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1.
J Chem Inf Model ; 64(5): 1682-1690, 2024 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-38417111

RESUMO

Epitranscriptomic mRNA modifications affect gene expression, with their altered balance detected in various cancers. YTHDF proteins contain the YTH reader domain recognizing the m6A mark on mRNA and represent valuable drug targets. Crystallographic structures have been determined for all three family members; however, discrepancies are present in the organization of the m6A-binding pocket. Here, we present new crystallographic structures of the YTH domain of YTHDF1, accompanied by computational studies, showing that this domain can exist in different stable conformations separated by a significant energetic barrier. During the transition, additional conformations are explored, with peculiar druggable pockets appearing and offering new opportunities for the design of YTH-interfering small molecules.


Assuntos
Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Maleabilidade , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Conformação Molecular
2.
J Biol Chem ; 298(12): 102652, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36444882

RESUMO

The serpin plasminogen activator inhibitor 1 (PAI-1) spontaneously undergoes a massive structural change from a metastable and active conformation, with a solvent-accessible reactive center loop (RCL), to a stable, inactive, or latent conformation, with the RCL inserted into the central ß-sheet. Physiologically, conversion to the latent state is regulated by the binding of vitronectin, which hinders the latency transition rate approximately twofold. The molecular mechanisms leading to this rate change are unclear. Here, we investigated the effects of vitronectin on the PAI-1 latency transition using all-atom path sampling simulations in explicit solvent. In simulated latency transitions of free PAI-1, the RCL is quite mobile as is the gate, the region that impedes RCL access to the central ß-sheet. This mobility allows the formation of a transient salt bridge that facilitates the transition; this finding rationalizes existing mutagenesis results. Vitronectin binding reduces RCL and gate mobility by allosterically rigidifying structural elements over 40 Å away from the binding site, thus blocking transition to the latent conformation. The effects of vitronectin are propagated by a network of dynamically correlated residues including a number of conserved sites that were previously identified as important for PAI-1 stability. Simulations also revealed a transient pocket populated only in the vitronectin-bound state, corresponding to a cryptic drug-binding site identified by crystallography. Overall, these results shed new light on PAI-1 latency transition regulation by vitronectin and illustrate the potential of path sampling simulations for understanding functional protein conformational changes and for facilitating drug discovery.


Assuntos
Inibidor 1 de Ativador de Plasminogênio , Vitronectina , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Vitronectina/química , Modelos Moleculares , Conformação Proteica , Solventes
3.
PLoS Comput Biol ; 16(9): e1007922, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946455

RESUMO

Prions are self-replicative protein particles lacking nucleic acids. Originally discovered for causing infectious neurodegenerative disorders, they have also been found to play several physiological roles in a variety of species. Functional and pathogenic prions share a common mechanism of replication, characterized by the ability of an amyloid conformer to propagate by inducing the conversion of its physiological, soluble counterpart. Since time-resolved biophysical experiments are currently unable to provide full reconstruction of the physico-chemical mechanisms responsible for prion replication, one must rely on computer simulations. In this work, we show that a recently developed algorithm called Self-Consistent Path Sampling (SCPS) overcomes the computational limitations of plain MD and provides a viable tool to investigate prion replication processes using state-of-the-art all-atom force fields in explicit solvent. First, we validate the reliability of SCPS simulations by characterizing the folding of a class of small proteins and comparing against the results of plain MD simulations. Next, we use SCPS to investigate the replication of the prion forming domain of HET-s, a physiological fungal prion for which high-resolution structural data are available. Our atomistic reconstruction shows remarkable similarities with a previously reported mechanism of mammalian PrPSc propagation obtained using a simpler and more approximate path sampling algorithm. Together, these results suggest that the propagation of prions generated by evolutionary distant proteins may share common features. In particular, in both these cases, prions propagate their conformation through a very similar templating mechanism.


Assuntos
Proteínas Fúngicas , Simulação de Dinâmica Molecular , Príons , Algoritmos , Biologia Computacional , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Príons/química , Príons/metabolismo , Conformação Proteica , Dobramento de Proteína
4.
Commun Biol ; 4(1): 62, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33437023

RESUMO

Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP and identified four different small molecule ligands for this conformer, all capable of selectively lowering the load of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Doenças Priônicas/tratamento farmacológico , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Dobramento de Proteína , Animais , Sítios de Ligação , Simulação por Computador , Retículo Endoplasmático/metabolismo , Fibroblastos , Células HEK293 , Humanos , Ligantes , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Reprodutibilidade dos Testes
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