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ACS Chemical Biology Aug 2018Peptide secondary and tertiary structure motifs frequently serve as inspiration for the development of protein-protein interaction (PPI) inhibitors. While a wide variety...
Peptide secondary and tertiary structure motifs frequently serve as inspiration for the development of protein-protein interaction (PPI) inhibitors. While a wide variety of strategies have been used to stabilize or imitate α-helices, similar strategies for β-sheet stabilization are more limited. Synthetic scaffolds that stabilize reverse turns and cross-strand interactions have provided important insights into β-sheet stability and folding. However, these templates occupy regions of the β-sheet that might impact the β-sheet's ability to bind at a PPI interface. Here, we present the hydrogen bond surrogate (HBS) approach for stabilization of β-hairpin peptides. The HBS linkage replaces a cross-strand hydrogen bond with a covalent linkage, conferring significant conformational and proteolytic resistance. Importantly, this approach introduces the stabilizing linkage in the buried β-sheet interior, retains all side chains for further functionalization, and allows efficient solid-phase macrocyclization. We anticipate that HBS stabilization of PPI β-sheets will enhance the development of β-sheet PPI inhibitors and expand the repertoire of druggable PPIs.
Topics: Cyclization; Hydrogen Bonding; Peptides; Protein Conformation, beta-Strand; Protein Engineering; Protein Stability
PubMed: 30005156
DOI: 10.1021/acschembio.8b00641 -
Proteins Nov 2020Protein domains exist by themselves or in combination with other domains to form complex multidomain proteins. Defining domain boundaries in proteins is essential for...
Protein domains exist by themselves or in combination with other domains to form complex multidomain proteins. Defining domain boundaries in proteins is essential for understanding their evolution and function but is not trivial. More specifically, partitioning domains that interact by forming a single β-sheet is known to be particularly troublesome for automatic structure-based domain decomposition pipelines. Here, we study edge-to-edge β-strand interactions between domains in a protein chain, to help define the boundaries for some more difficult cases where a single β-sheet spanning over two domains gives an appearance of one. We give a number of examples where β-strands belonging to a single β-sheet do not belong to a single domain and highlight the difficulties of automatic domain parsers on these examples. This work can be used as a baseline for defining domain boundaries in homologous proteins or proteins with similar domain interactions in the future.
Topics: Amino Acid Isomerases; Amino Acid Sequence; Animals; Bacteria; Binding Sites; Databases, Protein; Datasets as Topic; Humans; Models, Molecular; Penicillin-Binding Proteins; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Racemases and Epimerases; Thermodynamics
PubMed: 32543729
DOI: 10.1002/prot.25970 -
Molecules (Basel, Switzerland) Oct 202114-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition,...
14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer's and Parkinson's diseases, respectively, a process that is intimately linked to the diseases' progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of N-labeled Aβ and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.
Topics: 14-3-3 Proteins; Amyloid; Amyloid beta-Peptides; Humans; Molecular Chaperones; Protein Aggregates; Protein Binding; Protein Conformation; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Unfolding; alpha-Synuclein
PubMed: 34684701
DOI: 10.3390/molecules26206120 -
Structure (London, England : 1993) Aug 2019CD160 is a signaling molecule that interacts with herpes virus entry mediator (HVEM) and contributes to a wide range of immune responses, including T cell inhibition,...
CD160 is a signaling molecule that interacts with herpes virus entry mediator (HVEM) and contributes to a wide range of immune responses, including T cell inhibition, natural killer cell activation, and mucosal immunity. GPI-anchored and transmembrane isoforms of CD160 share the same ectodomain responsible for HVEM engagement, which leads to bidirectional signaling. Despite the importance of the CD160:HVEM signaling axis and its therapeutic relevance, the structural and mechanistic basis underlying CD160-HVEM engagement has not been described. We report the crystal structures of the human CD160 extracellular domain and its complex with human HVEM. CD160 adopts a unique variation of the immunoglobulin fold and exists as a monomer in solution. The CD160:HVEM assembly exhibits a 1:1 stoichiometry and a binding interface similar to that observed in the BTLA:HVEM complex. Our work reveals the chemical and physical determinants underlying CD160:HVEM recognition and initiation of associated signaling processes.
Topics: Antigens, CD; Binding Sites; Crystallography, X-Ray; GPI-Linked Proteins; HEK293 Cells; Humans; Models, Molecular; Protein Binding; Protein Conformation, beta-Strand; Protein Domains; Protein Folding; Receptors, Immunologic; Receptors, Tumor Necrosis Factor, Member 14
PubMed: 31230945
DOI: 10.1016/j.str.2019.05.010 -
Analytical Chemistry Apr 2022Conventional in vitro aggregation assays often involve tagging with extrinsic fluorophores, which can interfere with aggregation. We propose the use of intrinsic amyloid...
Conventional in vitro aggregation assays often involve tagging with extrinsic fluorophores, which can interfere with aggregation. We propose the use of intrinsic amyloid fluorescence lifetime probed using two-photon excitation and represented by model-free phasor plots as a label-free assay to characterize the amyloid structure. Intrinsic amyloid fluorescence arises from the structured packing of β-sheets in amyloids and is independent of aromatic-based fluorescence. We show that different amyloids [i.e., α-Synuclein (αS), β-Lactoglobulin (βLG), and TasA] and different polymorphic populations of αS (induced by aggregation in salt-free and salt buffers mimicking the intra-/extracellular environments) can be differentiated by their unique fluorescence lifetimes. Moreover, we observe that disaggregation of the preformed fibrils of αS and βLG leads to increased fluorescence lifetimes, distinct from those of their fibrillar counterparts. Our assay presents a medium-throughput method for rapid classification of amyloids and their polymorphs (the latter of which recent studies have shown lead to different disease pathologies) and for testing small-molecule inhibitory compounds.
Topics: Amyloid; Amyloidogenic Proteins; Fluorescence; Protein Conformation, beta-Strand; alpha-Synuclein
PubMed: 35333515
DOI: 10.1021/acs.analchem.1c05651 -
Proteins Aug 2020Peptide-protein docking is challenging due to the considerable conformational freedom of the peptide. CAPRI rounds 38-45 included two peptide-protein interactions, both...
Peptide-protein docking is challenging due to the considerable conformational freedom of the peptide. CAPRI rounds 38-45 included two peptide-protein interactions, both characterized by a peptide forming an additional beta strand of a beta sheet in the receptor. Using the Rosetta FlexPepDock peptide docking protocol we generated top-performing, high-accuracy models for targets 134 and 135, involving an interaction between a peptide derived from L-MAG with DLC8. In addition, we were able to generate the only medium-accuracy models for a particularly challenging target, T121. In contrast to the classical peptide-mediated interaction, in which receptor side chains contact both peptide backbone and side chains, beta-sheet complementation involves a major contribution to binding by hydrogen bonds between main chain atoms. To establish how binding affinity and specificity are established in this special class of peptide-protein interactions, we extracted PeptiDBeta, a benchmark of solved structures of different protein domains that are bound by peptides via beta-sheet complementation, and tested our protocol for global peptide-docking PIPER-FlexPepDock on this dataset. We find that the beta-strand part of the peptide is sufficient to generate approximate and even high resolution models of many interactions, but inclusion of adjacent motif residues often provides additional information necessary to achieve high resolution model quality.
Topics: Amino Acid Sequence; Animals; Binding Sites; Dyneins; Humans; Hydrogen Bonding; Ligands; Mice; Molecular Docking Simulation; Myelin-Associated Glycoprotein; Peptides; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Protein Multimerization; Proteins; Research Design; Software; Structural Homology, Protein; Thermodynamics
PubMed: 31891416
DOI: 10.1002/prot.25871 -
PloS One 2020Fibrillar aggregates of amyloid-β (Aβ) are the main component of plaques lining the cerebrovasculature in cerebral amyloid angiopathy. As the predominant Aβ isoform...
Fibrillar aggregates of amyloid-β (Aβ) are the main component of plaques lining the cerebrovasculature in cerebral amyloid angiopathy. As the predominant Aβ isoform in vascular deposits, Aβ40 is a valuable target in cerebral amyloid angiopathy research. However, the slow process of Aβ40 aggregation in vitro is a bottleneck in the search for Aβ-targeting molecules. In this study, we sought a method to accelerate the aggregation of Aβ40 in vitro, to improve experimental screening procedures. We evaluated the aggregating ability of bicine, a biological buffer, using various in vitro methods. Our data suggest that bicine promotes the aggregation of Aβ40 with high speed and reproducibility, yielding a mixture of aggregates with significant β-sheet-rich fibril formation and toxicity.
Topics: Amyloid beta-Peptides; Animals; Cell Line; Cell Survival; Cerebral Amyloid Angiopathy; Glycine; Humans; Mice; Neurons; Peptide Fragments; Protein Aggregates; Protein Conformation, beta-Strand
PubMed: 33048999
DOI: 10.1371/journal.pone.0240608 -
Macromolecular Bioscience Apr 2018Native silk fibroin (NSF) is a unique biomaterial with extraordinary mechanical and biochemical properties. These key characteristics are directly associated with the...
Native silk fibroin (NSF) is a unique biomaterial with extraordinary mechanical and biochemical properties. These key characteristics are directly associated with the physical transformation of unstructured, soluble NSF into highly organized nano- and microscale fibrils rich in β-sheet content. Here, it is shown that this NSF fibrillation process is accompanied by the development of intrinsic fluorescence in the visible range, upon near-UV excitation, a phenomenon that has not been investigated in detail to date. Here, the optical and fluorescence characteristics of NSF fibrils are probed and a route for potential applications in the field of self-assembled optically active biomaterials and systems is explored. In particular, it is demonstrated that NSF can be structured into autofluorescent microcapsules with a controllable level of β-sheet content and fluorescence properties. Furthermore, a facile and efficient fabrication route that permits arbitrary patterns of NSF microcapsules to be deposited on substrates under ambient conditions is shown. The resulting fluorescent NSF patterns display a high level of photostability. These results demonstrate the potential of using native silk as a new class of biocompatible photonic material.
Topics: Animals; Biocompatible Materials; Bombyx; Capsules; Fibroins; Fluorescence; Protein Conformation, beta-Strand; Silk
PubMed: 29377575
DOI: 10.1002/mabi.201700295 -
Colloids and Surfaces. B, Biointerfaces Jan 2022Cancer is one of the main causes of death worldwide. The current cancer treatment strategies often lack selectivity for cancer cells resulting in dose-limiting adverse...
Cancer is one of the main causes of death worldwide. The current cancer treatment strategies often lack selectivity for cancer cells resulting in dose-limiting adverse effects and reduced quality of life. Recently, anticancer peptides (ACPs) have emerged as an alternative treatment with higher selectivity, less adverse effects, and lower propensity for drug resistance. However, most of the current studies on the ACPs are focused on α-helical ACPs and there is lack of systematic studies on β-sheet forming ACPs. Herein we report the development of a new series of rationally designed short cationic amphiphilic β-sheet forming ACPs and their structure activity relationship. The peptides had the general formula (XYXY), with X representing hydrophobic amino acids (isoleucine (I) or leucine (L)), Y and Y representing cationic amino acids (arginine (R) or lysine (K)). The cytotoxicity of the designed ACPs in HCT 116 colorectal cancer, HeLa cervical cancer and human dermal fibroblast (HDF) cells was assessed by MTT test. The physicochemical properties of the peptides were characterized by various techniques including RP-HPLC, LC-MS, and Circular Dichroism (CD) spectroscopy. The surface activity of the peptides at the air-water interface and their interaction with the lipid monolayers as models for cell membranes were studied by Langmuir trough. The peptides consisting of I with R and K had selective anticancer activity while the combination of L and R diminished the anticancer activity of the peptides but rendered them more toxic to HDFs. The anticancer activity of the peptides was directed by their surface activity (amphiphilicity) and their secondary structure in hydrophobic surfaces including cancer cell membranes. The selectivity of the peptides for cancer cells was a result of their higher penetration into cancer cell membranes compared to normal cell membranes. The peptides exerted their anticancer activity by disrupting the mitochondrial membranes and eventually apoptosis. The results presented in this study provide an insight into the structure-activity relationship of this class of ACPs which can be employed as guidance to design new ACPs with improved anticancer activity and lower toxicity against normal cells.
Topics: Antineoplastic Agents; Humans; Peptides; Protein Conformation, beta-Strand; Protein Structure, Secondary; Quality of Life
PubMed: 34715505
DOI: 10.1016/j.colsurfb.2021.112165 -
Proceedings of the National Academy of... Mar 2020Peptide self-assembly, wherein molecule A associates with other A molecules to form fibrillar β-sheet structures, is common in nature and widely used to fabricate...
Peptide self-assembly, wherein molecule A associates with other A molecules to form fibrillar β-sheet structures, is common in nature and widely used to fabricate synthetic biomaterials. Selective coassembly of peptide pairs A and B with complementary partial charges is gaining interest due to its potential for expanding the form and function of biomaterials that can be realized. It has been hypothesized that charge-complementary peptides organize into alternating ABAB-type arrangements within assembled β-sheets, but no direct molecular-level evidence exists to support this interpretation. We report a computational and experimental approach to characterize molecular-level organization of the established peptide pair, CATCH. Discontinuous molecular dynamics simulations predict that CATCH(+) and CATCH(-) peptides coassemble but do not self-assemble. Two-layer β-sheet amyloid structures predominate, but off-pathway β-barrel oligomers are also predicted. At low concentration, transmission electron microscopy and dynamic light scattering identified nonfibrillar ∼20-nm oligomers, while at high concentrations elongated fibers predominated. Thioflavin T fluorimetry estimates rapid and near-stoichiometric coassembly of CATCH(+) and CATCH(-) at concentrations ≥100 μM. Natural abundance C NMR and isotope-edited Fourier transform infrared spectroscopy indicate that CATCH(+) and CATCH(-) coassemble into two-component nanofibers instead of self-sorting. However, C-C dipolar recoupling solid-state NMR measurements also identify nonnegligible AA and BB interactions among a majority of AB pairs. Collectively, these results demonstrate that strictly alternating arrangements of β-strands predominate in coassembled CATCH structures, but deviations from perfect alternation occur. Off-pathway β-barrel oligomers are also suggested to occur in coassembled β-strand peptide systems.
Topics: Amyloid; Computer Simulation; Nanofibers; Polymerization; Protein Conformation, beta-Strand; Protein Multimerization; Static Electricity
PubMed: 32071201
DOI: 10.1073/pnas.1912810117