RESUMEN
Although ß-hairpins are widespread in proteins, there is no tool to coax any small peptide to adopt a ß-hairpin conformation, regardless of sequence. Here, we report that δ-linked γ(R)-methyl-ornithine (δ MeOrn) provides an improved ß-turn template for inducing a ß-hairpin conformation in peptides. We developed a synthesis of protected δ MeOrn as a building block suitable for use in Fmoc-based solid-phase peptide synthesis. The synthesis begins with l-leucine and affords gram quantities of the Nα -Boc-Nδ -Fmoc-γ(R)-methyl-ornithine building block. X-ray crystallography confirms that the δ MeOrn turn unit adopts a folded structure in a macrocyclic ß-hairpin peptide. CD and NMR spectroscopy allow comparison of the δ MeOrn turn template to the δ-linked ornithine (δ Orn) turn template that we previously introduced and to the popular d-Pro-Gly turn template. These studies show that the folding of the δ MeOrn turn template is substantially better than that of δ Orn and is comparable to d-Pro-Gly.
Asunto(s)
Péptidos/química , Dicroismo Circular , Cristalografía por Rayos X , Resonancia Magnética Nuclear Biomolecular , Ornitina/química , Péptidos/síntesis química , Estructura Secundaria de Proteína , Técnicas de Síntesis en Fase SólidaRESUMEN
Oligomers of the ß-amyloid peptide, Aß, play a central role in the pathogenesis and progression of Alzheimer's disease. Trimers and higher-order oligomers composed of trimers are thought to be the most neurotoxic Aß oligomers. To gain insights into the structure and assembly of Aß oligomers, our laboratory has previously designed and synthesized macrocyclic peptides derived from Aß17-23 and Aß30-36 that fold to form ß-hairpins and assemble to form trimers. In this study, we found that mutating Phe20 to cyclohexylalanine (Cha) in macrocyclic Aß-derived peptides promotes crystallization of an Aß-derived peptide containing the Aß24-29 loop (peptide 3F20Cha) and permits elucidation of its structure and assembly by X-ray crystallography. X-ray crystallography shows that peptide 3F20Cha forms a hexamer. X-ray crystallography and SDS-PAGE further show that trimer 4F20Cha, a covalently stabilized trimer derived from peptide 3F20Cha, forms a dodecamer. Size exclusion chromatography shows that trimer 4F20Cha forms higher-order assemblies in solution. Trimer 4F20Cha exhibits cytotoxicity against the neuroblastoma cell line SH-SY5Y. These studies demonstrate the use of the F20Cha mutation to further stabilize oligomers of Aß-derived peptides that contain more of the native sequence and thus better mimic the oligomers formed by full-length Aß.
Asunto(s)
Péptidos beta-Amiloides/química , Fenilalanina/análogos & derivados , Fenilalanina/química , Secuencia de Aminoácidos , Péptidos beta-Amiloides/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Mutación , Conformación Proteica en Lámina beta , Multimerización de ProteínaRESUMEN
A key challenge in studying the biological and biophysical properties of amyloid-forming peptides is that they assemble to form heterogeneous mixtures of soluble oligomers and insoluble fibrils. Photolabile protecting groups have emerged as tools to control the properties of biomolecules with light. Blocking intermolecular hydrogen bonds that stabilize amyloid oligomers provides a general strategy to control the biological and biophysical properties of amyloid-forming peptides. In this paper we describe the design, synthesis, and characterization of macrocyclic ß-hairpin peptides that are derived from amyloidogenic peptides and contain the N-2-nitrobenzyl photolabile protecting group. Each peptide contains two heptapeptide segments from Aß16-36 or Aß17-36 constrained into ß-hairpins. The N-2-nitrobenzyl group is appended to the amide backbone of Gly33 to disrupt the oligomerization of the peptides by disrupting intermolecular hydrogen bonds. X-ray crystallography reveals that N-2-nitrobenzyl groups can either block assembly into discrete oligomers or permit formation of trimers, hexamers, and dodecamers. Photolysis of the N-2-nitrobenzyl groups with long-wave UV light unmasks the amide backbone and alters the assembly and the biological properties of the macrocyclic ß-hairpin peptides. SDS-PAGE studies show that removing the N-2-nitrobenzyl groups alters the assembly of the peptides. MTT conversion and LDH release assays show that decaging the peptides induces cytotoxicity. Circular dichroism studies and dye leakage assays with liposomes reveal that decaging modulates interactions of the peptides with lipid bilayers. Collectively, these studies demonstrate that incorporating N-2-nitrobenzyl groups into macrocyclic ß-hairpin peptides provides a new strategy to probe the structures and the biological properties of amyloid oligomers.
RESUMEN
Soluble oligomers of the ß-amyloid peptide, Aß, are associated with the progression of Alzheimer's disease. Although many small molecules bind to these assemblies, the details of how these molecules interact with Aß oligomers remain unknown. This paper reports that crystal violet, and other C3 symmetric triphenylmethane dyes, bind to C3 symmetric trimers derived from Aß17-36. Binding changes the color of the dyes from purple to blue, and causes them to fluoresce red when irradiated with green light. Job plot and analytical ultracentrifugation experiments reveal that two trimers complex with one dye molecule. Studies with several triphenylmethane dyes reveal that three N, N-dialkylamino substituents are required for complexation. Several mutant trimers, in which Phe19, Phe20, and Ile31 were mutated to cyclohexylalanine, valine, and cyclohexylglycine, were prepared to probe the triphenylmethane dye binding site. Size exclusion chromatography, SDS-PAGE, and X-ray crystallographic studies demonstrate that these mutations do not impact the structure or assembly of the triangular trimer. Fluorescence spectroscopy and analytical ultracentrifugation experiments reveal that the dye packs against an aromatic surface formed by the Phe20 side chains and is clasped by the Ile31 side chains. Docking and molecular modeling provide a working model of the complex in which the triphenylmethane dye is sandwiched between two triangular trimers. Collectively, these findings demonstrate that the X-ray crystallographic structures of triangular trimers derived from Aß can be used to guide the discovery of ligands that bind to soluble oligomers derived from Aß.
Asunto(s)
Péptidos beta-Amiloides/química , Colorantes/química , Compuestos de Tritilo/química , Sitios de UniónRESUMEN
The absence of high-resolution structures of amyloid oligomers constitutes a major gap in our understanding of amyloid diseases. A growing body of evidence indicates that oligomers of the ß-amyloid peptide Aß are especially important in the progression of Alzheimer's disease. In many Aß oligomers, the Aß monomer components are thought to adopt a ß-hairpin conformation. This paper describes the design and study of a macrocyclic ß-hairpin peptide derived from Aß16-36. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography studies show that the Aß16-36 ß-hairpin peptide assembles in solution to form hexamers, trimers, and dimers. X-ray crystallography reveals that the peptide assembles to form a hexamer in the crystal state and that the hexamer is composed of dimers and trimers. Lactate dehydrogenase release assays show that the oligomers formed by the Aß16-36 ß-hairpin peptide are toxic toward neuronally derived SH-SY5Y cells. Replica-exchange molecular dynamics demonstrates that the hexamer can accommodate full-length Aß. These findings expand our understanding of the structure, solution-phase behavior, and biological activity of Aß oligomers and may offer insights into the molecular basis of Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/química , Fragmentos de Péptidos/química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Oligomeric assemblies of the protein α-synuclein are thought to cause neurodegeneration in Parkinson's disease and related synucleinopathies. Characterization of α-synuclein oligomers at high resolution is an outstanding challenge in the field of structural biology. The absence of high-resolution structures of oligomers formed by α-synuclein impedes understanding the synucleinopathies at the molecular level. This paper reports the X-ray crystallographic structure of oligomers formed by a peptide derived from residues 36-55 of α-synuclein. The peptide 1a adopts a ß-hairpin structure, which assembles in a hierarchical fashion. Three ß-hairpins assemble to form a triangular trimer. Three copies of the triangular trimer assemble to form a basket-shaped nonamer. Two nonamers pack to form an octadecamer. Molecular modeling suggests that full-length α-synuclein may also be able to assemble in this fashion. Circular dichroism spectroscopy demonstrates that peptide 1a interacts with anionic lipid bilayer membranes, like oligomers of full-length α-synuclein. LDH and MTT assays demonstrate that peptide 1a is toxic toward SH-SY5Y cells. Comparison of peptide 1a to homologues suggests that this toxicity results from nonspecific interactions with the cell membrane. The oligomers formed by peptide 1a are fundamentally different than the proposed models of the fibrils formed by α-synuclein and suggest that α-Syn36-55, rather than the NAC, may nucleate oligomer formation.
Asunto(s)
Modelos Moleculares , Enfermedad de Parkinson/metabolismo , Péptidos/química , Conformación Proteica , alfa-Sinucleína/química , Membrana Celular/metabolismo , Cristalografía por Rayos X , Resonancia Magnética Nuclear Biomolecular , alfa-Sinucleína/farmacologíaRESUMEN
Amyloid diseases such as Alzheimer's disease, Parkinson's disease, and type II diabetes share common features of toxic soluble protein oligomers. There are no structures at atomic resolution of oligomers formed by full-length amyloidogenic peptides and proteins, and only a few structures of oligomers formed by peptide fragments. The paucity of structural information provides a fundamental roadblock to understanding the pathology of amyloid diseases and developing preventions or therapies. Here, we present the X-ray crystallographic structures of three families of oligomers formed by macrocyclic peptides containing a heptapeptide sequence derived from the amyloidogenic E chain of ß2-microglobulin (ß2m). Each macrocyclic peptide contains the heptapeptide sequence ß2m63-69 and a second heptapeptide sequence containing an N-methyl amino acid. These peptides form ß-sheets that further associate into hexamers, octamers, and dodecamers: the hexamers are trimers of dimers; the octamers are tetramers of dimers; and the dodecamers contain two trimer subunits surrounded by three pairs of ß-sheets. These structures illustrate a common theme in which dimer and trimer subunits further associate to form a hydrophobic core. The seven X-ray crystallographic structures not only illustrate a range of oligomers that a single amyloidogenic peptide sequence can form, but also how mutation can alter the size and topology of the oligomers. A cocrystallization experiment in which a dodecamer-forming peptide recruits a hexamer-forming peptide to form mixed dodecamers demonstrates that one species can dictate the oligomerization of another. These findings should also be relevant to the formation of oligomers of full-length peptides and proteins in amyloid diseases.
Asunto(s)
Amiloide/química , Fragmentos de Péptidos/química , Microglobulina beta-2/química , Amiloide/metabolismo , Línea Celular , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Multimerización de Proteína , Microglobulina beta-2/metabolismoRESUMEN
Small macrocycles with four or fewer amino acids are among the most potent natural products known, but there is currently no way to systematically generate such compounds. We describe a computational method for identifying ordered macrocycles composed of alpha, beta, gamma, and 17 other amino acid backbone chemistries, which we used to predict 14.9 million closed cycles composed of >42,000 monomer combinations. We chemically synthesized 18 macrocycles predicted to adopt single low-energy states and determined their x-ray or nuclear magnetic resonance structures; 15 of these were very close to the design models. We illustrate the therapeutic potential of these macrocycle designs by developing selective inhibitors of three protein targets of current interest. By opening up a vast space of readily synthesizable drug-like macrocycles, our results should considerably enhance structure-based drug design.
Asunto(s)
Amidas , Aminoácidos , Productos Biológicos , Diseño de Fármacos , Péptidos Cíclicos , Amidas/química , Aminoácidos/química , Productos Biológicos/síntesis química , Productos Biológicos/química , Productos Biológicos/farmacología , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacologíaRESUMEN
We describe an approach for designing high-affinity small molecule-binding proteins poised for downstream sensing. We use deep learning-generated pseudocycles with repeating structural units surrounding central binding pockets with widely varying shapes that depend on the geometry and number of the repeat units. We dock small molecules of interest into the most shape complementary of these pseudocycles, design the interaction surfaces for high binding affinity, and experimentally screen to identify designs with the highest affinity. We obtain binders to four diverse molecules, including the polar and flexible methotrexate and thyroxine. Taking advantage of the modular repeat structure and central binding pockets, we construct chemically induced dimerization systems and low-noise nanopore sensors by splitting designs into domains that reassemble upon ligand addition.
Asunto(s)
Aprendizaje Profundo , Unión Proteica , Proteínas , Bibliotecas de Moléculas Pequeñas , Sitios de Unión , Ligandos , Metotrexato/química , Simulación del Acoplamiento Molecular , Nanoporos , Multimerización de Proteína , Proteínas/química , Bibliotecas de Moléculas Pequeñas/química , Tiroxina/químicaRESUMEN
A general method for designing proteins to bind and sense any small molecule of interest would be widely useful. Due to the small number of atoms to interact with, binding to small molecules with high affinity requires highly shape complementary pockets, and transducing binding events into signals is challenging. Here we describe an integrated deep learning and energy based approach for designing high shape complementarity binders to small molecules that are poised for downstream sensing applications. We employ deep learning generated psuedocycles with repeating structural units surrounding central pockets; depending on the geometry of the structural unit and repeat number, these pockets span wide ranges of sizes and shapes. For a small molecule target of interest, we extensively sample high shape complementarity pseudocycles to generate large numbers of customized potential binding pockets; the ligand binding poses and the interacting interfaces are then optimized for high affinity binding. We computationally design binders to four diverse molecules, including for the first time polar flexible molecules such as methotrexate and thyroxine, which are expressed at high levels and have nanomolar affinities straight out of the computer. Co-crystal structures are nearly identical to the design models. Taking advantage of the modular repeating structure of pseudocycles and central location of the binding pockets, we constructed low noise nanopore sensors and chemically induced dimerization systems by splitting the binders into domains which assemble into the original pseudocycle pocket upon target molecule addition.
RESUMEN
Despite remarkable advances in the assembly of highly structured coordination polymers and metal-organic frameworks, the rational design of such materials using more conformationally flexible organic ligands such as peptides remains challenging. In an effort to make the design of such materials fully programmable, we first developed a computational design method for generating metal-mediated 3D frameworks using rigid and symmetric peptide macrocycles with metal-coordinating sidechains. We solved the structures of six crystalline networks involving conformationally constrained 6 to 12 residue cyclic peptides with C2, C3, and S2 internal symmetry and three different types of metals (Zn2+, Co2+, or Cu2+) by single-crystal X-ray diffraction, which reveals how the peptide sequences, backbone symmetries, and metal coordination preferences drive the assembly of the resulting structures. In contrast to smaller ligands, these peptides associate through peptide-peptide interactions without full coordination of the metals, contrary to one of the assumptions underlying our computational design method. The cyclic peptides are the largest peptidic ligands reported to form crystalline coordination polymers with transition metals to date, and while more work is required to develop methods for fully programming their crystal structures, the combination of high chemical diversity with synthetic accessibility makes them attractive building blocks for engineering a broader set of new crystalline materials for use in applications such as sensing, asymmetric catalysis, and chiral separation.
RESUMEN
Peptide and protein bioconjugation technologies have revolutionized our ability to site-specifically or chemoselectively install a variety of functional groups for applications in chemical biology and medicine, including the enhancement of bioavailability. Here, we introduce a site-specific bioconjugation strategy inspired by chemical ligation at serine that relies on a noncanonical amino acid containing a 1-amino-2-hydroxy functional group and a salicylaldehyde ester. More specifically, we harness this technology to generate analogues of glucagon-like peptide-1 that resemble Semaglutide, a long-lasting blockbuster drug currently used in the clinic to regulate glucose levels in the blood. We identify peptides that are more potent than unmodified peptide and equipotent to Semaglutide in a cell-based activation assay, improve the stability in human serum, and increase glucose disposal efficiency in vivo. This approach demonstrates the potential of "serine ligation" for various applications in chemical biology, with a particular focus on generating stabilized peptide therapeutics.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón , Serina , Péptido 1 Similar al Glucagón , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Glucosa , Humanos , Hipoglucemiantes , Péptidos/farmacologíaRESUMEN
The assembly of the ß-amyloid peptide, Aß, into soluble oligomers is associated with neurodegeneration in Alzheimer's disease. The Aß oligomers are thought to be composed of ß-hairpins. Here, the effect of shifting the residue pairing of the ß-hairpins on the structures of the oligomers that form is explored through X-ray crystallography. Three residue pairings were investigated using constrained macrocyclic ß-hairpins in which Aß30-36 is juxtaposed with Aß17-23, Aß16-22, and Aß15-21. The Aß16-22-Aß30-36 pairing forms a compact ball-shaped dodecamer composed of fused triangular trimers. This dodecamer may help explain the structures of the trimers and dodecamers formed by full-length Aß.