Architecture and Molecular Mechanism of PAN, the Archaeal Proteasome Regulatory ATPase

Abstract

In Archaea, an hexameric ATPase complex termed PAN promotes proteins unfolding and translocation into the 20 S proteasome. PAN is highly homologous to the six ATPases of the eukaryotic 19 S proteasome regulatory complex. Thus, insight into the mechanism of PAN function may reveal a general mode of action mutual to the eukaryotic 19 S proteasome regulatory complex. In this study we generated a three-dimensional model of PAN from tomographic reconstruction of negatively stained particles. Surprisingly, this reconstruction indicated that the hexameric complex assumes a two-ring structure enclosing a large cavity. Assessment of distinct three-dimensional functional states of PAN in the presence of adenosine 5-O-(thiotriphosphate) and ADP and in the absence of nucleotides outlined a possible mechanism linking nucleotide binding and hydrolysis to substrate recognition, unfolding, and translocation. A novel feature of the ATPase complex revealed in this study is a gate controlling the "exit port" of the regulatory complex and, presumably, translocation into the 20 S proteasome. Based on our structural and biochemical findings, we propose a possible model in which substrate binding and unfolding are linked to structural transitions driven by nucleotide binding and hydrolysis, whereas translocation into the proteasome only depends upon the presence of an unfolded substrate and binding but not hydrolysis of nucleotide.In eukaryotic cells most protein breakdown in the cytosol and nucleus is catalyzed by the 26 S proteasome. This ϳ2.5-MDa (1) complex degrades ubiquitin-conjugated and certain non-ubiquitinated proteins in an ATP-dependent manner (2, 3). The 26 S complex is composed of one or two 19 S regulatory particles situated at the ends of the cylindrical 20 S proteasome. Within the 26 S complex, proteins are hydrolyzed in the 20 S proteasome. Tagged substrates, however, first bind to the 19 S regulatory particle, which catalyzes their unfolding and translocation into the 20 S subcomplex (4, 5). The 19 S regulatory particle consists of at least 17 different subunits (1, 6). Nine of these subunits form a "lid," whereas the other eight subunits, including six ATPases, comprise the base of the 19 S particle. Electron microscopy (7-10) as well as cross-linking experiments (11,12) have demonstrated that the six homologous ATPases are associated with the ␣ rings of the 20 S particle.Unlike eukaryotes, Archaea and certain eubacteria contain homologous 20 S particles but lack ubiquitin. Their proteasomes degrade proteins in association with a hexameric ATPase ring complex termed PAN (13). PAN appears to be the evolutionary precursor of the 19 S base, predating the coupling of ubiquitination and proteolysis in eukaryotes (14). In addition, PAN recognizes the bacterial targeting sequence ssrA (in analogy to the polyubiquitin conjugates in eukaryotes) and efficiently unfolds and translocates globular substrates, like green fluorescent protein, when tagged with ssrA (15). In both PAN and the 19 S proteasome regulatory complexes, AT...

The Direction of Protein Entry into the Proteasome Determines the Variety of Products and Depends on the Force Needed to Unfold Its Two Termini

“…While the location of the ubiquitin tag on the target protein may perhaps influence directional preference (Prakash et al, 2004), it seems most likely that ATP-driven conformational changes or exposure of hydrophobic surfaces on the ATPase (Medalia et al, 2009) promote local unfolding and tight binding of the substrate’s less stable terminus, which then becomes the initiation site for translocation. Because the directionality seen with the archaeal PAN complex resembled that of the 26S proteasomes for several substrates, it seems most likely that the hexameric ATPases at the base of the 19S particle are both necessary and sufficient to bind and unfold the terminus of the protein.…”

“…This gated narrow orifice (~15Å) allows the entry only of unfolded polypeptides. The gate is formed by the α-subunits’ N-termini and is regulated by the associated 19S ATPase ring (Medalia et al, 2009; Rabl et al, 2008; Smith et al, 2005). Consequently, a globular substrate must undergo unfolding by the AAA ATPases of the 19S complex (Rpt1–6) prior to translocation and digestion.…”

Proteasomes and protein conjugation across domains of life

“…Proteasome-associated AAA+ proteins from all three domains of life (the eukaryotic 19S RP with Rpt1–Rpt6, archaeal Pan and actinobacterial Arc and Mpa) seem to interact with the ends of the CP cylinder, selectively bind and unfold substrate proteins, open the CP gate and facilitate the unidirectional, processive translocation of substrate proteins into the CP channel for proteolysis 38,47,48 . Among these processes, unfolding of substrate proteins and their subsequent translocation into the CP are both coupled to ATP hydrolysis.…”

Harnessing Proteasome Dynamics and Allostery in Drug Design

“…However, the idea of periodic lowering of the affinity between the core and the cap may still be viable. The reverse-chaperone and polypeptidetranslocase activities of the 19S base ATPases are expected to involve robust mechanical interactions between the substrate, the base, and the 20S core (61,88,124). Focusing on the base-core interface, the most striking effect of the base attachment is gate opening (123,124).…”