In eukaryotes, tripeptidyl peptidase II (TPPII) is a crucial component of the proteolytic cascade acting downstream of the 26S proteasome in the ubiquitin-proteasome pathway. It is an amino peptidase belonging to the subtilase family removing tripeptides from the free N terminus of oligopeptides. The 150-kDa subunits of Drosophila TPPII assemble into a giant proteolytic complex of 6 MDa with a remarkable architecture consisting of two segmented and twisted strands that form a spindle-shaped structure. A refined 3D model has been obtained by cryoelectron microscopy, which reveals details of the molecular architecture and, in conjunction with biochemical data, provides insight into the assembly mechanism. The building blocks of this complex are apparently dimers, within which the 150-kDa monomers are oriented head to head. Stacking of these dimers leads to the formation of twisted single strands, two of which comprise the fully assembled spindle. This spindle also forms when TPPII is heterologously expressed in Escherichia coli, demonstrating that no scaffolding protein is required for complex formation and length determination. Reciprocal interactions of the N-terminal part of subunits from neighboring strands are probably involved in the formation of the native quaternary structure, lending the TPPII spindle a stability higher than that of single strands.3D reconstruction ͉ cryoelectron microscopy ͉ scanning transmission electron microscopy I ntracellular proteolysis must be subject to spatial and temporal control to avoid havoc. Self-compartmentalization serves as a key stratagem, enabling the cell to confine proteolysis to selfassembled nanocompartments. As a consequence, a number of large homo-or heterooligomeric proteolytic complexes have evolved; the prime example is the 26S proteasome, which degrades ubiquitinated proteins in an ATP-dependent manner (1, 2). However, proteases that can degrade the peptides released by the proteasome further, such as the Tricorn protease in prokaryotes (3, 4) or tripeptidyl peptidase II (TPPII) in eukaryotes (5), are also very large megadalton complexes. TPPII has recently attracted attention because, in addition to its role in general intracellular protein turnover, it seems to be capable of complementing 26S proteasome function to some extent under conditions in which the latter is inhibited (6-9). TPPII plays a role in pathological conditions such as muscle sepsis (10, 11) and in apoptosis (12)(13)(14) and, furthermore, is instrumental in the N-terminal trimming that occurs in the late stages of antigenic peptide processing for the MHC class I system (15-17). A membrane-bound form exists in addition to cytosolic TPPII, and this form is responsible for the degradation of cholecystokinin (18,19), one of the most abundant neurotransmitters in the brain and an important signal factor for the peripheral and central nervous systems (20).TPPII has only been found thus far in eukaryotes, in which it exists as a large homooligomeric assembly with a molecular mass far beyond 1 MDa....

Tripeptidyl peptidase II (TPP II) is the largest known eukaryotic protease (6MDa). It is believed to act downstream of the 26S proteasome cleaving tripeptides from the N– termini of longer peptides and it is implicated in numerous cellular processes. Here we report the structure of Drosophila TPP II determined by a hybrid approach: The structure of the dimer was solved by x–ray crystallography and docked into the three– dimensional map of the holocomplex obtained by single-particle cryo-electron microscopy. The resulting structure reveals the compartmentalization of the active sites inside a system of chambers and suggests the existence of a molecular ruler determining the size of the cleavage products. Furthermore, the structure suggests a model for activation of TPP II involving the relocation of a flexible loop and a repositioning of the active–site serine, coupling it to holocomplex assembly and active site sequestration.

Tripeptidylpeptidase II (TPP II) is an exopeptidase of the subtilisin type of serine proteases, a key component of the protein degradation cascade in many eukaryotes, which cleaves tripeptides from the N terminus of proteasome-released products. The Drosophila TPP II is a large homooligomeric complex (ϳ6 MDa) that is organized in a unique repetitive structure with two strands each composed of ten stacked homodimers; two strands intertwine to form a spindleshaped structure. We report a novel procedure of preparing an active, structurally homogeneous TPP II holo-complex overexpressed in Escherichia coli. Assembly studies revealed that the specific activity of TPP II increases with oligomer size, which in turn is strongly concentration-dependent. At a TPP II concentration such as prevailing in Drosophila, equilibration of size and activity proceeds on a time scale of hours and leads to spindle formation at a TPP II concentration of >0.03 mg/ml. Before equilibrium is reached, activation lags behind assembly, suggesting that activation occurs in a twostep process consisting of (i) assembly and (ii) a subsequent conformational change leading to a switch from basal to full activity. We propose a model consistent with the hyperbolic increase of activity with oligomer size. Spindle formation by strand pairing causes both significant thermodynamic and kinetic stabilization. The strands inherently heterogeneous in length are thus locked into a discrete oligomeric state. Our data indicate that the unique spindle form of the holo-complex represents an assembly motif stabilizing a highly active state.