Chaperonins are biological nanomachines that help newly translated proteins to fold by rescuing them from kinetically trapped misfolded states. Protein folding assistance by the chaperonin machinery is obligatory in vivo for a subset of proteins in the bacterial proteome. Chaperonins are large oligomeric complexes, with unusual seven fold symmetry (group I) or eight/nine fold symmetry (group II), that form double-ring constructs, enclosing a central cavity that serves as the folding chamber. Dramatic large-scale conformational changes, that take place during ATP-driven cycles, allow chaperonins to bind misfolded proteins, encapsulate them into the expanded cavity and release them back into the cellular environment, regardless of whether they are folded or not. The theory associated with the iterative annealing mechanism, which incorporated the conformational free energy landscape description of protein folding, quantitatively explains most, if not all, the available data. Misfolded conformations are associated with low energy minima in a rugged energy landscape. Random disruptions of these low energy conformations result in higher free energy, less folded, conformations that can stochastically partition into the native state. Two distinct mechanisms of annealing action have been described. Group I chaperonins (GroEL homologues in eubacteria and endosymbiotic organelles), recognize a large number of misfolded proteins non-specifically and operate through highly coordinated cooperative motions. By contrast, the less well understood group II chaperonins (CCT in Eukarya and thermosome/TF55 in Archaea), assist a selected set of substrate proteins. Sequential conformational changes within a CCT ring are observed, perhaps promoting domain-by-domain substrate folding. Chaperonins are implicated in bacterial infection, autoimmune disease, as well as protein aggregation and degradation diseases. Understanding the chaperonin mechanism and the specific proteins they rescue during the cell cycle is important not only for the fundamental aspect of protein folding in the cellular environment, but also for effective therapeutic strategies.
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Deep Mutagenesis of a Transporter for Uptake of a Non-Native Substrate Identifies Conformationally Dynamic Regions
The serotonin transporter, SERT, catalyzes serotonin reuptake at the synapse to terminate neurotransmission via an alternating access mechanism, and SERT inhibitors are the most widely prescribed antidepressants. Here, deep mutagenesis is used to determine the effects of nearly all amino acid substitutions on human SERT surface expression and transport of the fluorescent substrate analogue APP+, identifying many mutations that enhance APP+ import. Comprehensive simulations of the entire ion-coupled import process reveal that while binding of the native substrate, serotonin, reduces free energy barriers between conformational states to promote SERT dynamics, the conformational free energy landscape in the presence of APP+ instead resembles Na+ bound-SERT, with a higher free energy barrier for transitioning to an inward-facing state. The deep mutational scan for SERT-catalyzed import of APP+ finds mutations that promote the necessary conformational changes that would otherwise be facilitated by the native substrate. Indeed, hundreds of gain-of-function mutations for APP+ import are found along the permeation pathway, most notably mutations that favor opening of a solvent-exposed intracellular vestibule. The mutagenesis data support the simulated mechanism in which the neurotransmitter and a symported sodium share a common cytosolic exit pathway to achieve coupling. Furthermore, the mutational landscape for SERT surface trafficking, which likely filters out misfolded sequences, reveals that residues along the permeation pathway are mutationally tolerant, providing plausible evolutionary pathways for changes in transporter properties while maintaining folded structure.
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- Award ID(s):
- 1845606
- NSF-PAR ID:
- 10407606
- Date Published:
- Journal Name:
- bioRxiv
- ISSN:
- 2692-8205
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Cellular import of D-xylose, the second most abundant sugar in typical lignocellulosic biomass, has been evidenced to be an energy-depriving process in bacterial biocatalysts. The sugar facilitator of Zymomonas mobilis, Glf, is capable of importing xylose at high rates without extra energy input, but is inhibited by D-glucose (the primary biomass sugar), potentially limiting the utility of this transporter for fermentation of sugar mixtures derived from lignocellulose. In this work we developed an Escherichia coli platform strain deficient in glucose and xylose transport to facilitate directed evolution of Glf to overcome glucose inhibition. Using this platform, we isolated nine Glf variants created by both random and site-saturation mutagenesis with increased xylose utilization rates ranging from 4.8-fold to 13-fold relative to wild-type Glf when fermenting 100 g l–1 glucose–xylose mixtures. Diverse point mutations such as A165M and L445I were discovered leading to released glucose inhibition. Most of these mutations likely alter sugar coordinating pocket for the 6-hydroxymethyl group of D-glucose. These discovered glucose-resistant Glf variants can be potentially used as energy-conservative alternatives to the native sugar transport systems of bacterial biocatalysts for fermentation of lignocellulose-derived sugars.
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Abstract Presenilin-1 (PS1) is the catalytic subunit of γ-secretase which cleaves within the transmembrane domain of over 150 peptide substrates. Dominant missense mutations in PS1 cause early-onset familial Alzheimer’s disease (FAD); however, the exact pathogenic mechanism remains unknown. Here we combined Gaussian accelerated molecular dynamics (GaMD) simulations and biochemical experiments to determine the effects of six representative PS1 FAD mutations (P117L, I143T, L166P, G384A, L435F, and L286V) on the enzyme-substrate interactions between γ-secretase and amyloid precursor protein (APP). Biochemical experiments showed that all six PS1 FAD mutations rendered γ-secretase less active for the endoproteolytic (ε) cleavage of APP. Distinct low-energy conformational states were identified from the free energy profiles of wildtype and PS1 FAD-mutant γ-secretase. The P117L and L286V FAD mutants could still sample the “Active” state for substrate cleavage, but with noticeably reduced conformational space compared with the wildtype. The other mutants hardly visited the “Active” state. The PS1 FAD mutants were found to reduce γ-secretase proteolytic activity by hindering APP residue L49 from proper orientation in the active site and/or disrupting the distance between the catalytic aspartates. Therefore, our findings provide mechanistic insights into how PS1 FAD mutations affect structural dynamics and enzyme-substrate interactions of γ-secretase and APP.more » « less
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Cyanobacteria are responsible for up to 80% of aquatic carbon dioxide fixation and have evolved specialized carbon concentrating mechanism to increase photosynthetic yield. As such, cyanobacteria are attractive targets for synethic biology and engineering approaches to address the demands of global energy security, food production, and climate change for an increasing world’s population. The bicarbonate transporter BicA is a sodium-dependent, low-affinity, high-flux bicarbonate symporter expressed in the plasma membrane of cyanobacteria. Despite extensive biochemical characterization of BicA, including the resolution of the BicA crystal structure, the dynamic understanding of the bicarbonate mechanism remains elusive. To this end, we have collected over 1 ms of all-atom molecular dynamics simulation data of the BicA dimer to elucidate the structural rearrangements involved in the substrate transport process. We further characterized the energetics of the cooperativity between BicA promoters and investigated potential mutations that are shown to decrease the free energy barrier of conformational transitions. In all, our study illuminates a detailed mechanistic understanding of the conformational dynamics of bicarbonate transporters and provide atomistic insights to engineering these transporters for enhanced photosynthetic production.more » « less
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