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We describe the discovery and structure of an undecapeptide natural product from a marine sponge, termed halichondamide A, that is morphed into a fused bicyclic ring topology via two disulfide bonds. Molecular dynamics simulations allow us to posit that the installation of one disulfide bond biases the intermediate peptide conformation and predisposes the formation of the second disulfide bond. The natural product was found to be mildly cytotoxic against liver and breast cancer cell lines.
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Artificial intelligence-driven advances in protein structure prediction in recent years have raised the question: has the protein structure-prediction problem been solved? Here, with a focus on nonglobular proteins, we highlight the many strengths and potential weaknesses of DeepMind's AlphaFold2 in the context of its biological and therapeutic applications. We summarize the subtleties associated with evaluation of AlphaFold2 model quality and reliability using the predicted local distance difference test (pLDDT) and predicted aligned error (PAE) values. We highlight various classes of proteins that AlphaFold2 can be applied to and the caveats involved. Concrete examples of how AlphaFold2 models can be integrated with experimental data in the form of small-angle X-ray scattering (SAXS), solution NMR, cryo-electron microscopy (cryo-EM) and X-ray diffraction are discussed. Finally, we highlight the need to move beyond structure prediction of rigid, static structural snapshots toward conformational ensembles and alternate biologically relevant states. The overarching theme is that careful consideration is due when using AlphaFold2-generated models to generate testable hypotheses and structural models, rather than treating predicted models as de facto ground truth structures.
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Modelos Moleculares , Conformación Proteica , Proteínas , Dispersión del Ángulo Pequeño , Proteínas/química , Difracción de Rayos X , Microscopía por Crioelectrón , Pliegue de Proteína , Inteligencia Artificial , HumanosRESUMEN
To biosynthesize ribosomally synthesized and post-translationally modified peptides (RiPPs), enzymes recognize and bind to the N-terminal leader region of substrate peptides which enables catalytic modification of the C-terminal core. Our current understanding of RiPP leaders is that they are short and largely unstructured. Proteusins are RiPP precursor peptides that defy this characterization as they possess unusually long leaders. Proteusin peptides have not been structurally characterized, and we possess scant understanding of how these atypical leaders engage with modifying enzymes. Here, we determine the structure of a proteusin peptide which shows that unlike other RiPP leaders, proteusin leaders are preorganized into a rigidly structured region and a smaller intrinsically disordered region. With residue level resolution gained from NMR titration experiments, the intermolecular peptide-protein interactions between proteusin leaders and a flavin-dependent brominase are mapped onto the disordered region, leaving the rigidly structured region of the proteusin leader to be functionally dispensable. Spectroscopic observations are biochemically validated to identify a binding motif in proteusin peptides that is conserved among other RiPP leaders as well. This study provides a structural characterization of the proteusin peptides and extends the paradigm of RiPP modification enzymes using not only unstructured peptides, but also structured proteins as substrates.
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Productos Biológicos , Ribosomas , Ribosomas/metabolismo , Péptidos/química , Procesamiento Proteico-Postraduccional , Catálisis , Compuestos Orgánicos/metabolismo , Productos Biológicos/químicaRESUMEN
The lasting threat of viral pandemics necessitates the development of tailorable first-response antivirals with specific but adaptive architectures for treatment of novel viral infections. Here, such an antiviral platform has been developed based on a mixture of hetero-peptides self-assembled into functionalized ß-sheets capable of specific multivalent binding to viral protein complexes. One domain of each hetero-peptide is designed to specifically bind to certain viral proteins, while another domain self-assembles into fibrils with epitope binding characteristics determined by the types of peptides and their molar fractions. The self-assembled fibrils maintain enhanced binding to viral protein complexes and retain high resilience to viral mutations. This method is experimentally and computationally tested using short peptides that specifically bind to Spike proteins of SARS-CoV-2. This platform is efficacious, inexpensive, and stable with excellent tolerability.
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COVID-19 , Humanos , Péptidos/química , SARS-CoV-2/metabolismo , Antivirales/farmacología , Proteínas Virales , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
De novo peptide design is a new frontier that has broad application potential in the biological and biomedical fields. Most existing models for de novo peptide design are largely based on sequence homology that can be restricted based on evolutionarily derived protein sequences and lack the physicochemical context essential in protein folding. Generative machine learning for de novo peptide design is a promising way to synthesize theoretical data that are based on, but unique from, the observable universe. In this study, we created and tested a custom peptide generative adversarial network intended to design peptide sequences that can fold into the ß-hairpin secondary structure. This deep neural network model is designed to establish a preliminary foundation of the generative approach based on physicochemical and conformational properties of 20 canonical amino acids, for example, hydrophobicity and residue volume, using extant structure-specific sequence data from the PDB. The beta generative adversarial network model robustly distinguishes secondary structures of ß hairpin from α helix and intrinsically disordered peptides with an accuracy of up to 96% and generates artificial ß-hairpin peptide sequences with minimum sequence identities around 31% and 50% when compared against the current NCBI PDB and nonredundant databases, respectively. These results highlight the potential of generative models specifically anchored by physicochemical and conformational property features of amino acids to expand the sequence-to-structure landscape of proteins beyond evolutionary limits.
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We applied solid- and solution-state nuclear magnetic resonance spectroscopy to examine the structure of multidomain peptides composed of self-assembling ß-sheet domains linked to bioactive domains. Bioactive domains can be selected to stimulate specific biological responses (e.g., via receptor binding), while the ß-sheets provide the desirable nanoscale properties. Although previous work has established the efficacy of multidomain peptides, molecular-level characterization is lacking. The bioactive domains are intended to remain solvent-accessible without being incorporated into the ß-sheet structure. We tested for three possible anticipated molecular-level consequences of introducing bioactive domains to ß-sheet-forming peptides: (1) the bioactive domain has no effect on the self-assembling peptide structure; (2) the bioactive domain is incorporated into the ß-sheet nanofiber; and (3) the bioactive domain interferes with self-assembly such that nanofibers are not formed. The peptides involved in this study incorporated self-assembling domains based on the (SL)6 motif and bioactive domains including a VEGF-A mimic (QK), an IGF-mimic (IGF-1c), and a de novo SARS-CoV-2 binding peptide (SBP3). We observed all three of the anticipated outcomes from our examination of peptides, illustrating the unintended structural effects that could adversely affect the desired biofunctionality and biomaterial properties of the resulting peptide hydrogel. This work is the first attempt to evaluate the structural effects of incorporating bioactive domains into a set of peptides unified by a similar self-assembling peptide domain. These structural insights reveal unmet challenges in the design of highly tunable bioactive self-assembling peptide hydrogels.
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Nanofibras , Péptidos , Conformación Proteica en Lámina beta , Péptidos/química , Nanofibras/química , Hidrogeles/química , Materiales BiocompatiblesRESUMEN
The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in viral replication and transcription and received great attention as a vital target for drug/peptide development. Therapeutic agents such as small-molecule drugs or peptides that interact with the Cys-His present in the catalytic site of Mpro are an efficient way to inhibit the protease. Although several emergency-approved vaccines showed good efficacy and drastically dropped the infection rate, evolving variants are still infecting and killing millions of people globally. While a small-molecule drug (Paxlovid) received emergency approval, small-molecule drugs have low target specificity and higher toxicity. Besides small-molecule drugs, peptide therapeutics are thus gaining increasing popularity as they are easy to synthesize and highly selective and have limited side effects. In this study, we investigated the therapeutic value of 67 peptides targeting Mpro using molecular docking. Subsequently, molecular dynamics (MD) simulations were implemented on eight protein-peptide complexes to obtain molecular-level information on the interaction between these peptides and the Mpro active site, which revealed that temporin L, indolicidin, and lymphocytic choriomeningitis virus (LCMV) GP1 are the best candidates in terms of stability, interaction, and structural compactness. These peptides were synthesized using the solid-phase peptide synthesis protocol, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and authenticated by mass spectrometry (MS). The in vitro fluorometric Mpro activity assay was used to validate the computational results, where temporin L and indolicidin were observed to be very active against SARS-CoV-2 Mpro with IC50 values of 38.80 and 87.23 µM, respectively. A liquid chromatography-MS (LC-MS) assay was developed, and the IC50 value of temporin L was measured at 23.8 µM. The solution-state nuclear magnetic resonance (NMR) structure of temporin L was determined in the absence of sodium dodecyl sulfate (SDS) micelles and was compared to previous temporin structures. This combined investigation provides critical insights and assists us to further develop peptide inhibitors of SARS-CoV-2 Mpro through structural guided investigation.
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COVID-19 , Péptido Hidrolasas , Humanos , SARS-CoV-2 , Simulación del Acoplamiento Molecular , Antivirales/farmacología , Inhibidores de Proteasas/farmacología , Simulación de Dinámica MolecularRESUMEN
The conformational landscapes of peptide/human leucocyte antigen (pHLA) protein complexes encompassing tumor neoantigens provide a rationale for target selection towards autologous T cell, vaccine, and antibody-based therapeutic modalities. Here, using complementary biophysical and computational methods, we characterize recurrent RAS55-64 Q61 neoepitopes presented by the common HLA-A*01:01 allotype. We integrate sparse NMR restraints with Rosetta docking to determine the solution structure of NRASQ61K/HLA-A*01:01, which enables modeling of other common RAS55-64 neoepitopes. Hydrogen/deuterium exchange mass spectrometry experiments alongside molecular dynamics simulations reveal differences in solvent accessibility and conformational plasticity across a panel of common Q61 neoepitopes that are relevant for recognition by immunoreceptors. Finally, we predict binding and provide structural models of NRASQ61K antigens spanning the entire HLA allelic landscape, together with in vitro validation for HLA-A*01:191, HLA-B*15:01, and HLA-C*08:02. Our work provides a basis to delineate the solution surface features and immunogenicity of clinically relevant neoepitope/HLA targets for cancer therapy.
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Antígenos de Neoplasias , Neoplasias , Humanos , Antígenos de Neoplasias/genética , Péptidos/metabolismo , Antígenos de Histocompatibilidad , Antígenos HLA-ARESUMEN
CD1 immunoreceptors are a non-classical major histocompatibility complex (MHC) that present antigens to T cells to elucidate immune responses against disease. The antigen repertoire of CD1 has been composed primarily of lipids until recently when CD1d-restricted T cells were shown to be activated by non-lipidic small molecules, such as phenyl pentamethyl dihydrobenzofuran sulfonate (PPBF) and related benzofuran sulfonates. To date structural insights into PPBF/CD1d interactions are lacking, so it is unknown whether small molecule and lipid antigens are presented and recognized through similar mechanisms. Furthermore, it is unknown whether CD1d can bind to and present a broader range of small molecule metabolites to T cells, acting out functions analogous to the MHC class I related protein MR1. Here, we perform in silico docking and molecular dynamics simulations to structurally characterize small molecule interactions with CD1d. PPBF was supported to be presented to T cell receptors through the CD1d F' pocket. Virtual screening of CD1d against more than 17,000 small molecules with diverse geometry and chemistry identified several novel scaffolds, including phytosterols, cholesterols, triterpenes, and carbazole alkaloids, that serve as candidate CD1d antigens. Protein-ligand interaction profiling revealed conserved residues in the CD1d F' pocket that similarly anchor small molecules and lipids. Our results suggest that CD1d could have the intrinsic ability to bind and present a broad range of small molecule metabolites to T cells to carry out its function beyond lipid antigen presentation.Communicated by Ramaswamy H. Sarma.
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Commensal bacteria associated with marine invertebrates are underappreciated sources of chemically novel natural products. Using mass spectrometry, we had previously detected the presence of peptidic natural products in obligate marine bacteria of the genus Microbulbifer cultured from marine sponges. In this report, the isolation and structural characterization of a panel of ureidohexapeptide natural products, termed the bulbiferamides, from Microbulbifer strains is reported wherein the tryptophan side chain indole participates in a macrocyclizing peptide bond formation. Genome sequencing identifies biosynthetic gene clusters encoding production of the bulbiferamides and implicates the involvement of a thioesterase in the indolic macrocycle formation. The structural diversity and widespread presence of bulbiferamides in commensal microbiomes of marine invertebrates point toward a possible ecological role for these natural products.
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Productos Biológicos , Poríferos , Animales , Productos Biológicos/química , Bacterias/genética , Poríferos/microbiología , Organismos Acuáticos , Acilación , IndolesRESUMEN
Chaperones tapasin and transporter associated with antigen processing (TAP)-binding protein related (TAPBPR) associate with the major histocompatibility complex (MHC)-related protein 1 (MR1) to promote trafficking and cell surface expression. However, the binding mechanism and ligand dependency of MR1/chaperone interactions remain incompletely characterized. Here in vitro, biochemical and computational studies reveal that, unlike MHC-I, TAPBPR recognizes MR1 in a ligand-independent manner owing to the absence of major structural changes in the MR1 α2-1 helix between empty and ligand-loaded molecules. Structural characterization using paramagnetic nuclear magnetic resonance experiments combined with restrained molecular dynamics simulations reveals that TAPBPR engages conserved surfaces on MR1 to induce similar adaptations to those seen in MHC-I/TAPBPR co-crystal structures. Finally, nuclear magnetic resonance relaxation dispersion experiments using 19F-labeled diclofenac show that TAPBPR can affect the exchange kinetics of noncovalent metabolites with the MR1 groove, serving as a catalyst. Our results support a role of chaperones in stabilizing nascent MR1 molecules to enable loading of endogenous or exogenous cargo.
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Antígenos de Histocompatibilidad Clase I , Inmunoglobulinas , Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/química , Inmunoglobulinas/química , Ligandos , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares , Péptidos/químicaRESUMEN
Chaperones Tapasin and TAP-binding protein related (TAPBPR) perform the important functions of stabilizing nascent MHC-I molecules (chaperoning) and selecting high-affinity peptides in the MHC-I groove (editing). While X-ray and cryo-EM snapshots of MHC-I in complex with TAPBPR and Tapasin, respectively, have provided important insights into the peptide-deficient MHC-I groove structure, the molecular mechanism through which these chaperones influence the selection of specific amino acid sequences remains incompletely characterized. Based on structural and functional data, a loop sequence of variable lengths has been proposed to stabilize empty MHC-I molecules through direct interactions with the floor of the groove. Using deep mutagenesis on two complementary expression systems, we find that important residues for the Tapasin/TAPBPR chaperoning activity are located on a large scaffolding surface, excluding the loop. Conversely, loop mutations influence TAPBPR interactions with properly conformed MHC-I molecules, relevant for peptide editing. Detailed biophysical characterization by solution NMR, ITC and FP-based assays shows that the loop hovers above the MHC-I groove to promote the capture of incoming peptides. Our results suggest that the longer loop of TAPBPR lowers the affinity requirements for peptide selection to facilitate peptide loading under conditions and subcellular compartments of reduced ligand concentration, and to prevent disassembly of high-affinity peptide-MHC-I complexes that are transiently interrogated by TAPBPR during editing.
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Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Antígenos/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Técnicas de Inactivación de Genes , Células HEK293 , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/aislamiento & purificación , Antígenos de Histocompatibilidad Clase I/ultraestructura , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/aislamiento & purificación , Inmunoglobulinas/ultraestructura , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/ultraestructura , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/ultraestructura , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mutación , Biblioteca de Péptidos , Unión Proteica/genética , Unión Proteica/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructuraRESUMEN
Methyl-specific isotope labeling is a powerful tool to study the structure, dynamics and interactions of large proteins and protein complexes by solution-state NMR. However, widespread applications of this methodology have been limited by challenges in obtaining confident resonance assignments. Here, we present Methyl Assignments Using Satisfiability (MAUS), leveraging Nuclear Overhauser Effect cross-peak data, peak residue type classification and a known 3D structure or structural model to provide robust resonance assignments consistent with all the experimental inputs. Using data recorded for targets with known assignments in the 10-45 kDa size range, MAUS outperforms existing methods by up to 25,000 times in speed while maintaining 100% accuracy. We derive de novo assignments for multiple Cas9 nuclease domains, demonstrating that the methyl resonances of multi-domain proteins can be assigned accurately in a matter of days, while reducing biases introduced by manual pre-processing of the raw NOE data. MAUS is available through an online web-server.
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Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodos , Algoritmos , Proteína 9 Asociada a CRISPR/ultraestructura , Isótopos de Carbono , Interleucina-2/química , Interleucina-2/aislamiento & purificación , Marcaje Isotópico/métodos , Resonancia Magnética Nuclear Biomolecular/instrumentación , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/ultraestructura , Streptococcus pyogenes/enzimología , TritioRESUMEN
The plasticity of naturally occurring protein structures, which can change shape considerably in response to changes in environmental conditions, is critical to biological function. While computational methods have been used for de novo design of proteins that fold to a single state with a deep free-energy minimum [P.-S. Huang, S. E. Boyken, D. Baker, Nature 537, 320-327 (2016)], and to reengineer natural proteins to alter their dynamics [J. A. Davey, A. M. Damry, N. K. Goto, R. A. Chica, Nat. Chem. Biol. 13, 1280-1285 (2017)] or fold [P. A. Alexander, Y. He, Y. Chen, J. Orban, P. N. Bryan, Proc. Natl. Acad. Sci. U.S.A. 106, 21149-21154 (2009)], the de novo design of closely related sequences which adopt well-defined but structurally divergent structures remains an outstanding challenge. We designed closely related sequences (over 94% identity) that can adopt two very different homotrimeric helical bundle conformations-one short (â¼66 Å height) and the other long (â¼100 Å height)-reminiscent of the conformational transition of viral fusion proteins. Crystallographic and NMR spectroscopic characterization shows that both the short- and long-state sequences fold as designed. We sought to design bistable sequences for which both states are accessible, and obtained a single designed protein sequence that populates either the short state or the long state depending on the measurement conditions. The design of sequences which are poised to adopt two very different conformations sets the stage for creating large-scale conformational switches between structurally divergent forms.
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Biología Computacional/métodos , Proteínas/química , Secuencia de Aminoácidos/genética , Aminoácidos/química , Conformación Molecular , Conformación Proteica , Ingeniería de Proteínas/métodos , Pliegue de ProteínaRESUMEN
The computational design of a symmetric protein homo-oligomer that binds a symmetry-matched small molecule larger than a metal ion has not yet been achieved. We used de novo protein design to create a homo-trimeric protein that binds the C3 symmetric small molecule drug amantadine with each protein monomer making identical interactions with each face of the small molecule. Solution NMR data show that the protein has regular three-fold symmetry and undergoes localized structural changes upon ligand binding. A high-resolution X-ray structure reveals a close overall match to the design model with the exception of water molecules in the amantadine binding site not included in the Rosetta design calculations, and a neutron structure provides experimental validation of the computationally designed hydrogen-bond networks. Exploration of approaches to generate a small molecule inducible homo-trimerization system based on the design highlight challenges that must be overcome to computationally design such systems.
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Amantadina/química , Ingeniería de Proteínas , Proteínas/química , Bibliotecas de Moléculas Pequeñas/química , Sitios de Unión/efectos de los fármacos , Química Computacional , Simulación por Computador , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Multimerización de Proteína/efectos de los fármacos , Proteínas/antagonistas & inhibidoresRESUMEN
The interplay between a highly polymorphic set of MHC-I alleles and molecular chaperones shapes the repertoire of peptide antigens displayed on the cell surface for T cell surveillance. Here, we demonstrate that the molecular chaperone TAP-binding protein related (TAPBPR) associates with a broad range of partially folded MHC-I species inside the cell. Bimolecular fluorescence complementation and deep mutational scanning reveal that TAPBPR recognition is polarized toward the α2 domain of the peptide-binding groove, and depends on the formation of a conserved MHC-I disulfide epitope in the α2 domain. Conversely, thermodynamic measurements of TAPBPR binding for a representative set of properly conformed, peptide-loaded molecules suggest a narrower MHC-I specificity range. Using solution NMR, we find that the extent of dynamics at "hotspot" surfaces confers TAPBPR recognition of a sparsely populated MHC-I state attained through a global conformational change. Consistently, restriction of MHC-I groove plasticity through the introduction of a disulfide bond between the α1/α2 helices abrogates TAPBPR binding, both in solution and on a cellular membrane, while intracellular binding is tolerant of many destabilizing MHC-I substitutions. Our data support parallel TAPBPR functions of 1) chaperoning unstable MHC-I molecules with broad allele-specificity at early stages of their folding process, and 2) editing the peptide cargo of properly conformed MHC-I molecules en route to the surface, which demonstrates a narrower specificity. Our results suggest that TAPBPR exploits localized structural adaptations, both near and distant to the peptide-binding groove, to selectively recognize discrete conformational states sampled by MHC-I alleles, toward editing the repertoire of displayed antigens.
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Antígenos de Histocompatibilidad Clase I , Chaperonas Moleculares , Péptidos , Disulfuros/química , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoglobulinas/química , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Resonancia Magnética Nuclear Biomolecular , Péptidos/química , Péptidos/metabolismo , Conformación Proteica , Dominios ProteicosRESUMEN
Intrinsically disordered transcription factor transactivation domains (TADs) function through structural plasticity, adopting ordered conformations when bound to transcriptional co-regulators. Many transcription factors contain a negative regulatory domain (NRD) that suppresses recruitment of transcriptional machinery through autoregulation of the TAD. We report the solution structure of an autoinhibited NRD-TAD complex within FoxM1, a critical activator of mitotic gene expression. We observe that while both the FoxM1 NRD and TAD are primarily intrinsically disordered domains, they associate and adopt a structured conformation. We identify how Plk1 and Cdk kinases cooperate to phosphorylate FoxM1, which releases the TAD into a disordered conformation that then associates with the TAZ2 or KIX domains of the transcriptional co-activator CBP. Our results support a mechanism of FoxM1 regulation in which the TAD undergoes switching between disordered and different ordered structures.
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Activación Enzimática , Proteína Forkhead Box M1/química , Proteína Forkhead Box M1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fragmentos de Péptidos/metabolismo , Fosforilación , Unión Proteica , Conformación Proteica , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Sialoglicoproteínas/metabolismo , Quinasa Tipo Polo 1RESUMEN
ß-sheet proteins carry out critical functions in biology, and hence are attractive scaffolds for computational protein design. Despite this potential, de novo design of all-ß-sheet proteins from first principles lags far behind the design of all-α or mixed-αß domains owing to their non-local nature and the tendency of exposed ß-strand edges to aggregate. Through study of loops connecting unpaired ß-strands (ß-arches), we have identified a series of structural relationships between loop geometry, side chain directionality and ß-strand length that arise from hydrogen bonding and packing constraints on regular ß-sheet structures. We use these rules to de novo design jellyroll structures with double-stranded ß-helices formed by eight antiparallel ß-strands. The nuclear magnetic resonance structure of a hyperthermostable design closely matched the computational model, demonstrating accurate control over the ß-sheet structure and loop geometry. Our results open the door to the design of a broad range of non-local ß-sheet protein structures.
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Ingeniería de Proteínas/métodos , Proteínas/química , Secuencia de Aminoácidos , Simulación por Computador , Enlace de Hidrógeno , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Conformación Proteica en Lámina beta , Pliegue de Proteína , Estabilidad Proteica , Proteínas/genéticaRESUMEN
Antigen presentation is a cellular process that involves a number of steps, beginning with the production of peptides by proteolysis or aberrant synthesis and the delivery of peptides to cellular compartments where they are loaded on MHC class I (MHC-I) or MHC class II (MHC-II) molecules. The selective loading and editing of high-affinity immunodominant antigens is orchestrated by molecular chaperones: tapasin/TAP-binding protein, related for MHC-I and HLA-DM for MHC-II. Once peptide/MHC (pMHC) complexes are assembled, following various steps of quality control, they are delivered to the cell surface, where they are available for identification by αß receptors on CD8+ or CD4+ T lymphocytes. In addition, recognition of cell surface peptide/MHC-I complexes by natural killer cell receptors plays a regulatory role in some aspects of the innate immune response. Many of the components of the pathways of antigen processing and presentation and of T cell receptor (TCR)-mediated signaling have been studied extensively by biochemical, genetic, immunological, and structural approaches over the past several decades. Until recently, however, dynamic aspects of the interactions of peptide with MHC, MHC with molecular chaperones, or of pMHC with TCR have been difficult to address experimentally, although computational approaches such as molecular dynamics (MD) simulations have been illuminating. Studies exploiting X-ray crystallography, cryo-electron microscopy, and multidimensional nuclear magnetic resonance (NMR) spectroscopy are beginning to reveal the importance of molecular flexibility as it pertains to peptide loading onto MHC molecules, the interactions between pMHC and TCR, and subsequent TCR-mediated signals. In addition, recent structural and dynamic insights into how molecular chaperones define peptide selection and fine-tune the MHC displayed antigen repertoire are discussed. Here, we offer a review of current knowledge that highlights experimental data obtained by X-ray crystallography and multidimensional NMR methodologies. Collectively, these findings strongly support a multifaceted role for protein plasticity and conformational dynamics throughout the antigen processing and presentation pathway in dictating antigen selection and recognition.