RESUMO
Many peptide hormones form an α-helix on binding their receptors1-4, and sensitive methods for their detection could contribute to better clinical management of disease5. De novo protein design can now generate binders with high affinity and specificity to structured proteins6,7. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion8 to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar-affinity binders can be generated to helical peptide targets by either refining designs generated with other methods, or completely de novo starting from random noise distributions without any subsequent experimental optimization. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimize by partial diffusion both natural and designed proteins, should be broadly useful.
Assuntos
Desenho Assistido por Computador , Aprendizado Profundo , Peptídeos , Proteínas , Técnicas Biossensoriais , Difusão , Glucagon/química , Glucagon/metabolismo , Medições Luminescentes , Espectrometria de Massas , Hormônio Paratireóideo/química , Hormônio Paratireóideo/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Proteínas/química , Proteínas/metabolismo , Especificidade por Substrato , Modelos MolecularesRESUMO
We have developed Differential Specificity and Energy Landscape (DiSEL) analysis to comprehensively compare DNA-protein interactomes (DPIs) obtained by high-throughput experimental platforms and cutting edge computational methods. While high-affinity DNA binding sites are identified by most methods, DiSEL uncovered nuanced sequence preferences displayed by homologous transcription factors. Pairwise analysis of 726 DPIs uncovered homolog-specific differences at moderate- to low-affinity binding sites (submaximal sites). DiSEL analysis of variants of 41 transcription factors revealed that many disease-causing mutations result in allele-specific changes in binding site preferences. We focused on a set of highly homologous factors that have different biological roles but "read" DNA using identical amino acid side chains. Rather than direct readout, our results indicate that DNA noncontacting side chains allosterically contribute to sculpt distinct sequence preferences among closely related members of transcription factor families.
Assuntos
DNA/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , Técnica de Seleção de Aptâmeros , TermodinâmicaRESUMO
The rod of sarcomeric myosins directs thick filament assembly and is characterized by the insertion of four skip residues that introduce discontinuities in the coiled-coil heptad repeats. We report here that the regions surrounding the first three skip residues share high structural similarity despite their low sequence homology. Near each of these skip residues, the coiled-coil transitions to a nonclose-packed structure inducing local relaxation of the superhelical pitch. Moreover, molecular dynamics suggest that these distorted regions can assume different conformationally stable states. In contrast, the last skip residue region constitutes a true molecular hinge, providing C-terminal rod flexibility. Assembly of myosin with mutated skip residues in cardiomyocytes shows that the functional importance of each skip residue is associated with rod position and reveals the unique role of the molecular hinge in promoting myosin antiparallel packing. By defining the biophysical properties of the rod, the structures and molecular dynamic calculations presented here provide insight into thick filament formation, and highlight the structural differences occurring between the coiled-coils of myosin and the stereotypical tropomyosin. In addition to extending our knowledge into the conformational and biological properties of coiled-coil discontinuities, the molecular characterization of the four myosin skip residues also provides a guide to modeling the effects of rod mutations causing cardiac and skeletal myopathies.
Assuntos
Aminoácidos/química , Miosinas Cardíacas/química , Miosinas Cardíacas/metabolismo , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/metabolismo , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Sequência de Aminoácidos , Humanos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Maleabilidade , Estabilidade Proteica , Estrutura Secundária de Proteína , Sequências Repetitivas de Aminoácidos , Sarcômeros/metabolismo , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
The KIX domain of CBP is a transcriptional coactivator. Concomitant binding to the activation domain of proto-oncogene protein c-Myb and the transactivation domain of the trithorax group protein mixed lineage leukemia (MLL) transcription factor lead to the biologically active ternary MLLâ¶KIXâ¶c-Myb complex which plays a role in Pol II-mediated transcription. The binding of the activation domain of MLL to KIX enhances c-Myb binding. Here we carried out molecular dynamics (MD) simulations for the MLLâ¶KIXâ¶c-Myb ternary complex, its binary components and KIX with the goal of providing a mechanistic explanation for the experimental observations. The dynamic behavior revealed that the MLL binding site is allosterically coupled to the c-Myb binding site. MLL binding redistributes the conformational ensemble of KIX, leading to higher populations of states which favor c-Myb binding. The key element in the allosteric communication pathways is the KIX loop, which acts as a control mechanism to enhance subsequent binding events. We tested this conclusion by in silico mutations of loop residues in the KIXâ¶MLL complex and by comparing wild type and mutant dynamics through MD simulations. The loop assumed MLL binding conformation similar to that observed in the KIXâ¶c-Myb state which disfavors the allosteric network. The coupling with c-Myb binding site faded, abolishing the positive cooperativity observed in the presence of MLL. Our major conclusion is that by eliciting a loop-mediated allosteric switch between the different states following the binding events, transcriptional activation can be regulated. The KIX system presents an example how nature makes use of conformational control in higher level regulation of transcriptional activity and thus cellular events.
Assuntos
Proteína de Ligação a CREB/química , Modelos Químicos , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide/química , Proteínas Proto-Oncogênicas c-myb/química , Ativação Transcricional , Sítios de Ligação , Proteína de Ligação a CREB/ultraestrutura , Simulação por Computador , Histona-Lisina N-Metiltransferase , Proteína de Leucina Linfoide-Mieloide/ultraestrutura , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myb/ultraestruturaRESUMO
The human lymphotactin (hLtn) is a protein that features two native states both of which are physiologically relevant: it is a monomer (hLtn10) at 10 °C with 200 mM salt and a dimer (hLtn40) at 40 °C and without salt. Here we focus on the networks of electrostatic and hydrophobic interactions that display substantial changes upon the conversion from hLtn10 to hLtn40 since they are expected to modulate the relative stability of the two folds. In addition to the Arg 23-Arg 43 interaction discussed in previous work, we find several other like-charge pairs that are likely important to the stability of hLtn10. Free energy perturbation calculations are carried out to explicitly evaluate the contribution of the Arg 23-Arg 43 interaction to the hLtn10 stability. hLtn40 features a larger number of salt bridges, and a set of hydrophobic residues undergo major changes in the solvent accessible surface area between hLtn10 and hLtn40, pointing to their importance to the relative stability of the two folds. We also discuss the use of explicit and implicit solvent simulations for characterizing the conformational ensembles under different solution conditions.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Linfocinas/química , Sialoglicoproteínas/química , Eletricidade Estática , Humanos , Simulação de Dinâmica Molecular , Estabilidade Proteica , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
The aryl hydrocarbon receptor (AHR) is a transcription factor that responds to diverse ligands and plays a critical role in toxicology, immune function, and cardiovascular physiology. The structural basis of the AHR for ligand promiscuity and preferences is critical for understanding AHR function. Based on the structure of a closely related protein HIF2α, we modeled the AHR ligand binding domain (LBD) bound to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(a)pyrene (BaP) and identified residues that control ligand preferences by shape and H-bond potential. Mutations to these residues, particularly Q377 and G298, resulted in robust and opposite changes in the potency of TCDD and BaP and up to a 20-fold change in the ratio of TCDD/BaP efficacy. The model also revealed a flexible "belt" structure; molecular dynamic (MD) simulation suggested that the "belt" and several other structural elements in the AHR-LBD are more flexible than HIF2α and likely contribute to ligand promiscuity. Molecular docking of TCDD congeners to a model of human AHR-LBD ranks their binding affinity similar to experimental ranking of their toxicity. Our study reveals key structural basis for prediction of toxicity and understanding the AHR signaling through diverse ligands.