Curated articles

Evaluation Statement (14 June 2024)

The study by He et al. explores the structure and mechanisms of the human mitochondrial RNA splicing 2 (MRS2) protein, predicted to form Mg2+-selective channels in the mitochondrial inner membrane based on homology to the CorA family of prokaryotic Mg2+ channels. The authors use an innovative biochemical strategy to express MRS2 and perform single particle reconstructions in the absence and presence of key divalent cations. High resolution reconstructions of the pentameric channel reveal binding sites for Mg2+ and Ca2+, and electrophysiological investigations suggest that MRS2 is a Ca2+-regulated, cation-selective, Mg2+-permeable channel, in contrast to the Mg2+-regulated, Mg2+-selective CorA channel. This is an important study with interesting structural and functional observations, which will motivate further investigations of a potential role for MRS2 in mitochondrial Ca2+ signaling.

Biophysics Colab recommends this study to scientists interested in the structure, function and regulation of cation channels as well as those working on mitochondrial transport.

Biophysics Colab has evaluated this study as one that meets the following criteria:

- Rigorous methodology

- Transparent reporting

- Appropriate interpretation

(This evaluation refers to the version of record for this work, which is linked to and has been revised from the original preprint following peer review.)

Evaluation statement (17 January 2024)

The study by Bassetto Jr. et al. presents an elegant and pioneering technique to rapidly manipulate membrane temperature by up to 10 ºC in less than 1.5 ms, thereby enabling high temporal resolution of the temperature dependence of ion channel currents. The approach combines the cut-open oocyte voltage clamp technique with laser illumination to heat the sub-membrane melanosome layer. Temperature is quantified from observed changes in membrane capacitance. Recordings of Kir1.1, TRPM8, and TRPV1 channels are used to validate the effectiveness of the technique. A limitation is that, in its current form, the technique can be used only on melanosome-containing Xenopus oocyte membranes.

Biophysics Colab recommends this study to scientists working on the temperature dependence of ion channels and other membrane proteins.

Biophysics Colab has evaluated this study as one that meets the following criteria:

• Rigorous methodology
• Transparent reporting
• Appropriate interpretation

(This evaluation refers to the version of record for this work, which is linked to and has been revised from the original preprint following peer review.)

Evaluation statement (8 March 2024)

Seljeset et al. investigate the mechanism by which NMDA receptors are activated by co-agonists glutamate and glycine. By mutating residue Asp732 in the glycine-binding site, they generate receptors activated by glutamate, and not glycine, but inhibited by glycine antagonists. Conventional and unnatural amino acid mutagenesis reveals that Asp732 interacts with nearby residues to influence channel gating as well as ligand binding. Furthermore, a homomeric receptor from Trichoplax adhaerens, which has a tyrosine in the homologous position, displays constitutive activity that becomes glycine-dependent when the tyrosine is mutated to aspartate. The study is valuable because it reveals the importance of position 732 for controlling ligand potency and channel activity in glutamate receptors, which should lead to a better understanding of how these receptors are primed for channel opening.

Biophysics Colab recommends this study to scientists interested in the structure and function of glutamate receptors

Biophysics Colab has evaluated this study as one that meets the following criteria:

• Rigorous methodology
• Transparent reporting
• Appropriate interpretation

(This evaluation refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

Evaluation statement (17 January 2024; revised 31 January 2024)

Feng and colleagues investigate the molecular basis of lipid scrambling in a fungal member of the TMEM16 family of Ca²⁺-dependent lipid scramblases. These proteins possess a groove in their 3D structure that has been implicated in lipid scrambling, which the authors investigate in the absence and presence of Ca²⁺ using a combination of cryo-EM structure determination, mutagenesis and functional assays. Their closed-groove structure reveals a continuous file of lipid molecules around the catalytic groove region, providing a structural basis for lipid interaction with the protein. Additionally, the authors capture three novel states of TMEM16, completing the picture of conformational transitions that this protein undergoes. Strikingly, the authors show that both structure and distribution of the protein’s conformations depend on lipid composition and nanodisc scaffold protein.

Biophysics Colab considers this to be exceptional work and recommends it to scientists interested in plasma membrane lipid homeostasis and cryoEM.

(This evaluation by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

Evaluation statement (10 January 2024)

Driggers et al. is an elegant study that reports the structure of an open KATP channel complex formed from the Q52R diabetes mutation of the pore-forming subunit Kir 6.2, the sulfonylurea receptor (SUR1), and long-chain phosphatidylinositol 4,5-bisphosphate (PIP2) – a key lipid that stabilizes the open state of the channel and regulates inhibition by intracellular ATP. The structure reveals one PIP2 site related to that seen in other Kir channels as well as a second unanticipated one where the lipid snuggles into the interface between Kir6.2 and a region of SUR1 previously implicated in promoting the open state of KATP. This important finding helps to explain how PIP2 exerts such a profound regulatory influence on KATP.

Biophysics Colab considers this to be a convincing study and recommends it to scientists working on KATP and other membrane proteins regulated by PIP2.

(This evaluation by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

Evaluation statement (1 September 2023)

Flores-Aldama and colleagues set out to identify molecular determinants of fast inactivation in the TRPV6 ion channel, a mechanism not observed in the closely related TRPV5 channel. The work focuses on a helix-loop-helix (HLH) motif, located at the interface between several important regions for channel gating. Using molecular dynamics simulations and analysis of mutations, the authors identify pairs of amino acid residues in a structural triad formed by the HLH, S2-S3 linker, and transmembrane domains, which show different conformations in the available TRPV5 and TRPV6 cryo-EM structures. An important aspect of the study is that some of the structural hypotheses were derived from an evolutionary analysis of sequences from orthologues of both channels, demonstrating the value of this type of analysis.

Biophysics Colab considers this to be a convincing study and recommends it to scientists interested in the molecular determinants of ion channel gating.

(This evaluation by Biophysics Colab refers to the version of record for this work, which is linked to and has been revised from the original preprint following peer review.)

Evaluation statement (22 August 2023)

Bansal et al. present an atomistic view of the transition cascade of the class F GPCR Smoothened (Smo). The extensive long-range molecular dynamics simulations together with stochastic modelling provide theoretical insight into Smo activation and how this is modulated by different ligands. The work identifies testable hypotheses for functional studies of Smo and other class F GPCRs. Future simulations of regions beyond the seven-transmembrane bundle, particularly the cysteine-rich domain, will afford a more complete understanding of receptor activation.

Biophysics Colab considers this to be a convincing computational study and recommends it to scientists interested in the conformational dynamics of class F GPCRs.

(This evaluation by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

Evaluation statement (24 May 2023)

Yang et al. present valuable information about ligand interactions with the serotonin transporter SERT, innovatively purified from pig brain using Fab fragments. The approach of using natively expressed SERT is notable for its potential insight into binding of endogenous membrane components such as lipids. Data distinguishing binding of the psychostimulants methamphetamine and cocaine add to our knowledge of substrate and inhibitor interactions with SERT and allow direct comparison with the closely related dopamine transporter DAT. The authors carefully state the limitations of their findings, including the possibility that the monomeric transporter stable in detergent micelles might exist in a multimeric state in native membranes.

Biophysics Colab considers this to be a convincing study and recommends it to scientists interested in the structure, mechanism and ligand interactions of neurotransmitter transporters.

(This evaluation by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

eLife assessment

The authors describe the dynamics underlying allostery of the adenosine A1 receptor, providing valuable insights into the receptor's activation pathway. The enhanced sampling molecular dynamics simulations of available structural data, followed by network analysis, reveal transient conformational states and communication between functional regions. The authors carefully state the limitations of their work, including the restricted convergence of the free energy landscape and missing water-mediated hydrogen bond coordination. Collectively, they provide a convincing framework for advancing rational design strategies of specific modulators with desired modes of action.

[Editors' note: this was originally reviewed and assessed by Biophysics Colab]

Evaluation statement (16 June 2023)

Maria-Solano and Choi present the dynamics underlying allostery of the adenosine A1 receptor, providing valuable insights into the receptor's activation pathway. The enhanced sampling molecular dynamics simulations of available structural data, followed by network analysis, reveal transient conformational states and communication between functional regions. The authors carefully state the limitations of their work, including the restricted convergence of the free energy landscape and missing water-mediated hydrogen bond coordination. Collectively, the findings provide a convincing framework to advance rational design strategies of specific modulators with desired modes of action.

Biophysics Colab considers this to be a convincing study and recommends it to scientists interested in the structural dynamics, allosteric pathway activations, and free energy landscapes of GPCRs.

(This evaluation by Biophysics Colab refers to version 5 of this preprint, which has been revised in response to peer review of versions 3 and 4.)

Endorsement statement (22 December 2022)

The preprint by Suarez-Delgado et al. explores the mechanisms by which the Hv1 voltage-activated proton channel is dependent upon transmembrane voltage and pH by incorporating the small fluorescent non-canonical amino acid Anap into the S4 helix and monitoring its fluorescence. Anap spectra suggest the fluorophore resides in an aqueous environment and moves relative to a quenching aromatic residue (F150) in the S2 helix upon depolarization. Two kinetically distinct components of fluorescence change support the presence of at least three conformational states in the activation pathway of Hv1. Measurements using different pH gradients suggest that S4 movement and channel opening are similarly affected by pH gradients. This is the first study to incorporate Anap into Hv1, and provide a rigorous and thorough characterization of how the fluorophore can be used to explore mechanisms of gating and regulation, paving the way for future studies. The work will be of interest to physiologists and biophysicists investigating membrane protein mechanisms using non-canonical fluorescent amino acids.

(This endorsement by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

Endorsement statement (6 December 2022)

The preprint by Yang et al. asks how the shape of the membrane influences the localization of mechanosensitive Piezo channels. The authors use a creative approach involving methods that distort the plasma membrane by generating blebs and artificial filopodia. They convincingly show that curvature of the lipid environment influences Piezo1 localization, such that increased curvature causes channel depletion, and that application of the chemical modulator Yoda1 is sufficient to allow channels to enter filopodia. The study provides support for a provocative “flattening model” of Yoda1 action, and should inspire future studies by researchers interested in mechanosensitive channels and membrane curvature.

(This endorsement by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

eLife assessment

This fundamental study presents solid evidence for T1r (sweet /umami) taste receptors as chloride (Cl-) receptors, based on a combination of state-of-the-art techniques to demonstrate that T1r receptors from Medaka fish bind chloride and that this binding induces a conformational change in the heteromeric receptor. This conformational change leads to low-concentration chloride-specific action potential firing in nerves from neurons containing these receptors in mice, results that represent an important advance in our understanding of the logic of taste perception.

Endorsement statement (17 November 2022)

The preprint by Atsumi et al. describes how chloride binding to sweet- and umami-sensing proteins (T1R taste receptors) can evoke taste sensation. The authors use an elegant combination of structural, biophysical and electrophysiological approaches to locate a chloride binding site in the ligand-binding domain of medaka fish T1r2a/3 receptors. They convincingly show that low mM concentrations of chloride induce conformational changes and, using single fiber recordings, establish that mouse chorda tympani nerves are activated by chloride in a T1R-dependent manner. This suggests that chloride binding to sweet receptors could mediate the commonly reported sweet taste sensation following ingestion of low concentrations of table salt. The findings will be of broad relevance to those studying taste sensation and ligand recognition in GPCRs.

(This endorsement by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

Endorsement statement (3 October 2022)

The preprint by Galles et al. reports the generation of pyrrolysine-based aminoacyl-tRNA synthetases capable of incorporating fluorinated phenylalanine non-canonical amino acids into proteins expressed in either bacteria or mammalian cells. For the most extensively characterized synthetases, fluorinated phenylalanine derivatives were successfully incorporated into GFP and two membrane proteins (CFTR and Nav1.5) at expression levels adequate for biochemical studies, suggesting that the approach could be combined with multiple different structural and biophysical techniques. The work provides a valuable tool that will enable the functional role of cation-pi interactions to be interrogated in both soluble and integral membrane proteins.

(This endorsement by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

Endorsement statement (5 August 2022)

Peter et al. describe the first experimentally validated structural model of a canonical member of the TRAP family of transporters, Haemophilus influenzae (Hi)SiaPQM, which transports sialic acid into bacteria. By elegantly combining a cryo-EM structure of the HiSiaQM dimer, AlphaFoldmodels, and sequence, biochemical, and mutational analyses, the authors shed light on the fold and domain organization of the tripartite HiSiaPQM holocomplex. The authors also propose a structure-based model for its transport mechanism: substrate recognition is "outsourced" to the substrate binding protein (P protein) by the QM proteins, which in turn use an elevator mechanism to transport sialic acid across the membrane. The work is rigorous and convincing, and it presents valuable findings that will be of interest to scientists investigating transporters with an elevator-type mechanism as well as membrane transport more generally.

(This endorsement by Biophysics Colab refers to the version of record for this work, which is linked to and has been revised from the original preprint following peer review.)

Endorsement statement (28 June 2022)

Sauer et al. describe two cryo-EM structures of the Na⁺-dependent dicarboxylate transporter VcINDY in two inward-facing states. The high-quality structural data, complemented by NMR-inspired analysis, functional assays and cysteine accessibility measurements, reveal crucial conformational changes induced by Na⁺ binding to apo VcINDY that result in formation of the substrate-binding site. This is a strong manuscript that provides an important contribution to our understanding of the transport mechanism in the SLC13/DASS family of transporters, several members of which have critical physiological functions. The work will be of interest to researchers working on this and other ion-coupled transporter families.

(This endorsement by Biophysics Colab refers to the version of record for this work, which is linked to and has been revised from the original preprint following peer review.)

Biophysics Colab

Endorsement statement (11 May 2022)

Falzone et al. report important new cryo-EM structures of the fungal calcium-activated lipid scramblase afTMEM16, as well as the functional impact of mutations and different lipid membrane compositions on lipid scrambling. Individual lipids are beautifully resolved around the subunit cavity involved in lipid scrambling in one of the highest resolution structures of a TMEM16 protein solved to date, enabling the role of these lipid-interacting residues to be interrogated. Collectively, the results suggest that afTMEM16 catalyzes lipid scrambling by thinning the membrane rather than providing a hydrophilic permeation pathway for lipids. The work represents an important contribution that will be of interest to scientists investigating the mechanisms of lipid scrambling and how membrane proteins interact with their lipid environment.

(This endorsement by Biophysics Colab refers to the version of record for this work, which is linked to and has been revised from the original preprint following peer review.)

Endorsement statement (27 April 2022)

The preprint by Fiedorczuk and Chen presents structures of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in complex with type I correctors, a class of drug currently used to treat cystic fibrosis by targeting CFTR folding and stability. The strength of the paper lies in the consistency of the structural data with maturation and binding assays, as well as with much of the existing literature. Overall, the work represents a rigorous investigation of the mechanism of these drugs, and will be of interest to those who study cystic fibrosis, protein folding, and drug design.

(This endorsement refers to version 1 of this preprint, which was peer reviewed by Biophysics Colab.)

Endorsement statement (30 August 2021)

The preprint by Patel et al. describes the development of a single molecule approach for studying individual ligand binding events in membrane proteins within native lipid environments. The approach represents an elegant way to investigate the dynamics of ligand binding, and potential relationships with conformational changes, in molecules embedded within physiological membranes. The work makes an important contribution that will be of interest to scientists working on molecular mechanisms in ion channels and other membrane proteins.

(This endorsement by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

Endorsement statement (21 September 2021)

The preprint by Heitkamp and Börsch describes visualization of the fast ATP-dependent subunit rotation in reconstituted FoF1-ATP synthase using single-molecule FRET techniques. Using a highly innovative method for trapping single molecules, the authors were able to see the static and dynamic disorder of enzymes in solution, not possible in previous studies. The work makes important contributions to both understanding the structural dynamics of FoF1-ATP synthase and the development of methodologies to study single-molecule dynamics of other proteins in solution.

(This endorsement refers to version 5 of this preprint, which was peer reviewed by Biophysics Colab.)