|Giant Protease TPP II’s Structure, Mechanism Uncovered|
Tripeptidyl peptidase II (TPP II), the largest known eukaryotic enzyme that breaks down proteins (a protease), is implicated in numerous cellular processes including the degradation of the endogenous satiety agent cholecystokinin–8, making TPP II a target in the treatment of obesity. To gain insight into this molecular machine’s mechanisms of activation and proteolysis, researchers from Berkeley Lab, the University of California, Berkeley, and the Max Planck Institute of Biochemistry combined single-particle cryo-electron microscopy and x-ray crystallography at ALS Beamline 8.2.2.
Earlier single-particle cryo-electron microscopy investigations showed the overall structure of the TPP II holocomplex to be a spindle-shaped assembly composed of two twisted strands, with each strand containing 10 repeat segments (dimers). Functional studies show the dimers are effectively inactive, but the specific activity of TPP II increases with the number of assembled dimers, indicating a contact–induced activation mechanism.
The structure of the TPP II holocomplex is highly dynamic in vitro, and crystallization of the complete assembly has remained elusive. To address this challenge, Drosophila TPP II spindle assemblies were dissociated into dimers in the presence of detergent. The dimers were crystallized and their structure determined by x-ray crystallography; the holocomplex model was prepared by docking the dimers into the electron-microscopy-derived envelope.
The resulting structure reveals the compartmentalization of the active sites inside a system of chambers with small openings, restricting substrate access to short unfolded peptides, and suggesting the existence of a molecular ruler limiting the size of cleavage products. Furthermore, the structure supports a model of TPP II assembly-dependent activation involving the relocation of a flexible loop and the repositioning of the active-site serine, coupling it to holocomplex assembly and active-site sequestration.
TPP II monomers comprise three general domains: (i) a subtilisin-like domain containing a long insert (~200 amino acids) typical of all TPP II homologues, (ii) a central domain rich in β–sheet structure, and (iii) a C–terminal domain containing a 12-helix bundle.
TPP II dimers are formed by monomers—generally shaped like bottomless “bowls” with a curved protrusion—oriented back-to-back, with the opening at each bowl’s bottom blocked by the adjoining monomer. The monomers’ protrusions, formed by the C-terminal domain and part of the subtilisin-like domain’s insert, abut to form the dimer’s “handle.”
The overall arrangement of the catalytic residues in the crystal structure of the TPP II dimer is similar to that of other subtilisin (a nonspecific protease) family members, but one of the active-site residues (serine) is displaced ~5 Å away from where it would be expected based on its equivalent in subtilisin. Three residues at the C-terminal end of a loop, adjacent to the active-site serine, are bound to the TPP II substrate binding cleft, rendering the enzyme unable to bind substrate. Two glutamate residues at one end of the substrate-binding cleft limit how many residues can be accommodated there, creating a molecular ruler limiting exoproteolytic products to tripeptides. At the same time, these residues orient substrates so the tripeptides are removed exclusively from N-termini.
The stacking of TPP II dimers into proteolytically active strands forms a network of chambers within the holocomplex. At each dimer–dimer interface, a suite of chambers compartmentalizes the active sites. Any substrate to be degraded by TPP II must diffuse over a long distance to reach an active site; however, this is counterbalanced by the presence of multiple pathways, improving access to the active sites.
The structure of the TPP II holocomplex suggests a model for the assembly-dependent activation of TPP II, in which a loop adjacent to the active-site serine plays a key role as an activation switch: TPP II dimers are inactive when the active-site serine is displaced. At the same time, a three-residue segment of the loop is bound to the substrate-binding cleft, which precludes substrate binding the cleft and provides an autoinhibitory mechanism. As the dimers form strands, the loop is repositioned to participate in dimer–dimer contacts, and concomitantly, the active-site serines are repositioned to proteolytically active conformations. Thus, the priming of the active sites is coupled to their sequestration from the bulk environment into chambers.
Research conducted by C.K. Chuang and B.K. Jap (Berkeley Lab and UC Berkeley); B. Rockel, G. Seyit, A.-M. Schonegge, J. Peters, and W. Baumeister (Max Planck Institute of Biochemistry); P.J. Walian (Berkeley Lab); and P.H. Zwart (ALS).
Research funding: U.S. Department of Energy (DOE), Office of Basic Energy Sciences (BES); National Institutes of Health; and Deutsche Forschungsgemeinschaft. Operation of the ALS is supported by DOE BES.
Publication about this research: C.K. Chuang, B. Rockel, G. Seyit, P.J. Walian, A.-M. Schonegge, J. Peters, P.H. Zwart, W. Baumeister, and B.K. Jap, "Hybrid molecular structure of the giant protease tripeptidyl peptidase II," Nat. Struct. Mol. Biol. 17, 990 (2010).
ALS Science Highlight #222