Pablo Mesa

Chaperonins: two rings for folding

Chaperonins are ubiquitous chaperones found in Eubacteria, eukaryotic organelles (group I), Archaea and the eukaryotic cytosol (group II). They all share a common structure and a basic functional mechanism. Although a large amount of information has been gathered for the simpler group I, much less is known about group II chaperonins. Recent crystallographic and electron microscopy structures have provided new insights into the mechanism of these chaperonins and revealed important differences between group I and II chaperonins, mainly in the molecular rearrangements that take place during the functional cycle. These differences are evident for the most complex chaperonin, the eukaryotic cytosolic CCT, which highlights the uniqueness of this important molecular machine.

Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin

Protein folding is assisted by molecular chaperones. CCT (chaperonin containing TCP-1, or TRiC) is a 1-MDa oligomer that is built by two rings comprising eight different 60-kDa subunits. This chaperonin regulates the folding of important proteins including actin, α-tubulin and β-tubulin. We used an electron density map at 5.5 Å resolution to reconstruct CCT, which showed a substrate in the inner cavities of both rings. Here we present the crystal structure of the open conformation of this nanomachine in complex with tubulin, providing information about the mechanism by which it aids tubulin folding. The structure showed that the substrate interacts with loops in the apical and equatorial domains of CCT. The organization of the ATP-binding pockets suggests that the substrate is stretched inside the cavity. Our data provide the basis for understanding the function of this chaperonin.

Molecular architecture of a multifunctional MCM complex

DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of them is the replicative helicase, which is required for initiation and elongation phases. A MCM helicase found as a prophage in the genome of Bacillus cereus is fused with a primase domain constituting an integrative arrangement of two essential activities for replication. We have isolated this helicase–primase complex (BcMCM) showing that it can bind DNA and displays not only helicase and primase but also DNA polymerase activity. Using single-particle electron microscopy and 3D reconstruction, we obtained structures of BcMCM using ATPγS or ADP in the absence and presence of DNA. The complex depicts the typical hexameric ring shape. The dissection of the unwinding mechanism using site-directed mutagenesis in the Walker A, Walker B, arginine finger and the helicase channels, suggests that the BcMCM complex unwinds DNA following the extrusion model similarly to the E1 helicase from papillomavirus.

Directly from the source: endogenous preparations of molecular machines

Purification from a source enriched in large macromolecular machines with basic cellular function is still the method of choice in many cases. Such complexes occur in sufficiently high copy numbers in the cell and can be isolated using classical protein purification protocols. Although advanced DNA recombinant technologies and sophisticated overexpression strategies are available, many complexes like the ribosome, RNA polymerase II and membrane protein complexes involved in photosynthesis or in oxidative phosphorylation can only be purified from a rich source. Here, we review recent accomplishments and limitations in applying this strategy.

Crystallization of the Bacillus subtilis SPP1 bacteriophage helicase loader protein G39P.

The essential helicase loader protein G39P encoded by Bacillus subtilis SPP1 phage has been overproduced in Escherichia coli and purified. The wild-type protein has been crystallized by the hanging-drop vapour-diffusion method in a primitive hexagonal space group, probably P6(1)22/P6(5)22, but the crystals diffract to only 3.4 A and are poorly reproducible. Mass-spectrometric analysis has revealed marked proteolytic cleavage from the C-terminus and the presence of a major species corresponding to deletion of the 14 C-terminal residues. Thus, a new variant of the protein (G39P112) has been engineered that corresponds to a 14-residue C-terminal truncation. The G39P112 variant has also been crystallized but now in a primitive orthorhombic form, probably P2(1)2(1)2 or P2(1)2(1)2(1), with unit-cell parameters a = 85.6, b = 89.7, c = 47.6 A, with diffraction to 2.4 A on a synchrotron source and with greatly improved reproducibility. Calculation of V(M) values for this G39P112 variant suggests the presence of three monomers in the asymmetric unit, corresponding to a solvent content of about 47%. A selenomethionine-incorporated form of the protein has been produced and a full three-wavelength MAD data collection undertaken.