Group II chaperonins are ATP-dependent ring-shaped complexes that bind nonnative polypeptides and facilitate protein folding in archaea and eukaryotes. A built-in lid encapsulates substrate proteins ...within the central chaperonin chamber. Here, we describe the fate of the substrate during the nucleotide cycle of group II chaperonins. The chaperonin substrate-binding sites are exposed, and the lid is open in both the ATP-free and ATP-bound prehydrolysis states. ATP hydrolysis has a dual function in the folding cycle, triggering both lid closure and substrate release into the central chamber. Notably, substrate release can occur in the absence of a lid, and lid closure can occur without substrate release. However, productive folding requires both events, so that the polypeptide is released into the confined space of the closed chamber where it folds. Our results show that ATP hydrolysis coordinates the structural and functional determinants that trigger productive folding.
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► ATP hydrolysis has a dual function in the folding cycle of group II chaperonins ► ATP hydrolysis triggers substrate release by creating a new lateral subunit interface ► Closing the built-in lid over the chaperonin chamber requires ATP hydrolysis ► Folding requires both events, so the polypeptide is released in the closed chamber
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The hydrogenase maturation proteins HypF and HypE catalyze the synthesis of the CN ligands of the active site iron of the NiFe‐hydrogenases using carbamoylphosphate as a substrate. HypE protein from ...Escherichia coli was purified from a transformant overexpressing the hypE gene from a plasmid. Purified HypE in gel filtration experiments behaves predominantly as a monomer. It does not contain statistically significant amounts of metals or of cofactors absorbing in the UV and visible light range. The protein displays low intrinsic ATPase activity with ADP and phosphate as the products, the apparent Km being 25 µm and the kcat 1.7 × 10−3 s−1. Removal of the C‐terminal cysteine residue of HypE which accepts the carbamoyl moiety from HypF affected the Km (47 µm) but not significantly the kcat (2.1 × 10−3 s−1). During the carbamoyltransfer reaction, HypE and HypF enter a complex which is rather tight at stoichiometric ratios of the two proteins. A mutant HypE variant was generated by amino acid replacements in the nucleoside triphosphate binding region, which showed no intrinsic ATPase activity. The variant was active as an acceptor in the transcarbamoylation reaction but did not dehydrate the thiocarboxamide to the thiocyanate. The results obtained with the HypE variants and also with mutant HypF forms are integrated to explain the complex reaction pattern of protein HypF.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
The fungus Ustilago maydis is a pathogen that establishes a biotrophic interaction with Zea mays. The interaction with the plant host is largely governed by more than 300 novel, secreted protein ...effectors, of which only four have been functionally characterized. Prerequisite to examine effector function is to know where effectors reside after secretion. Effectors can remain in the extracellular space, i.e. the plant apoplast (apoplastic effectors), or can cross the plant plasma membrane and exert their function inside the host cell (cytoplasmic effectors). The U. maydis effectors lack conserved motifs in their primary sequences that could allow a classification of the effectome into apoplastic/cytoplasmic effectors. This represents a significant obstacle in functional effector characterization. Here we describe our attempts to establish a system for effector classification into apoplastic and cytoplasmic members, using U. maydis for effector delivery.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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TRiC/CCT is a mammalian chaperonin made up of eight distinct polypeptides arranged in two rings. TRiC changes conformation in an ATP‐dependent manner which is critical to promote ...protein folding in the eukaryotic cell. Unlike prokaryotic chaperonins, substrate encapsulation within the central chamber of TRiC is triggered by ATP‐induced closure of a built‐in lid.
Our single particle cryo‐EM studies, including both reference‐free 2D analyses and 3D reconstructions, show the conformation variations of TRiC in different nucleotide states in the protein folding cycle. The observed changes are consistent with various biochemical evidences. Based on comparative modeling in combination with flexible fitting, pseudoatomic models have been built for each conformational state. Interestingly, in the most physiological related ATP hydrolysis transition (ADP‐AlFx) state, TRiC adopts an one ring open and one ring closed conformation. The closed ring is obviously expanded comparing to that in the closed (ATP‐AlFx) state. Notably, the inter‐domain motions in TRiC appear differently from those of prokaryotic chaperonins, despite their overall structural similarity.
This research is supported through NIH Roadmap Nanomedicine Initiative grant (PN2EY016525).
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
TRiC is a eukaryotic chaperonin essential for de novo folding of ~10% newly synthesized cytosolic proteins, many of which cannot be folded by other chaperones. This is likely linked to TRiC's unique ...subunit organization, whereby each ring consists of eight different subunits in an arrangement that remains uncertain. Using single particle cryo‐EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7 Å resolution, which is the highest resolution asymmetric cryo‐EM structure to date. This revealed the existence of a two‐fold axis between its two rings. A subsequent two‐fold symmetrized map yielded a 4.0 Å resolution structure that evinces the densities of a large fraction of side‐chains, loops and insertions, which permitted unambiguous identification of all eight individual subunits. Independent biochemical near‐neighbor analysis supports our TRiC subunit arrangement. This allowed the optimization of a Cα backbone model of the entire TRiC complex from the homology models against the cryo‐EM density. A refined atomic model for one subunit showed ~95% of the dihedral angles in the allowable regions of the Ramachandran plot. Our model reveals an unevenly distributed positively charged wall lining the closed folding chamber of TRiC is strikingly different from those of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.
Supported by Nanomedicine Development Center (PN1EY016525) and NCRR (P41RR02250).
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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