Combinatorial Methods Solve a Difficult Structural Problem to Reveal How Chaperonins Work in Eukaryotes
Speaker: Michael Levitt, Stanford University
Department: Chemical & Biological Engineering
Location: Friend Center Convocation Room 113
Date/Time: Wednesday, November 20, 2013, 4:00 p.m. - 5:00 p.m.
Eukaryotic group II Chaperonin TRiC or CCT is a 0.95 megadalton protein complex that is essential for the correct and efficient folding of cytosolic polypeptides. The closed form is a 16 nm sphere made of two hemi-spherical rings of 8 subunits (~550 residues/subunit) that rotate to open a central folding chamber. In eukaryotes, 8 different genes encode the subunits of this ATP-powered nanomachine. The high sequence identity of subunits made the 40,320 (=8 factorial) possible arrangements indistinguishable in previous cryo-electron microscopy and crystallographic analysis.
First we use cross-linking, mass spectrometry and combinatorial homology modeling. We react bovine TRiC under native conditions with a lysine-specific cross-linker, follow up with trypsin digestion, and use mass spectrometry to identify 63 cross-linked pairs providing distance restraints. Independently of the cross-link set, we construct all 40,320 homology models of the TRiC particle. When we compared each model with the cross-link set, we discovered that one model is significantly more compatible than any other model. Bootstrapping analysis confirms that this model is 10 times more likely to result from this cross-link set than the next best-fitting model.
Second, we re-examine the 3.8 Å resolution X-ray data of yeast TRiC. Our method of R-Value Exploration (RVX) exhaustively tests all 2,580,460 possible models. This unbiased analysis singles out with overwhelming significance one model, which is fully consistent with our previous biochemical data and refines to a much lower Rfree value than reported previously with the same X-ray data. With four-fold averaging, our structure reveals remarkably resolved details of the unique conformation of each subunit, and suggests a mechanism for the initiation of transition to the open state. More generally, we expect RVX to resolve ambiguity in low-resolution crystallographic studies.