binding Specificity through Computational Peptide Screening

The binding of short disordered peptide stretches to globular protein domains is important for a wide range of cellular processes, including signal transduction, protein transport, and immune response. The often promiscuous nature of these interactions and the conformational flexibility of the peptide chain, sometimes even when bound, make the binding specificity of this type of protein interaction a challenge to understand. Here we develop and test a Monte Carlo-based procedure for calculating protein-peptide binding thermodynamics for many sequences in a single run. The method explores both peptide sequence and conformational space simultaneously by simulating a joint probability distribution which, in particular, makes searching through peptide sequence space computationally efficient. To test our method, we apply it to 3 different peptide-binding protein domains and test its ability to capture the experimentally determined specificity profiles. Insight into the molecular underpinnings of the observed specificities is obtained by analyzing the peptide conformational ensembles of a large number of binding-competent sequences. We also explore the possibility of using our method to discover new peptide-binding pockets on protein structures.     

Active Peptide Sequences in the Cell Binding Domain of Thrombospondins

Methods for measuring the attachment of viable cells to immobilized peptides to identify sequences possessing cell binding activity are described. The 212-residue C-terminal cell binding domain (CBD) of human thrombospondin-1 (TS1 or TS) was thought to contain a site or sites for the attachment of a number of cell types based on inhibition of cell attachment to TS1 with monoclonal antibodies. The presence of such a site was confirmed by expression of this region of TS1 in Escherichia coli and demonstration of its cell binding activity independent of other known cell attachment sites in TS1. To further define active sequences within the CBD, a set of eight overlapping peptides that spanned the 212-residue domain was synthesized with standard techniques. Several of these long (30-mer) peptides were found to be insoluble in aqueous buffers; thus, they could not be tested as soluble inhibitors of the attachment of cells to TS1, a standard approach to identification of active cell binding sequences. Using chemically derivatized plates prepared by Costar Corp., we devised assays to test the direct attachment of cells to the peptides immobilized on plastic wells. We also found that the peptides were adsorbed to underivatized plastic when solubilized in 6 M guanidine hydrochloride. Even rather short peptides (5 residues) were efficiently immobilized with these methods. Peptides immobilized directly on plastic gave higher levels of cell attachment than the same peptides conjugated to a carrier protein and then immobilized. Using these methods, two of the 30-mers were found to support cell attachment. The active sequences within the 30-mers were then narrowed down to a hexamer in one case and a pentamer, both containing the tripeptide VVM. Shorter, soluble peptides containing these active sequences act synergistically to inhibit the attachment of cells to TS1 and its recombinant CBD.     

Importance of Cry1 δ-Endotoxin Domain II Loops for Binding Specificity in Heliothis virescens (L.)

We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens. Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with ¹²⁵I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H. virescens. Cry1Ac competed with high affinity (competition constant [K_com] = 1.1 nM) for ¹²⁵I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for ¹²⁵I-Cry1Ab binding sites, though the K_com values ranged from 179 to 304 nM. Cry1Ab competed for ¹²⁵I-Cry1Ac binding sites (K_com = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the ¹²⁵I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H. virescens. Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H. virescens. The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H. virescens.     

Crucial Roles of Single Residues in Binding Affinity,Specificity, and Promiscuity in the Cellulosomal Cohesin-Dockerin Interface

Interactions between cohesin and dockerin modules play a crucial role in the assembly of multienzyme cellulosome complexes. Although intraspecies cohesin and dockerin modules bind in general with high affinity but indiscriminately, cross-species binding is rare. Here, we combined ELISA-based experiments with Rosetta-based computational design to evaluate the contribution of distinct residues at the Clostridium thermocellum cohesin-dockerin interface to binding affinity, specificity, and promiscuity. We found that single mutations can show distinct and significant effects on binding affinity and specificity. In particular, mutations at cohesin position Asn³⁷ show dramatic variability in their effect on dockerin binding affinity and specificity: the N37A mutant binds promiscuously both to cognate (C. thermocellum) as well as to non-cognate Clostridium cellulolyticum dockerin. N37L in turn switches binding specificity: compared with the wild-type C. thermocellum cohesin, this mutant shows significantly increased preference for C. cellulolyticum dockerin combined with strongly reduced binding to its cognate C. thermocellum dockerin. The observation that a single mutation can overcome the naturally observed specificity barrier provides insights into the evolutionary dynamics of this system that allows rapid modulation of binding specificity within a high affinity background.     

Peptide Binding to a PDZ Domain by Electrostatic Steering via Nonnative Salt Bridges

We have captured the binding of a peptide to a PDZ domain by unbiased molecular dynamics simulations. Analysis of the trajectories reveals on-pathway encounter complex formation, which is driven by electrostatic interactions between negatively charged carboxylate groups in the peptide and positively charged side chains surrounding the binding site. In contrast, the final stereospecific complex, which matches the crystal structure, features completely different interactions, namely the burial of the hydrophobic side chain of the peptide C-terminal residue and backbone hydrogen bonds. The simulations show that nonnative salt bridges stabilize kinetically the encounter complex during binding. Unbinding follows the inverse sequence of events with the same nonnative salt bridges in the encounter complex. Thus, in contrast to protein folding, which is driven by native interactions, the binding of charged peptides can be steered by nonnative interactions, which might be a general mechanism, e.g., in the recognition of histone tails by bromodomains.     

Phosphorylation of the LIR Domain of SCOC Modulates ATG8 Binding Affinity and Specificity

Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood.We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic.Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.     

Computer Aided Study of Ligand Binding With Catalytic Domain of Avian Sarcoma Virus Integrase and Its Ligand Binding Loops

Abstract

We have carried out 1 nanosecond (ns) Molecular Dynamics (MD) simulations of the drug Y3 (4-acetylamino-5-hydroxynaphthalene-2, 7-disulfonic acid) complexed with catalytic domain of Avian sarcoma virus Integrase (ASV-IN), both in vacuum and in the presence of explicit solvent. Starting models were obtained on the basis of PDB co-ordinates (1A5X) of ASV-IN-Y3 complex, by Lubkowski et al [1]. Mn²⁺ cation was present in the active site. To neutralize the positive charge in the presence of explicit solvent, eight Cl^? anions were added. Energy Minimization (EM) and MD simulations, for both the systems, were carried out using Sander's module of AMBER5.0 [2] with all atom force field. Analysis of ligand- protein interaction in both environments is discussed in the paper. We also carried out 1 ns MD simulation on two flexible loops—L1 (Gly54-Gln62) and L2 (Trp138-Met155) playing crucial role in interaction of IN with the drug [3], under differing environmental conditions (vacuum, aqueous and organic solvent methanol). Comparison of the conformational changes in the loops, monomer and dimer is presented in the paper. Our results showed that the conformation of the loop region was closest to crystallographic data in case of monomer and constrained loops in aqueous environment. However, the dimer in vacuum was more stable than monomer. The β sheet structure of the monomer in aqueous environment was unstable. Latter also took long time for equilibration. The box formed by loops L1 and L2 from two sub units IINA and INB) of the dimer satisfies prerequisites for ligand recognition site and seems to be the functional biological unit.     

Accurate PDZ/Peptide Binding Specificity with Additive and Polarizable Free Energy Simulations

PDZ domains contain 80–100 amino acids and bind short C-terminal sequences of target proteins. Their specificity is essential for cellular signaling pathways. We studied the binding of the Tiam1 PDZ domain to peptides derived from the C-termini of its Syndecan-1 and Caspr4 targets. We used free energy perturbation (FEP) to characterize the binding energetics of one wild-type and 17 mutant complexes by simulating 21 alchemical transformations between pairs of complexes. Thirteen complexes had known experimental affinities. FEP is a powerful tool to understand protein/ligand binding. It depends, however, on the accuracy of molecular dynamics force fields and conformational sampling. Both aspects require continued testing, especially for ionic mutations. For six mutations that did not modify the net charge, we obtained excellent agreement with experiment using the additive, AMBER ff99SB force field, with a root mean square deviation (RMSD) of 0.37 kcal/mol. For six ionic mutations that modified the net charge, agreement was also good, with one large error (3 kcal/mol) and an RMSD of 0.9 kcal/mol for the other five. The large error arose from the overstabilization of a protein/peptide salt bridge by the additive force field. Four of the ionic mutations were also simulated with the polarizable Drude force field, which represents the first test of this force field for protein/ligand binding free energy changes. The large error was eliminated and the RMS error for the four mutations was reduced from 1.8 to 1.2 kcal/mol. The overall accuracy of FEP indicates it can be used to understand PDZ/peptide binding. Importantly, our results show that for ionic mutations in buried regions, electronic polarization plays a significant role.     

Characterization of a Peptide that Specifically Blocks the Ras Binding Domain of p75

The neurotrophin receptor p75 interacts with the GTPase Ras. Unstimulated it inactivates Ras while ligand binding induces Ras activation. We developed an inhibitory peptide (ip75RBD) which interferes with the binding domain of Ras of the intracellular domain of p75. ip75RBD inhibits the binding of Ras to the receptor in vitro. It is membrane-permeable and inhibits ligand-induced Ras activation via p75 in vivo but does not influence Ras activation by the stimulated receptor tyrosine kinases Trk and the epidermal growth factor receptor EGFR. The activation of the neutral sphingomyelinase by stimulated p75 is slightly delayed but not inhibited by the peptide. p75-mediated neuronal death induced by NGF or aggregated beta-amyloid_1–42 is reduced. We conclude that ip75RBD specifically blocks the Ras binding site of p75 and can be used to analyze p75-induced Ras signaling.     

Sequence-Altered Peptide Adopts Optimum Conformation for Modification-Dependent Binding of the Yeast tRNAPhe Anticodon Domain

Amino acid contributions to protein recognition of naturally modified RNAs are not understood. Circular dichroism spectra and predictive software suggested that peptide tF2 (S1ISPW5GFSGL10 LRWSY15), selected from a phage display library to bind the modified anticodon domain of yeast tRNAPhe (ASL), adopted a beta-sheet structure. Ala residues incorporated at positions Pro4 and Gly6, both predicted to be involved in a turn, did not alter the peptide binding affinity for the ASLPhe, although major changes in the peptide's CD spectra were observed. Substitutions at three positions Pro4, Gly6, and Gly9, the latter not predicted to be in a turn, reduced the peptide's binding affinity to 4% of that of the unsubstituted tF2 and strongly influenced the peptide's secondary structure. The results suggest that peptides with different conformations, but similar affinities, adopt the optimal binding conformation, indicative of a structurally adaptive model of binding in which the modified RNA serves as a scaffold.     

Membrane Binding by the Endophilin N-BAR Domain

The structure of the endophilin N-terminal amphipathic helix Bin/Amphiphysin/Rvs-homology (N-BAR) domain is unique because of an additional insert helix under the arch of the N-BAR dimer. The structure of this additional helix has not been fully resolved in crystallographic studies, and thus presents a challenge to molecular-level analysis. Large-scale molecular-dynamics simulations were therefore employed to investigate the interaction of a single endophilin N-BAR with a lipid bilayer. Various possible configurations of the additional insert helix under the top of the arch of the endophilin N-BAR were modeled to examine their effect on membrane bending. A residue-residue and residue-lipid headgroup distance analysis, similar to that performed with electron paramagnetic resonance spectroscopy, revealed that the insert helix remains perpendicular to the long axis of the N-BAR over the duration of the simulations. It was also found that the degree of membrane bending is directly related to the orientation of the additional insert helix, and that the perpendicular configuration generates the largest curvature consistent with mutation experiments. In addition, the angle formed between the two N-BAR monomers at the top of the arch is sensitive to the orientation of the insert helices. A membrane sensing-binding-bending mechanism is proposed to describe the process of an endophilin N-BAR interaction with a membrane.     

The Specificity of Peptide Chain Extension by N-Carboxyanhydrides

We have used amino acids activated by carbonyldiimidazole to study the enantiospecificity of peptide elongation in aqueoussolution. Peptide `primers' Glu₁₀ and Ala₃Glu₁₀were elongated with the enantiomers of arginine, glutamic acid,asparagine, phenylalanine, serine and valine. The homochiral addition was always the more efficient reaction; the enantiospecificity was large in some cases but very small in others. In every case Ala₃Glu₁₀ was elongated more efficiently than Glu₁₀.     

Specificity of Milk Peptide Utilization by Lactococcus lactis

To study the substrate specificity of the oligopeptide transport system of Lactococcus lactis for its natural substrates, the growth of L. lactis MG1363 was studied in a chemically defined medium containing milk peptides or a tryptic digest of α_s2-casein as the source of amino acids. Peptides were separated into acidic, neutral, and basic pools by solid-phase extraction or by cation-exchange liquid chromatography. Their ability to sustain growth and the time course of their utilization demonstrated the preferential use of hydrophobic basic peptides with molecular masses ranging between 600 and 1,100 Da by L. lactis MG1363 and the inability to use large, acidic peptides. These peptide utilization preferences reflect the substrate specificity of the oligopeptide transport system of the strain, since no significant cell lysis was inferred. Considering the free amino acid content of milk and these findings on peptide utilization, it was demonstrated that the cessation of growth of L. lactis MG1363 in milk was due to deprivation of leucine and methionine.     

Solution Structure and Peptide Binding of the PTB Domain from the AIDA1 Postsynaptic Signaling Scaffolding Protein

AIDA1 links persistent chemical signaling events occurring at the neuronal synapse with global changes in gene expression. Consistent with its role as a scaffolding protein, AIDA1 is composed of several protein-protein interaction domains. Here we report the NMR structure of the carboxy terminally located phosphotyrosine binding domain (PTB) that is common to all AIDA1 splice variants. A comprehensive survey of peptides identified a consensus sequence around an NxxY motif that is shared by a number of related neuronal signaling proteins. Using peptide arrays and fluorescence based assays, we determined that the AIDA1 PTB domain binds amyloid protein precursor (APP) in a similar manner to the X11/Mint PTB domain, albeit at reduced affinity (∼10 µM) that may allow AIDA1 to effectively sample APP, as well as other protein partners in a variety of cellular contexts.     

Surface Loops in a Single SH2 Domain Are Capable of Encoding the Spectrum of Specificity of the SH2 Family,

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Highlights

• •Surface loops play an essential role in SH2 domain specificity.
• •Diverse specificities may be obtained from a single SH2 domain by combinatorial mutations in the EF and BG loops.
• •The specificity of a loop mutant correlates with the sequence characteristics of the bait peptide used in its isolation.