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.     

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.     

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.     

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.     

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.     

Low Energy Conformations for S100 Binding Peptide from the Negative Regulatory Domain of p53

We have computed the low energy conformations of the negative regulatory domain of p53, residues 374–388 using Empirical Conformational Energies of Peptides Program including solvation and computed the statistical weights of distinct conformational states. We find that there are two high probability conformations, one an α-helix from Lys 374–Lys 381, followed by another helical structure involving Lys 382–Glu 388 (statistical weight of 0.48) and an all-α-helix for the entire sequence (statistical weight of 0.23). Both structure are superimposable on the NMR structure of this sequence bound to the S100 protein. The global minimum structure (statistical weight of 0.014) is a beta structure from Gly 374–Arg 379 followed by an α-helix from His 380–Glu 388. Based on these results, we propose a possible strategy for enhancement of p53 anti-tumor activity in cancer cells. Since the structure of this sequence bound to the sirtuin protein, Sir2, is a β-sheet, we further propose that the global minimum may be an intermediate on the α-β structure transition.     

Rescue of Amyloid-Beta-Induced Inhibition of Nicotinic Acetylcholine Receptors by a Peptide Homologous to the Nicotine Binding Domain of the Alpha 7 Subtype

Alzheimer''s disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD.