Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum

Decreased susceptibilities of Plasmodium falciparum towards the endoperoxide antimalarial artemisinin are linked to mutations of residue C580 of Kelch13, which is the homologue of the redox sensor Keap1 in vertebrates. Here, we addressed whether mutations alter the artemisinin susceptibility by modifying the redox properties of Kelch13 or by compromising its native fold or abundance. Using selection-linked integration and the glmS ribozyme, efficient down-regulation of Kelch13 resulted in ring-stage survival rates around 40%. While the loss of a potential disulfide bond between residues C580 and C532 had no effect on the artemisinin suceptibility, the thiol group of C473 could not be replaced. We also established a protocol for the production of recombinant Kelch13. In contrast to cysteine-to-serine replacements, common field mutations resulted in misfolded and insoluble protein. In summary, not the redox properties but impaired folding of Kelch13, resulting in a decreased Kelch13 abundance, is the central parameter for mutant selection.

Metamorphic proteins: the Janus proteins of structural biology

The structural paradigm that the sequence of a protein encodes for a unique three-dimensional native fold does not acknowledge the intrinsic plasticity encapsulated in conformational free energy landscapes. Metamorphic proteins are a recently discovered class of biomolecules that illustrate this plasticity by folding into at least two distinct native state structures of comparable stability in the absence of ligands or cofactors to facilitate fold-switching. The expanding list of metamorphic proteins clearly shows that these proteins are not mere aberrations in protein evolution, but may have actually been a consequence of distinctive patterns in selection pressure such as those found in virus–host co-evolution. In this review, we describe the structure–function relationships observed in well-studied metamorphic protein systems, with specific focus on how functional residues are sequestered or exposed in the two folds of the protein. We also discuss the implications of metamorphosis for protein evolution and the efforts that are underway to predict metamorphic systems from sequence properties alone.

Unfolded and intermediate states of PrP play a key role in the mechanism of action of an antiprion chaperone

Prion and prion-like diseases involve the propagation of misfolded protein conformers. Small-molecule pharmacological chaperones can inhibit propagated misfolding, but how they interact with disease-related proteins to prevent misfolding is often unclear. We investigated how pentosan polysulfate (PPS), a polyanion with antiprion activity in vitro and in vivo, interacts with mammalian prion protein (PrP) to alter its folding. Calorimetry showed that PPS binds two sites on natively folded PrP, but one PPS molecule can bind multiple PrP molecules. Force spectroscopy measurements of single PrP molecules showed PPS stabilizes not only the native fold of PrP but also many different partially folded intermediates that are not observed in the absence of PPS. PPS also bound tightly to unfolded segments of PrP, delaying refolding. These observations imply that PPS can act through multiple possible modes, inhibiting misfolding not only by stabilizing the native fold or sequestering natively folded PrP into aggregates, as proposed previously, but also by binding to partially or fully unfolded states that play key roles in mediating misfolding. These results underline the likely importance of unfolded states as critical intermediates on the prion conversion pathway.

Non-native fold of the putative VPS39 zinc finger domain

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.

Non-native fold of the putative VPS39 zinc finger domain

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.

Protein Unfolding in Freeze Frames: Intermediates of Ubiquitin and Lysozyme Revealed by Variable Temperature Ion Mobility-Mass Spectrometry

As experimentalists, we normally rely on assessing observables. However sometimes, the most fascinating phenomena are not noticeable directly. An example of such is our data and the corresponding interpretation presented in this manuscript. We have designed and constructed a ion mobility mass spectrometer (acs.analchem.6b01812) capable of taking ion mobility measurements over a temperature range from 150-500K. We chose to benchmark this new instrument, using the small proteins Ubiquitin and Lysozyme extensively studied as a “model proteins” in many in-silico, -solution and -vacuo studies focusing on conformational dynamics.In this work, we activate and subsequently thermally equilibrate the protein ions at several temperatures prior to collision cross section measurement. For Ubiquitin at 300K and above, the protein unfolds in a “step-wise” fashion as previously reported (by David Clemmer) and for other proteins including lysozyme, and cytochrome c by us and also by Martin Jarrold. However, to our surprise, activation and equilibration of ubiquitin at 150K yields a plethora of highly extended forms of the protein. We attribute these as kinetically trapped unfolding intermediates. Since the activation process is the same at both temperatures we infer that the unfolding must always proceed via these extended intermediate forms, which then converge to commonly reported conformations. Intriguingly, this “convergence” appears to occur mostly below the temperature of irreversible conformational thermal transition of Ubiquitin reported in many solution phase studies. For Lysozyme the same experiment is performed and similar results are obtained although we cannot activate the gaseous ensemble too far from the native fold and the activation barrier to refolding is low enough to allow it to be re-accessed on the experimental timescale we use.

Protein Unfolding in Freeze Frames: Intermediates of Ubiquitin and Lysozyme Revealed by Variable Temperature Ion Mobility-Mass Spectrometry

<p>As experimentalists, we normally rely on assessing observables. However sometimes, the most fascinating phenomena are not noticeable directly. An example of such is our data and the corresponding interpretation presented in this manuscript. We have designed and constructed a ion mobility mass spectrometer (acs.analchem.6b01812) capable of taking ion mobility measurements over a temperature range from 150-500K. We chose to benchmark this new instrument, using the small proteins Ubiquitin and Lysozyme extensively studied as a “model proteins” in many <i>in-silico</i>, -<i>solution</i> and -<i>vacuo</i> studies focusing on conformational dynamics.In this work, we activate and subsequently thermally equilibrate the protein ions at several temperatures prior to collision cross section measurement. For Ubiquitin at 300K and above, the protein unfolds in a “step-wise” fashion as previously reported (by David Clemmer) and for other proteins including lysozyme, and cytochrome c by us and also by Martin Jarrold. However, to our surprise, activation and equilibration of ubiquitin at 150K yields a plethora of highly extended forms of the protein. We attribute these as kinetically trapped unfolding intermediates. Since the activation process is the same at both temperatures we infer that the unfolding must always proceed <i>via</i> these extended intermediate forms, which then converge to commonly reported conformations. Intriguingly, this “convergence” appears to occur mostly below the temperature of irreversible conformational thermal transition of Ubiquitin reported in many solution phase studies. For Lysozyme the same experiment is performed and similar results are obtained although we cannot activate the gaseous ensemble too far from the native fold and the activation barrier to refolding is low enough to allow it to be re-accessed on the experimental timescale we use.</p>

Protein Unfolding in Freeze Frames: Intermediates of Ubiquitin and Lysozyme Revealed by Variable Temperature Ion Mobility-Mass Spectrometry

<p>As experimentalists, we normally rely on assessing observables. However sometimes, the most fascinating phenomena are not noticeable directly. An example of such is our data and the corresponding interpretation presented in this manuscript. We have designed and constructed a ion mobility mass spectrometer (acs.analchem.6b01812) capable of taking ion mobility measurements over a temperature range from 150-500K. We chose to benchmark this new instrument, using the small proteins Ubiquitin and Lysozyme extensively studied as a “model proteins” in many <i>in-silico</i>, -<i>solution</i> and -<i>vacuo</i> studies focusing on conformational dynamics.In this work, we activate and subsequently thermally equilibrate the protein ions at several temperatures prior to collision cross section measurement. For Ubiquitin at 300K and above, the protein unfolds in a “step-wise” fashion as previously reported (by David Clemmer) and for other proteins including lysozyme, and cytochrome c by us and also by Martin Jarrold. However, to our surprise, activation and equilibration of ubiquitin at 150K yields a plethora of highly extended forms of the protein. We attribute these as kinetically trapped unfolding intermediates. Since the activation process is the same at both temperatures we infer that the unfolding must always proceed <i>via</i> these extended intermediate forms, which then converge to commonly reported conformations. Intriguingly, this “convergence” appears to occur mostly below the temperature of irreversible conformational thermal transition of Ubiquitin reported in many solution phase studies. For Lysozyme the same experiment is performed and similar results are obtained although we cannot activate the gaseous ensemble too far from the native fold and the activation barrier to refolding is low enough to allow it to be re-accessed on the experimental timescale we use.</p>

Cotranslational Folding of Proteins on the Ribosome

Many proteins in the cell fold cotranslationally within the restricted space of the polypeptide exit tunnel or at the surface of the ribosome. A growing body of evidence suggests that the ribosome can alter the folding trajectory in many different ways. In this review, we summarize the recent examples of how translation affects folding of single-domain, multiple-domain and oligomeric proteins. The vectorial nature of translation, the spatial constraints of the exit tunnel, and the electrostatic properties of the ribosome-nascent peptide complex define the onset of early folding events. The ribosome can facilitate protein compaction, induce the formation of intermediates that are not observed in solution, or delay the onset of folding. Examples of single-domain proteins suggest that early compaction events can define the folding pathway for some types of domain structures. Folding of multi-domain proteins proceeds in a domain-wise fashion, with each domain having its role in stabilizing or destabilizing neighboring domains. Finally, the assembly of protein complexes can also begin cotranslationally. In all these cases, the ribosome helps the nascent protein to attain a native fold and avoid the kinetic traps of misfolding.