RESUMO
A methionine-rich low complexity (LC) domain is found within a C-terminal region of the TDP43 RNA-binding protein. Self-association of this domain leads to the formation of labile cross-ß polymers and liquid-like droplets. Treatment with H2O2 caused phenomena of methionine oxidation and droplet melting that were reversed upon exposure of the oxidized protein to methionine sulfoxide reductase enzymes. Morphological features of the cross-ß polymers were revealed by H2O2-mediated footprinting. Equivalent TDP43 LC domain footprints were observed in polymerized hydrogels, liquid-like droplets, and living cells. The ability of H2O2 to impede cross-ß polymerization was abrogated by the prominent M337V amyotrophic lateral sclerosis-causing mutation. These observations may offer insight into the biological role of TDP43 in facilitating synapse-localized translation as well as aberrant aggregation of the protein in neurodegenerative diseases.
Assuntos
Ataxina-2/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Células HEK293 , Humanos , Polimerização , Domínios Proteicos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The apoptotic effector caspase-6 (CASP6) has been clearly identified as a drug target due to its strong association with neurodegeneration and axonal pruning events as well as its crucial roles in Huntington disease and Alzheimer disease. CASP6 activity is suppressed by ARK5-mediated phosphorylation at Ser(257) with an unclear mechanism. In this work, we solved crystal structures of ΔproCASP6S257E and p20/p10S257E, which mimicked the phosphorylated CASP6 zymogen and activated CASP6, respectively. The structural investigation combined with extensive biochemical assay and molecular dynamics simulation studies revealed that phosphorylation on Ser(257) inhibited self-activation of CASP6 zymogen by "locking" the enzyme in the TEVD(193)-bound "inhibited state." The structural and biochemical results also showed that phosphorylation on Ser(257) inhibited the CASP6 activity by steric hindrance. These results disclosed the inhibition mechanism of CASP6 phosphorylation and laid the foundation for a new strategy of rational CASP6 drug design.
Assuntos
Caspase 6/química , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Serina/química , Substituição de Aminoácidos , Caspase 6/genética , Caspase 6/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Humanos , Modelos Moleculares , Mutação , Fosforilação , Serina/genética , Serina/metabolismoRESUMO
Protein domains of low sequence complexity do not fold into stable, three-dimensional structures. Nevertheless, proteins with these sequences assist in many aspects of cell organization, including assembly of nuclear and cytoplasmic structures not surrounded by membranes. The dynamic nature of these cellular assemblies is caused by the ability of low-complexity domains (LCDs) to transiently self-associate through labile, cross-ß structures. Mechanistic studies useful for the study of LCD self-association have evolved over the past decade in the form of simple assays of phase separation. Here, we have used such assays to demonstrate that the interactions responsible for LCD self-association can be dictated by labile protein structures poised close to equilibrium between the folded and unfolded states. Furthermore, missense mutations causing Charcot-Marie-Tooth disease, frontotemporal dementia, and Alzheimer's disease manifest their pathophysiology in vitro and in cultured cell systems by enhancing the stability of otherwise labile molecular structures formed upon LCD self-association.
Assuntos
Doença de Alzheimer , Doença de Charcot-Marie-Tooth , Proteínas de Ligação a DNA , Demência Frontotemporal , Doença de Alzheimer/genética , Células Cultivadas , Doença de Charcot-Marie-Tooth/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Demência Frontotemporal/genética , Humanos , Mutação de Sentido Incorreto , Domínios Proteicos , Dobramento de Proteína , Estabilidade ProteicaRESUMO
The detailed basis of walking by dimeric molecules of kinesin along microtubules has remained unclear, partly because available structural methods have been unable to capture microtubule-bound intermediates of this process. Utilizing novel electron cryomicroscopy methods, we solved structures of microtubule-attached, dimeric kinesin bound to an ATP analog. We find that under these conditions, the kinesin dimer can attach to the microtubule with either one or two motor domains, and we present sub-nanometer resolution reconstructions of both states. The former structure reveals a novel kinesin conformation that revises the current understanding of how ATP binding is coupled to forward stepping of the motor. The latter structure indicates how tension between the two motor domains keeps their cycles out of phase in order to stimulate directional motility. The methods presented here pave the way for future structural studies of a variety of challenging macromolecules that bind to microtubules and other filaments.