Probing the cooperativity of Thermoplasma acidophilum proteasome core particle gating by NMR spectroscopy.
Proc Natl Acad Sci U S A
; 114(46): E9846-E9854, 2017 11 14.
Article
em En
| MEDLINE
| ID: mdl-29087330
ABSTRACT
The 20S proteasome core particle (20S CP) plays an integral role in cellular homeostasis by degrading proteins no longer required for function. The process is, in part, controlled via gating residues localized to the ends of the heptameric barrel-like CP structure that occlude substrate entry pores, preventing unregulated degradation of substrates that might otherwise enter the proteasome. Previously, we showed that the N-terminal residues of the α-subunits of the CP from the archaeon Thermoplasma acidophilum are arranged such that, on average, two of the seven termini are localized inside the lumen of the proteasome, thereby plugging the entry pore and functioning as a gate. However, the mechanism of gating remains unclear. Using solution NMR and a labeling procedure in which a series of mixed proteasome rings are prepared such that the percentage of gate-containing subunits is varied, we address the energetics of gating and establish whether gating is a cooperative process involving the concerted action of residues from more than a single protomer. Our results establish that the intrinsic probability of a gate entering the lumen favors the in state by close to 20-fold, that entry of each gate is noncooperative, with the number of gates that can be accommodated inside the lumen a function of the substrate entry pore size and the bulkiness of the gating residues. Insight into the origin of the high affinity for the in state is obtained from spin-relaxation experiments. More generally, our approach provides an avenue for dissecting interactions of individual protomers in homo-oligomeric complexes.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Endopeptidases
/
Thermoplasma
/
Espectroscopia de Ressonância Magnética
/
Proteínas Arqueais
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article