Summary TRiC/CCT is a highly conserved and essential chaperonin that uses ATP cycling to facilitate folding of approximately 10% of the eukaryotic proteome. This 1 MDa hetero-oligomeric complex consists of two stacked rings of eight paralogous subunits each. Previously proposed TRiC models differ substantially in their subunit arrangements and ring register. Here, we integrate chemical crosslinking, mass spectrometry and combinatorial modeling to reveal the definitive subunit arrangement of TRiC. In vivo disulfide mapping provided additional validation for the crosslinking-derived arrangement as the definitive TRiC topology. This subunit arrangement allowed the refinement of a structural model using existing X-ray diffraction data. The new structure explains all available crosslink experiments, provides a rationale for previously unexplained structural features and reveals a surprising asymmetry of charges within the chaperonin folding chamber.

The eukaryotic chaperonin, TRiC/CCT (TRiC, TCP-1 ring complex; CCT, chaperonin containing TCP-1), uses a built-in lid to mediate protein folding in an enclosed central cavity. Recent structural data suggest an effective size limit for the TRiC folding chamber of ∼70 kDa, but numerous chaperonin substrates are substantially larger. Using artificial fusion constructs with actin, an obligate chaperonin substrate, we show that TRiC can mediate folding of large proteins by segmental or domain-wise encapsulation. Single or multiple protein domains up to ∼70 kDa are stably enclosed by stabilizing the ATP-hydrolysis transition state of TRiC. Additional domains, connected by flexible linkers that pass through the central opening of the folding chamber, are excluded and remain accessible to externally added protease. Experiments with the physiological TRiC substrate hSnu114, a 109-kDa multidomain protein, suggest that TRiC has the ability to recognize domain boundaries in partially folded intermediates. In the case of hSnu114, this allows the selective encapsulation of the C-terminal ∼45-kDa domain and segments thereof, presumably reflecting a stepwise folding mechanism. The capacity of the eukaryotic chaperonin to overcome the size limitation of the folding chamber may have facilitated the explosive expansion of the multidomain proteome in eukaryotes.folding cage | molecular chaperone | Snu114 homolog | 116 kDa U5 small nuclear ribonucleoprotein component

Background: Heme attachment to cytochrome c is a catalyzed post-translational modification.Results: We identify a ternary complex of the cytochrome c biogenesis protein CcmE, heme, and a cytochrome, and demonstrate its functional significance.Conclusion: The complex is a trapped catalytic intermediate at the point of heme transfer from the cytochrome biogenesis apparatus to the cytochrome.Significance: An insight into biosynthesis of heme proteins.

ABC transporters are fascinating examples of fine-tuned molecular machines that use the energy from ATP hydrolysis to translocate a multitude of substrates across biological membranes. While structural details have emerged on many members of this large protein superfamily, a number of functional details are still under debate. High resolution structures yield valuable insights into protein function, but it is the combination of structural, functional and dynamic insights that facilitates a complete understanding of the workings of their complex molecular mechanisms. NMR is a technique well-suited to investigate proteins in atomic resolution while taking their dynamic properties into account. It thus nicely complements other structural techniques, such as X-ray crystallography, that have contributed high-resolution data to the architectural understanding of ABC transporters. Here, we describe the heterologous expression of LmrA, an ABC exporter from Lactococcus lactis, in Escherichia coli. This allows for more flexible isotope labeling for nuclear magnetic resonance (NMR) studies and the easy study of LmrA's multidrug resistance phenotype. We use a combination of solid-state magic angle spinning (MAS) on the reconstituted transporter and solution NMR on its isolated nucleotide binding domain to investigate consequences of nucleotide binding to LmrA. We find that nucleotide binding affects the protein globally, but that NMR is also able to pinpoint local dynamic effects to specific residues, such as the Walker A motif's conserved lysine residue.

Graphical AbstractHighlights d Eukaryotic elongation factor 2 (eEF2) is a chaperonedependent multi-domain protein d Hgh1 functions as a chaperone for eEF2 and cooperates with the chaperonin TRiC d The crystal structure shows that Hgh1 is a highly conserved armadillo repeat protein d Hgh1 binds to domain III of eEF2 and recruits TRiC for eEF2 folding