Abstract:ClC chloride channels and transporters are important for chloride homeostasis in species from bacteria to human. Mutations in ClC proteins cause genetically inherited diseases, some of which are likely to have folding defects. The ClC proteins present a challenging and unusual biological folding problem because they are large membrane proteins possessing a complex architecture with many re-entrant helices that go only part way through membrane and loop back out. Here we were able to examine the unfolding of th… Show more
“…In addition, 3/4TMH protodomains provide cohesive energetically stable supersecondary structural units that can self-assemble. The biophysical evidence is only now beginning to emerge (Min et al 2018).…”
Section: Resultsmentioning
confidence: 99%
“…The latter could in fact be described as a pseudo-quaternary organization of protodomains (Myers-Turnbull et al 2014;Youkharibache 2019). A recent biophysical study on the ClC chloride transporter found that the transporter is made up of two halves that fold independently as stable subunits, suggesting an evolutionary history of a stable protodomain that duplicated (Min et al 2018).…”
The polytopic helical membrane proteome is dominated by proteins containing seven transmembrane helices (7TMHs). They cannot be grouped under a monolithic fold or superfold. However, a parallel structural analysis of folds around that magic number of seven in distinct protein superfamilies (SWEET, PnuC, TRIC, FocA, Aquaporin, GPCRs) reveals a common homology, not in their structural fold, but in their systematic pseudo-symmetric construction during their evolution. Our analysis leads to guiding principles of intragenic duplication and pseudo-symmetric assembly of ancestral transmembrane helical protodomains, consisting of 3 (or 4) helices. A parallel deconstruction and reconstruction of these domains provides a structural and mechanistic framework for their evolutionary paths. It highlights the conformational plasticity inherent to fold formation itself, the role of structural as well as functional constraints in shaping that fold, and the usefulness of protodomains as a tool to probe convergent vs divergent evolution. In the case of FocA vs. Aquaporin, this protodomain analysis sheds new light on their potential divergent evolution at the protodomain level followed by duplication and parallel evolution of the two folds. GPCR domains, whose function does not seem to require symmetry, nevertheless exhibit structural pseudosymmetry. Their construction follows the same protodomain assembly as any other pseudo-symmetric protein suggesting their potential evolutionary origins. Interestingly, all the 6/7/8TMH pseudo-symmetric folds in this study also assemble as oligomeric forms in the membrane, emphasizing the role of symmetry in evolution, revealing self-assembly and co-evolution not only at the protodomain level but also at the domain level.
“…In addition, 3/4TMH protodomains provide cohesive energetically stable supersecondary structural units that can self-assemble. The biophysical evidence is only now beginning to emerge (Min et al 2018).…”
Section: Resultsmentioning
confidence: 99%
“…The latter could in fact be described as a pseudo-quaternary organization of protodomains (Myers-Turnbull et al 2014;Youkharibache 2019). A recent biophysical study on the ClC chloride transporter found that the transporter is made up of two halves that fold independently as stable subunits, suggesting an evolutionary history of a stable protodomain that duplicated (Min et al 2018).…”
The polytopic helical membrane proteome is dominated by proteins containing seven transmembrane helices (7TMHs). They cannot be grouped under a monolithic fold or superfold. However, a parallel structural analysis of folds around that magic number of seven in distinct protein superfamilies (SWEET, PnuC, TRIC, FocA, Aquaporin, GPCRs) reveals a common homology, not in their structural fold, but in their systematic pseudo-symmetric construction during their evolution. Our analysis leads to guiding principles of intragenic duplication and pseudo-symmetric assembly of ancestral transmembrane helical protodomains, consisting of 3 (or 4) helices. A parallel deconstruction and reconstruction of these domains provides a structural and mechanistic framework for their evolutionary paths. It highlights the conformational plasticity inherent to fold formation itself, the role of structural as well as functional constraints in shaping that fold, and the usefulness of protodomains as a tool to probe convergent vs divergent evolution. In the case of FocA vs. Aquaporin, this protodomain analysis sheds new light on their potential divergent evolution at the protodomain level followed by duplication and parallel evolution of the two folds. GPCR domains, whose function does not seem to require symmetry, nevertheless exhibit structural pseudosymmetry. Their construction follows the same protodomain assembly as any other pseudo-symmetric protein suggesting their potential evolutionary origins. Interestingly, all the 6/7/8TMH pseudo-symmetric folds in this study also assemble as oligomeric forms in the membrane, emphasizing the role of symmetry in evolution, revealing self-assembly and co-evolution not only at the protodomain level but also at the domain level.
“…Note that forces become negative values when a certain terminal COM moves more than a target position (defined by the dummy point) due to the thermal fluctuation. This could be considered to be evidence that the pulling speed in this work is low enough to probe a realistic response (although a pulling speed of 500 mm/s in this study is still much higher than those used in experiments whose usual highest pulling speed reaches 0.01 mm/s in atomic force microscopy 36 , 37 , or at most 0.1 mm/s 38 ). Figure 5 Force profiles as a function of D EE for different systems, ( A ) (red) and (blue) and ( B ) (green) and (black).…”
von Willebrand Factor (vWF) is a large multimeric protein that binds to platelets and collagen in blood clotting. vWF A2 domain hosts a proteolytic site for ADAMTS13 (A Disintegrin and Metalloprotease with a ThromboSpondin type 1 motif, member 13) to regulate the size of vWF multimers. This regulation process is highly sensitive to force conditions and protein-glycan interactions as the process occurs in flowing blood. There are two sites on A2 domain (N1515 and N1574) bearing various N-linked glycan structures. In this study, we used molecular dynamics (MD) simulation to study the force-induced unfolding of A2 domain with and without a single N-linked glycan type on each site. The sequential pullout of β-strands was used to represent a characteristic unfolding sequence of A2. This unfolding sequence varied due to protein-glycan interactions. The force-extension and total energy-extension profiles also show differences in magnitude but similar characteristic shapes between the systems with and without glycans. Systems with N-linked glycans encountered higher energy barriers for full unfolding and even for unfolding up to the point of ADAMTS13 cleavage site exposure. Interestingly, there is not much difference observed for A2 domain structure itself with and without glycans from standard MD simulations, suggesting roles of N-glycans in A2 unfolding through long-ranged protein-glycan interactions.
“…Since the publication of the force spectroscopy experiments on GlpG 3 , the same method has been applied to a designed α -helical transmembrane protein with four transmembrane helices 5 and a large chloride transporter, ClC, that has a complex topology and two independently metastable subdomains 4 . In the case of the designed transmembrane protein, unfolding occurs cooperatively in a single step and refolding after force-induced unfolding was found to be reliable and to occur in two steps.…”
Membrane protein folding mechanisms and rates are notoriously hard to determine. A recent force spectroscopy study of the folding of an α-helical membrane protein, GlpG, showed that the folded state has a very high kinetic stability and a relatively low thermodynamic stability. Here, we simulate the spontaneous insertion and folding of GlpG into a bilayer. An energy landscape analysis of the simulations suggests that GlpG folds via sequential insertion of helical hairpins. The rate-limiting step involves simultaneous insertion and folding of the final helical hairpin. The striking features of GlpG’s experimentally measured landscape can therefore be explained by a partially inserted metastable state, which leads us to a reinterpretation of the rates measured by force spectroscopy. Our results are consistent with the helical hairpin hypothesis but call into question the two-stage model of membrane protein folding as a general description of folding mechanisms in the presence of bilayers.
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