Related Experiment Video
Updated: Aug 7, 2026
X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
Published on: May 13, 2020
Size matters for the tripeptidylpeptidase II complex from Drosophila: The 6-MDa spindle form stabilizes the activated
Gönül Seyit1, Beate Rockel, Wolfgang Baumeister
1Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
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.
Insights
Tripeptidylpeptidase II (TPP II), a serine protease, forms a spindle-shaped complex in Drosophila. Its activity and structure are concentration-dependent, with the spindle form stabilizing high activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Tripeptidylpeptidase II (TPP II) is a serine protease crucial for protein degradation in eukaryotes.
- It cleaves tripeptides from N termini of proteasome-released products.
- Drosophila TPP II forms a large, spindle-shaped homooligomeric complex.
Purpose of the Study:
- To develop a method for preparing active, homogeneous Drosophila TPP II holo-complex.
- To investigate the relationship between TPP II assembly, size, concentration, and activity.
- To elucidate the structural basis of TPP II's high activity.
Main Methods:
- Overexpression of Drosophila TPP II in Escherichia coli.
- Preparation of active, structurally homogeneous holo-complex.
- Assembly studies to analyze oligomer size, concentration, and activity relationships.
Main Results:
- A novel procedure yielded active, homogeneous TPP II holo-complex from E. coli.
- TPP II activity increases with oligomer size in a concentration-dependent manner.
- Spindle formation, occurring at > or =0.03 mg/ml, stabilizes the complex and enhances activity.
Conclusions:
- The spindle structure of TPP II is an assembly motif that stabilizes a highly active state.
- Activation is a two-step process: assembly followed by a conformational change.
- Oligomer size and spindle formation are critical for TPP II's thermodynamic and kinetic stability.
