Two-substrate association with the 20S proteasome at single-molecule level

Abstract

The bipartite structure of the proteasome raises the question of functional significance. A rational design for unraveling mechanistic details of the highly symmetrical degradation machinery from Thermoplasma acidophilum pursues orientated immobilization at metal-chelating interfaces via affinity tags fused either around the pore apertures or at the sides. End-on immobilization of the proteasome demonstrates that one pore is sufficient for substrate entry and product release. Remarkably, a 'deadend' proteasome can process only one substrate at a time. In contrast, the side-on immobilized and free proteasome can bind two substrates, presumably one in each antechamber, with positive cooperativity as analyzed by surface plasmon resonance and single-molecule crosscorrelation spectroscopy. Thus, the two-stroke engine offers the advantage of speeding up degradation without enhancing complexity.

20S Proteasomes Have the Potential to Keep Substrates in Store for Continual Degradation

“…Binding of the first would therefore trigger binding of the second, third, and fourth substrate molecules. This is in accord with a previous proposal for cooperative binding of a much smaller protein (insulin); however, only binding in both antechambers, and not the central cavity, was considered (35). Additionally, atomic force microscope measurements have indicated a two-state model of allosteric transition that is dependent on substrate binding (40).…”

“…We also noted the formation of dimeric forms of the proteasome after loss of ␣-subunits and substrate, a process not observed for the free proteasome. This implies that a conformational change is induced upon substrate binding and translocation, as suggested by other studies (35)(36)(37), and that this allosteric transition persists even after loss of ␣-subunit and substrate. The finding that the FIGURE 7.…”

Splicing of Distant Peptide Fragments Occurs in the Proteasome by Transpeptidation and Produces the Spliced Antigenic Peptide Derived from Fibroblast Growth Factor-5

“…Such trans-splicing is conceivable in theory, because it was shown that the inner channel of the proteasome, with a diameter of 13 Å (17), can accommodate more than one polypeptide chain (18,19). The proteasome was also able to bind and process two substrates simultaneously: one in each catalytic chamber (20). To test this hypothesis, we designed plasmids encoding full-length FGF-5 proteins containing a point mutation in fragment NTYAS or PRFK.…”

Kontrolle der Aktivität des 20S‐Proteasoms durch synthetische Torwächter

“…[7] Hier zeigen wir erstmals den Einsatz von kleinen synthetischen Verbindungen, die gewissermaßen als Torwächter die proteolytische Aktivität des Proteasomkomplexes kontrollieren. Ein multivalenter Chelatorkopf (multivalent chelator head, MCH) mit vier NTA-Gruppen (tetrakisNTA, Schema 1 c) wird zur Vernetzung der Histidin-Markierungen (His-Tags) verwendet, die entweder an den N-terminalen Regionen der a-Untereinheiten rund um die beiden Öff-nungen (aN-His 6 -Proteasom) oder an den C-Termini der bUntereinheiten an der Seite des Proteasoms (bC-His 6 -Proteasom, Schema 1 b) lokalisiert sind.…”