Structural Basis for Species Selectivity in the HIV-1 gp120-CD4 Interaction: Restoring Affinity to gp120 in Murine CD4 Mimetic Peptides

The first step of HIV-1 infection involves interaction between the viral glycoprotein gp120 and the human cellular receptor CD4. Inhibition of the gp120-CD4 interaction represents an attractive strategy to block HIV-1 infection. In an attempt to explore the known lack of affinity of murine CD4 to gp120, we have investigated peptides presenting the putative gp120-binding site of murine CD4 (mCD4). Molecular modeling indicates that mCD4 protein cannot bind gp120 due to steric clashes, while the larger conformational flexibility of mCD4 peptides allows an interaction. This finding is confirmed by experimental binding assays, which also evidenced specificity of the peptide-gp120 interaction. Molecular dynamics simulations indicate that the mCD4-peptide stably interacts with gp120 via an intermolecular β-sheet, while an important salt-bridge formed by a C-terminal lysine is lost. Fixation of the C-terminus by introducing a disulfide bridge between the N- and C-termini of the peptide significantly enhanced the affinity to gp120

The diversity and utility of amyloid fibrils formed by short amyloidogenic peptides

Amyloidogenic peptides are well known for their involvement in diseases such as type 2 diabetes and Alzheimer's disease. However, more recently, amyloid fibrils have been shown to provide scaffolding and protection as functional materials in a range of organisms from bacteria to humans. These roles highlight the incredible tensile strength of the cross-β amyloid architecture. Many amino acid sequences are able to self-assemble to form amyloid with a cross-β core. Here we describe our recent advances in understanding how sequence contributes to amyloidogenicity and structure. For example, we describe penta- and hexapeptides that assemble to form different morphologies; a 12mer peptide that forms fibrous crystals; and an eight-residue peptide originating from α-synuclein that has the ability to form nanotubes. This work provides a wide range of peptides that may be exploited as fibrous bionanomaterials. These fibrils provide a scaffold upon which functional groups may be added, or templated assembly may be performed

A novel D-amino acid peptide with therapeutic potential (ISAD1) inhibits aggregation of neurotoxic disease-relevant mutant Tau and prevents Tau toxicity in vitro

Background: Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder that mainly affects older adults. One of the pathological hallmarks of AD is abnormally aggregated Tau protein that forms fibrillar deposits in the brain. In AD, Tau pathology correlates strongly with clinical symptoms, cognitive dysfunction, and neuronal death. Methods: We aimed to develop novel therapeutic D-amino acid peptides as Tau fibrillization inhibitors. It has been previously demonstrated that D-amino acid peptides are protease stable and less immunogenic than L-peptides, and these characteristics may render them suitable for in vivo applications. Using a phage display procedure against wild type full-length Tau (TauFL), we selected a novel Tau binding L-peptide and synthesized its D-amino acid version ISAD1 and its retro inversed form, ISAD1rev, respectively. Results: While ISAD1rev inhibited Tau aggregation only moderately, ISAD1 bound to Tau in the aggregation-prone PHF6 region and inhibited fibrillization of TauFL, disease-associated mutant full-length Tau (TauFLΔK, TauFL-A152T, TauFL-P301L), and pro-aggregant repeat domain Tau mutant (TauRDΔK). ISAD1 and ISAD1rev induced the formation of large high molecular weight TauFL and TauRDΔK oligomers that lack proper Thioflavin-positive β-sheet conformation even at lower concentrations. In silico modeling of ISAD1 Tau interaction at the PHF6 site revealed a binding mode similar to those known for other PHF6 binding peptides. Cell culture experiments demonstrated that ISAD1 and its inverse form are taken up by N2a-TauRDΔK cells efficiently and prevent cytotoxicity of externally added Tau fibrils as well as of internally expressed TauRDΔK. Conclusions: ISAD1 and related peptides may be suitable for therapy development of AD by promoting off-pathway assembly of Tau, thus preventing its toxicity. Keywords: Alzheimer’s disease; D-amino acid peptides; Phage display; Tau aggregation inhibitors; Therap

Structure-based synthetic mimicry of discontinuous protein binding sites: inhibitors of the interaction of Mena EVH1 domain with proline-rich ligands.

The Mena EVH1 domain, a protein-interaction module involved in actin-based cell motility, recognizes proline-rich ligand motifs, which are also present in the sequence of the surface protein ActA of Listeria monocytogenes. The interaction of ActA with host Mena EVH1 enables the bacterium to actively recruit host actin in order to spread into neighboring cells. Based on the crystal structure of Mena EVH1 in complex with a polyproline peptide ligand, we have generated a range of assembled peptides presenting the Mena EVH1 fragments that make up its discontinuous binding site for proline-rich ligands. Some of these peptides were found to inhibit the interaction of Mena EVH1 with the ligand pGolemi. One of them was further characterized at the level of individual amino acid residues; this yielded information on the contribution of individual positions of the peptides to the interaction with the ligand and identified sites for future structure optimization

Influence of nonenzymatic posttranslational modifications on constitution, oligomerization and receptor binding of S100A12.

This study examined the effect of methylglyoxal (MGO)-derived nonenzymatic posttranslational modifications (nePTMs) on the binding affinity of S100A12 to its natural receptor for advanced glycation end-products (RAGE). Binding of MGO-modified S100A12 to RAGE decreased significantly with increasing MGO concentration and incubation time. Ca(2+)-induced S100A12 hexamerization was impaired only at higher MGO concentrations indicating that the loss of affinity is not predominantly caused by disturbance of ligand oligomerization. nePTM mapping showed carboxyethylation of lysine (CEL) and the N-terminus without preferential modification sites. Besides, hydroimidazolone, hemiaminals, argpyrimidine, and tetrahydropyrimidine rapidly formed at R21. Even at the highest modification rate, hexamerization of synthesized CEL-S100A12 was unaffected and RAGE-binding only slightly impaired. Thus, nePTMs at R21 seem to be the major cause of MGO-induced impairment of S100A12 oligomerization and RAGE binding

Versatile C3-symmetric scaffolds and their use for covalent stabilization of the foldon trimer

C 3-Symmetric trimesic acid scaffolds, functionalized with bromoacetyl, aminooxyacetyl and azidoacetyl moieties, respectively, were synthesized and compared regarding their utility for the trivalent presentation of peptides using three different chemoselective ligation reactions, i.e. thioether and oxime formation, as well as the “click” reaction. The latter ligation method was then used to covalently stabilize the trimer of foldon, a 27 amino acid trimerization domain of bacteriophage T4 fibritin, by linking the three foldon monomers to the triazido-functionalized trimesic acid scaffold. This reaction dramatically enhanced the thermal stability of the trimer, while maintaining the correct fold, as demonstrated by CD spectroscopy and X-ray crystal structure analysis, respectively, of the foldon–scaffold conjugates

The solution structure of pGolemi, a high affinity Mena EVH1 binding miniature protein, suggests explanations for paralog-specific binding to Ena/VASP homology (EVH) 1 domains

Ena/VASP homology 1 (EVH1) domains are polyproline binding domains that are present in a wide range of adaptor proteins, among them Ena/VASP proteins involved in actin remodeling and axonal guidance. The interaction of ActA, a transmembrane protein from the food-borne pathogen Listeria monocytogenes, with EVH1 domains has been shown to be crucial for recruitment of the host's actin skeleton and, as a consequence, for the infectivity of this bacterium. We present the structure of a synthetic high-affinity Mena EVH1 ligand, pGolemi, capable of paralog-specific binding, solved by NMR spectroscopy. This peptide shares the common pancreatic peptide fold with its scaffold, avian pancreatic peptide, but shows pivotal differences in the amino-terminus. The interplay of spatial fixation and flexibility appears to be the reason for its high affinity towards Mena EVH1. Combined with earlier investigations, our structural data shed light on the specificity determinants of pGolemi and the importance of additional binding epitopes around the residues Thr74 and Phe32 on EVH1 domains regulating paralog specificity. Our results are expected to facilitate the design of other high-affinity, paralog-specific EVH1 domain ligands, and serve as a fundament for the investigation of the molecular mode of action of EVH1 domains