RESUMEN
The 20S proteasome (core particle, CP) is a multifunctional protease complex and composed of four heptameric subunit rings arranged in a hollow, barrel-shaped structure. Here, we report the crystal structure of the CP from Archaeoglobus fulgidus at 2.25A resolution. The analysis of the structure of early and late assembly intermediates of this CP gives new insights in the maturation of archaebacterial CPs and indicates similarities to assembly intermediates observed in eukaryotes. We also show a striking difference in mechanism and regulation of substrate access between eukaryotic and archaebacterial 20S proteasomes. While eukaryotic CPs are auto-inhibited by the N-terminal tails of the outer alpha-ring by imposing topological closure with a characteristic sequence motif (YDR-motif) and show regulatory gating this segment is disordered in the CP and differently structured in the alpha(7)-sub-complex of A.fulgidus leaving a pore leading into the particle with a diameter of 13A. Mutagenesis and functional studies indicate the absence of regulatory gating in the archaeal 20S proteasome.
Asunto(s)
Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Archaeoglobus fulgidus/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
PepV from Lactobacillus delbrueckii, a dinuclear zinc peptidase, has been characterized as an unspecific amino dipeptidase. The crystal structure of PepV in complex with the phosphinic inhibitor AspPsi[PO(2)CH(2)]AlaOH, a dipeptide substrate mimetic, reveals a "catalytic domain" and a "lid domain," which together form an internal active site cavity that traps the inhibitor. The catalytic domain is topologically similar to catalytic domains from amino- and carboxypeptidases. However, the lid domain is unique among the related enzymes. In contrast to the other related exopeptidases, PepV recognizes and fixes the dipeptide backbone, while the side chains are not specifically probed and can vary, rendering it a nonspecific dipeptidase. The cocrystallized inhibitor illustrates the two roles of the two catalytic zinc ions, namely stabilization of the tetrahedral intermediate and activation of the catalytic water molecule.