Abstract
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 (approximately 6 MDa) that is organized in a unique repetitive structure with two strands each composed of ten stacked homodimers; two strands intertwine to form a spindle-shaped 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 > or =0.03 mg/ml. Before equilibrium is reached, activation lags behind assembly, suggesting that activation occurs in a two-step 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.
Highlights
Intracellular proteolysis is one of the vital functions of life
Monomers of Tripeptidylpeptidase II (TPP II) and, likewise, of the archaeal subtilases pyrolysin [19] and STABLE [20] are much larger than the subtilisins, which essentially consist of the canonical catalytic domain, and homology is restricted to the N-terminal half of TPP II
Concentration Determines Size Determines Activity—In this work we have investigated the relationship between enzyme concentration, oligomeric state, and activity for the TPP II complex whose architecture and activation is, to our knowledge, fundamentally different from all large proteolytic complexes described far
Summary
Strains and Cloning—E. coli strain BL21(DE3) was used for expression of TPP II. From a Drosophila expressed sequence tag clone (Umea Drosophila Stock Center, University of Umea, Sweden) the structural gene for TPP II was cloned between the NdeI and the NotI sites of the vector pET-30b (Novagen). Functional cross-linker bis(sulfosuccinimidyl)suberate (Pierce) Because the expression yield of soluble protein significantly dissolved in Milli-Q grade water (Millipore) to a concentration depended on the conditions of growth and induction, we optiof 50 mM was added in five increments during 10 min to a final concentration of 2.5 mM. After another 5 min, 20 mM Tris/Cl, pH 8.5, was added to bind excess reagent, and the samples were prepared for SDS-PAGE (see above). Staining was performed by incubation on a drop of 2% uranyl acetate for 1 min
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