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eLife Assessment

This manuscript presents useful insights into the molecular basis underlying the positive cooperativity between the co-transported substrates (galactoside sugar and sodium ion) in the melibiose transporter MelB. Building on years of previous studies, this work improves on the resolution of previously published structures and reports the presence of a water molecule in the sugar binding site that would appear to be key for its recognition, introduces further structures bound to different substrates, and utilizes HDX-MS to further understand the positive cooperativity between sugar and the co-transported sodium cation. Although the experimental work is solid, the presentation of the data lacks clarity, and in particular, the HDX-MS data interpretation requires further explanation in both methodology and discussion, as well as a clearer description of the new insight that is obtained in relation to previous studies. The work will be of interest to biologists and biochemists working on cation-coupled symporters, which mediate the transport of a wide range of solutes across cell membranes.

Reviewer #1 (Public review):

While the structure of the melibiose permease in both outward and inward-facing forms has been solved previously, there remain unanswered questions regarding its mechanism. Hariharan et al set out to address this with further crystallographic studies complemented with ITC and hydrogen-deuterium exchange (HDX) mass spectrometry. They first report 4 different crystal structures of galactose derivatives to explore molecular recognition, showing that the galactose moiety itself is the main source of specificity. Interestingly, they observe a water-mediated hydrogen bonding interaction with the protein and suggest that this water molecule may be important in binding.

The results from the crystallography appear sensible, though the resolution of the data is low, with only the structure with NPG better than 3Å. However, it is a bit difficult to understand what novel information is being brought out here and what is known about the ligands. For instance, are these molecules transported by the protein or do they just bind? They measure the affinity by ITC, but draw very few conclusions about how the affinity correlates with the binding modes. Can the protein transport the trisaccharide raffinose?

The HDX also appears to be well done; however, in the manuscript as written, it is difficult to understand how this relates to the overall mechanism of the protein and the conformational changes that the protein undergoes.

Reviewer #2 (Public review):

This manuscript from Hariharan, Shi, Viner, and Guan presents x-ray crystallographic structures of membrane protein MelB and HDX-MS analysis of ligand-induced dynamics. This work improves on the resolution of previously published structures, introduces further sugar-bound structures, and utilises HDX to explore in further depth the previously observed positive cooperatively to cotransported cation Na+. The work presented here builds on years of previous study and adds substantial new details into how Na+ binding facilitates melibiose binding and deepens the fundamental understanding of the molecular basis underlying the symport mechanism of cation-coupled transporters. However, the presentation of the data lacks clarity, and in particular, the HDX-MS data interpretation requires further explanation in both methodology and discussion.

Comments on Crystallography and biochemical work:

(1) It is not clear what Figure 2 is comparing. The text suggests this figure is a comparison of the lower resolution structure to the structure presented in this work; however, the figure legend does not mention which is which, and both images include a modelled water molecule that was not assigned due to poor resolution previously, as stated by the authors, in the previously generated structure. This figure should be more clearly explained.

(2) It is slightly unclear what the ITC measurements add to this current manuscript. The authors comment that raffinose exhibiting poor binding affinity despite having more sugar units is surprising, but it is not surprising to me. No additional interactions can be mapped to these units on their structure, and while it fits into the substrate binding cavity, the extra bulk of additional sugar units is likely to reduce affinity. In fact, from their listed ITC measurements, this appears to be the trend. Additionally, the D59C mutant utilised here in structural determination is deficient in sodium/cation binding. The reported allostery of sodium-sugar binding will likely influence the sugar binding motif as represented by these structures. This is clearly represented by the authors' own ITC work. The ITC included in this work was carried out on the WT protein in the presence of Na+. The authors could benefit from clarifying how this work fits with the structural work or carrying out ITC with the D59C mutant, or additionally, in the absence of sodium.

Comments on HDX-MS work:

While the use of HDX-MS to deepen the understanding of ligand allostery is an elegant use of the technique, this reviewer advises the authors to refer to the Masson et al. (2019) recommendations for the HDX-MS article (https://doi.org/10.1038/s41592-019-0459-y) on how to best present this data. For example:

(1) The Methodology includes a lipid removal step. Based on other included methods, I assumed that the HDX-MS was being carried out in detergent-solubilised protein samples. I therefore do not see the need for a lipid removal step that is usually included for bilayer reconstituted samples. I note that this methodology is the same as previously used for MelB. It should be clarified why this step was included, if it was in fact used, aka, further details on the sample preparation should be included.

(2) A summary of HDX conditions and results should be given as recommended, including the mean peptide length and average redundancy per state alongside other included information such as reaction temperature, sequence coverage, etc., as prepared for previous publications from the authors, i.e., Hariharan et al., 2024.

(3) Uptake plots per peptide for the HDX-MS data should be included as supporting information outside of the few examples given in Figure 6.

(4) A reference should be given to the hybrid significance testing method utilised. Additionally, as stated by Hageman and Weis (2019) (doi:10.1021/acs.analchem.9b01325), the use of P < 0.05 greatly increases the likelihood of false positive ΔD identifications. While the authors include multiple levels of significance, what they refer to as high and lower significant results, this reviewer understands that working with dynamic transporters can lead to increased data variation; a statement of why certain statistical criteria were chosen should be included, and possibly accompanied by volcano plots. The legend of Figure 6 should include what P value is meant by * and ** rather than statistically significant and highly statistically significant.

(5) Line 316 states a significant difference in seen in dynamics, how is significance measured here? There is no S.D. given in Table S4. Can the authors further comment on the potential involvement in solvent accessibility and buried helices that might influence the overall dynamics outside of their role in sugar vs sodium binding? An expected low rate of exchange suggests that dynamics are likely influenced by solvent accessibility or peptide hydrophobicity? The increased dynamics at peptides covering the Na binding site on overall more dynamic helices suggests that there is no difference between the dynamics of each site.

(6) Previously stated HDX-MS results of MelB (Hariharan et al., 2024) state that the transmembrane helices are less dynamic than polypeptide termini and loops with similar distributions across all transmembrane bundles. The previous data was obtained in the presence of sodium. Does this remove the difference in dynamics in the sugar-binding helices and the cation-binding helices? Including this comparison would support the statement that the sodium-bound MelB is more stable than the Apo state, along with the lack of deprotection observed in the differential analysis.

(7) Have the authors considered carrying out an HDX-MS comparison between the WT and the D59C mutant? This may provide some further information on the WT structure (particularly a comparison with sugar-bound). This could be tied into a nice discussion of their structural data.

(8) Have the authors considered utilising Li+ to infer how cation selectivity impacts the allostery? Do they expect similar stabilisation of a higher-affinity sugar binding state with all cations?

(9) MD of MelB suggests all transmembrane helices are reorientated during substrate translocation, yet substrate and cotransporter ligand binding only significantly impacts a small number of helices. Can the authors comment on the ensemble of states expected from each HDX experiment? The data presented here instead shows overall stabilisation of the transporter. This data can be compared to that of HDX on MFS sugar cation symporter XylE, where substrate binding induces a transition to OF state. There is no discussion of how this HDX data compares to previous MFS sugar transporter HDX. The manuscript could benefit from this comparison rather than a comparison to LacY. It is unlikely that there are universal mechanisms that can be inferred even from these model proteins. Highlighting differences instead between these transport systems provides broader insights into this protein class. Doi: 10.1021/jacs.2c06148 and 10.1038/s41467-018-06704-1.

(10) Additionally, the recent publication of SMFS data (by the authors: doi:10.1016/j.str.2022.11.011) states the following: "In the presence of either melibiose or a coupling Na+-cation, however, MelB increasingly populates the mechanically less stable state which shows a destabilized middle-loop C3." And "In the presence of both substrate and co-substrate, this mechanically less stable state of MelB is predominant.". It would benefit the authors to comment on these data in contrast to the HDX obtained here. Additionally, is the C3 loop covered, and does it show the destabilization suggested by these studies? HDX can provide a plethora of results that are missing from the current analysis on ligand allostery. The authors instead chose to reference CD and thermal denaturation methods as comparisons.

Reviewer #3 (Public review):

Summary:

The melibiose permease from Salmonella enterica serovar Typhimurium (MelBSt) is a member of the Major Facilitator Superfamily (MFS). It catalyzes the symport of a galactopyranoside with Na⁺, H⁺, or Li⁺, and serves as a prototype model system for investigating cation-coupled transport mechanisms. In cation-coupled symporters, a coupling cation typically moves down its electrochemical gradient to drive the uphill transport of a primary substrate; however, the precise role and molecular contribution of the cation in substrate binding and translocation remain unclear. In a prior study, the authors showed that the binding affinity for melibiose is increased in the presence of Na+ by about 8-fold, but the molecular basis for the cooperative mechanism remains unclear. The objective of this study was to better understand the allosteric coupling between the Na+ and melibiose binding sites. To verify the sugar-recognition specific determinants, the authors solved the outward-facing crystal structures of a uniport mutant D59C with four sugar ligands containing different numbers of monosaccharide units (α-NPG, melibiose, raffinose, or α-MG). The structure with α-NPG bound has improved resolution (2.7 Å) compared to a previously published structure and to those with other sugars. These structures show that the specificity is clearly directed toward the galactosyl moiety. However, the increased affinity for α-NPG involves its hydrophobic phenyl group, positioned at 4 Å-distance from the phenyl group of Tyr26 forms a strong stacking interaction. Moreover, a water molecule bound to OH-4 in the structure with α-NPG was proposed to contribute to the sugar recognition and appears on the pathway between the two specificity-determining pockets. Next, the authors analyzed by hydrogen-to-deuterium exchange coupled to mass spectrometry (HDX-MS) the changes in structural dynamics of the transporter induced by melibiose, Na+, or both. The data support the conclusion that the binding of the coupling cation at a remote location stabilizes the sugar-binding residues to switch to a higher-affinity state. Therefore, the coupling cation in this symporter was proposed to be an allosteric activator.

Strengths:

(1) The manuscript is generally well written.

(2) This study builds on the authors' accumulated knowledge of the melibiose permease and integrates structural and HDX-MS analyses to better understand the communication between the sodium ion and sugar binding sites. A high sequence coverage was obtained for the HDX-MS data (86-87%), which is high for a membrane protein.

Weaknesses:

(1) I am not sure that the resolution of the structure (2.7 Å) is sufficiently high to unambiguously establish the presence of a water molecule bound to OH-4 of the α-NPG sugar. In Figure 2, the density for water 1 is not obvious to me, although it is indeed plausible that water mediates the interaction between OH4/OH6 and the residues Q372 and T373.

(2) Site-directed mutagenesis could help strengthen the conclusions of the authors. Would the mutation(s) of Q372 and/or T373 support the water hypothesis by decreasing the affinity for sugars? Mutations of Thr 121, Arg 295, combined with functional and/or HDX-MS analyses, may also help support some of the claims of the authors regarding the allosteric communication between the two substrate-binding sites.

(3) The main conclusion of the authors is that the binding of the coupling cation stabilizes those dynamic sidechains in the sugar-binding pocket, leading to a high-affinity state. This is visible when comparing panels c and a from Figure S5. However, there is both increased protection (blue, near the sugar) and decreased protection in other areas (red). The latter was less commented, could the increased flexibility in these red regions facilitate the transition between inward- and outward-facing conformations? The HDX changes induced by the different ligands were compared to the apo form (see Figure S5). It might be worth it for data presentation to also analyze the deuterium uptake difference by comparing the conditions sodium ion+melibiose vs melibiose alone. It would make the effect of Na+ on the structural dynamics of the melibiose-bound transporter more visible. Similarly, the deuterium uptake difference between sodium ion+melibiose vs sodium ion alone could be analyzed too, in order to plot the effect of melibiose on the Na+-bound transporter.

(4) For non-specialists, it would be beneficial to better introduce and explain the choice of using D59C for the structural analyses.

(5) In Figure 5a, deuterium changes are plotted as a function of peptide ID number. It is hardly informative without making it clearer which regions it corresponds to. Only one peptide is indicated (213-226), I would recommend indicating more of them in areas where deuterium changes are substantial.

(6) From prior work of the authors, melibiose binding also substantially increases the affinity of the sodium ion. Can the authors interpret this observation based on the HDX data?

Author response:

Reviewer #1:

While the structure of the melibiose permease in both outward and inward-facing forms has been solved previously, there remain unanswered questions regarding its mechanism. Hariharan et al set out to address this with further crystallographic studies complemented with ITC and hydrogen-deuterium exchange (HDX) mass spectrometry.

They first report 4 different crystal structures of galactose derivatives to explore molecular recognition, showing that the galactose moiety itself is the main source of specificity. Interestingly, they observe a water-mediated hydrogen bonding interaction with the protein and suggest that this water molecule may be important in binding.

We appreciate the understanding of our work presented in this manuscript by this reviewer.

The results from the crystallography appear sensible, though the resolution of the data is low, with only the structure with NPG better than 3Å. However, it is a bit difficult to understand what novel information is being brought out here and what is known about the ligands. For instance, are these molecules transported by the protein or do they just bind? They measure the affinity by ITC, but draw very few conclusions about how the affinity correlates with the binding modes. Can the protein transport the trisaccharide raffinose?

The four structures with a bound sugar of different sizes aimed to identify the binding motif on both the primary substrate (sugar) and the transporter (MelB_St). Although the resolutions of the structures complexed with melibiose, raffinose, or a-MG are relatively low, the size and shape of the densities at each structure are consistent with the corresponding sugar molecules, which provide valuable data for determining the pose of the bound sugar. Additionally, there is another a-NPG-bound structure at a higher resolution of 2.7 Å. Therefore, our new data support the published binding site with the galactosyl moiety as the main interacting group. The identified water-1 in this study further confirms the orientation of C4-OH. Notably, this transporter does not recognize or transport glucosides where the orientation of C4-OH at the glucopyranosyl ring is opposite. We will provide stronger data to support the water-1.

Regarding the raffinose question, we should have clearly introduced the historical background. Bacterial disaccharide transporters have broad specificity, allowing them to work on a group of sugars with shared structural elements; for example, one sugar molecule can be transported by several transporters. As reported in the literature, the galactosides melibiose, lactose, and raffinose can be transported by both LacY and MelB of E. coli. We did not test whether MelB_St can transport the a-NPG and raffinose. To address this issue and strengthen our conclusions, we plan to conduct additional experiments to gather evidence of the translocation of these sugars by MelB_St.

The HDX also appears to be well done; however, in the manuscript as written, it is difficult to understand how this relates to the overall mechanism of the protein and the conformational changes that the protein undergoes.

Previously, we used HDX-MS to examine the conformational transition between inward- and outward-facing conformations using a conformation-specific nanobody to trap MelB_St in an inward-facing state, as structurally resolved by cryoEM single-particle analysis and published in eLife 2024. That study identified dynamic regions that may be involved in the conformational transitions; however, there was no sugar present. We also solved and published the crystal structure of the apo D59C MelB_St. The sugar-bound and apo states are virtually identical. To address the positive cooperativity of binding between the sugar and co-transport cations observed in biophysical analysis, in this study, we utilize HDX-MS to analyze the structural dynamics induced by melibiose, Na⁺, or both, focusing on the binding residues at the sugar-binding and cation-binding pockets. The results suggest that the coupling cation stabilizes sugar-binding residues at helices I and V, contributing to affinity but not specificity.

Since MelB_St favors the outward-facing conformation, and simulations on the free-energy landscape suggest that the highest affinity of the sugar-bound state is also at an outward-facing state, MelB_St in both the apo and bound states tend to remain in the outward-facing conformation. We will include a section comparing these differences. Thank you to this reviewer for the critical insight.

Reviewer #2:

This manuscript from Hariharan, Shi, Viner, and Guan present x-ray crystallographic structures of membrane protein MelB and HDX-MS analysis of ligand-induced dynamics. This work improves on the resolution of previously published structures, introduces further sugar-bound structures, and utilises HDX to explore in further depth the previously observed positive cooperatively to cotransported cation Na⁺. The work presented here builds on years of previous study and adds substantial new details into how Na⁺ binding facilitates melibiose binding and deepens the fundamental understanding of the molecular basis underlying the symport mechanism of cation-coupled transporters. However, the presentation of the data lacks clarity, and in particular, the HDX-MS data interpretation requires further explanation in both methodology and discussion.

We thank this reviewer for taking the time to read our previous articles related to this manuscript.

Comments on Crystallography and biochemical work:

(1) It is not clear what Figure 2 is comparing. The text suggests this figure is a comparison of the lower resolution structure to the structure presented in this work; however, the figure legend does not mention which is which, and both images include a modelled water molecule that was not assigned due to poor resolution previously, as stated by the authors, in the previously generated structure. This figure should be more clearly explained.

This figure shows a stereo view of a density map created in cross-eye style to demonstrate its quality. We will update this figure with a higher-resolution map, and the density for Wat-1 is clearly visible. This also addresses Reviewer-3’s comment regarding the map resolution.

(2) It is slightly unclear what the ITC measurements add to this current manuscript. The authors comment that raffinose exhibiting poor binding affinity despite having more sugar units is surprising, but it is not surprising to me. No additional interactions can be mapped to these units on their structure, and while it fits into the substrate binding cavity, the extra bulk of additional sugar units is likely to reduce affinity. In fact, from their listed ITC measurements, this appears to be the trend. Additionally, the D59C mutant utilised here in structural determination is deficient in sodium/cation binding. The reported allostery of sodium-sugar binding will likely influence the sugar binding motif as represented by these structures. This is clearly represented by the authors' own ITC work. The ITC included in this work was carried out on the WT protein in the presence of Na⁺. The authors could benefit from clarifying how this work fits with the structural work or carrying out ITC with the D59C mutant, or additionally, in the absence of sodium.

While raffinose and a-MG have been reported as substrates of MelB in E. coli, binding data are unavailable; additionally, for MelB_St, we lack data on the binding of two of the four sugars or sugar analogs. We performed a label-free binding assay using ITC to address this concern with the WT MelB_St. We will also perform the binding assay with the D59C MelB_St, since sugar binding has been structurally analyzed with this mutant, as pointed out by this reviewer. Along with other new functional results, we will prepare a new Figure 1 on functional analysis, which will also address the comment regarding extra bulk at the non-galactosyl moiety with poor affinity.

This D59C uniport mutant exhibits increased thermostability, making it a valuable tool for crystal structure determination, especially since the wild type (WT) is difficult to crystallize at high quality. Asp59 is the only site that responds to the binding of all coupling cations: Na⁺, Li⁺, or H⁺. Notably, this mutant selectively abolishes cation binding and cotransport. However, it still maintains intact sugar binding with slightly higher affinity and preserves the conformational transition, as demonstrated by an electroneutral transport reaction, the melibiose exchange, and fermentation assays with intact cells. Therefore, the structural data derived from this mutant are significant and offer important mechanistic insights into sugar transport. We will provide additional details during the revision.

Comments on HDX-MS work:

While the use of HDX-MS to deepen the understanding of ligand allostery is an elegant use of the technique, this reviewer advises the authors to refer to the Masson et al. (2019) recommendations for the HDX-MS article (https://doi.org/10.1038/s41592-019-0459-y) on how to best present this data. For example:

All authors appreciate this reviewer’s comments and suggestions, which will be incorporated into the revision.

(1) The Methodology includes a lipid removal step. Based on other included methods, I assumed that the HDX-MS was being carried out in detergent-solubilised protein samples. I therefore do not see the need for a lipid removal step that is usually included for bilayer reconstituted samples. I note that this methodology is the same as previously used for MelB. It should be clarified why this step was included, if it was in fact used, aka, further details on the sample preparation should be included.

Yes, a lipid/detergent removal step was applied in this study and in previous studies and this information was clearly described in Methods.

(2) A summary of HDX conditions and results should be given as recommended, including the mean peptide length and average redundancy per state alongside other included information such as reaction temperature, sequence coverage, etc., as prepared for previous publications from the authors, i.e., Hariharan et al., 2024.

We will update the Table S2. Thank you.

(3) Uptake plots per peptide for the HDX-MS data should be included as supporting information outside of the few examples given in Figure 6.

We will prepare the plots in supplementary information.

(4) A reference should be given to the hybrid significance testing method utilised. Additionally, as stated by Hageman and Weis (2019) (doi:10.1021/acs.analchem.9b01325), the use of P < 0.05 greatly increases the likelihood of false positive ΔD identifications. While the authors include multiple levels of significance, what they refer to as high and lower significant results, this reviewer understands that working with dynamic transporters can lead to increased data variation; a statement of why certain statistical criteria were chosen should be included, and possibly accompanied by volcano plots. The legend of Figure 6 should include what P value is meant by * and ** rather than statistically significant and highly statistically significant.

We appreciate this comment and will cite this article on the hybrid significance method. We will include volcano plots for each dataset. We fully acknowledge that using a cutoff of P < 0.05 can increase the likelihood of false-positive identifications. However, given the complexity of the samples analyzed in this study, we believe that some important changes may have been excluded due to higher variability within the dataset. By applying multiple levels of statistical testing, we determined that P < 0.05 represents a suitable threshold for this study. The threshold values were marked in the residual plots and explained in the text. For Figure 6, we have revised it by showing the P value directly.

(5) Line 316 states a significant difference in seen in dynamics, how is significance measured here? There is no S.D. given in Table S4. Can the authors further comment on the potential involvement in solvent accessibility and buried helices that might influence the overall dynamics outside of their role in sugar vs sodium binding? An expected low rate of exchange suggests that dynamics are likely influenced by solvent accessibility or peptide hydrophobicity? The increased dynamics at peptides covering the Na binding site on overall more dynamic helices suggests that there is no difference between the dynamics of each site.

Table S4 was created to provide an overall view of the dynamic regions. If we understand correctly, this reviewer asked us to comment on the effect of solvent accessibility or hydrophobic regions on the overall dynamics outside the binding residues of the peptides that carry binding residues. Since the HDX rate is influenced by two linked factors: solvent accessibility and hydrogen-bonding interactions that reflect structural dynamics, poor solvent accessibility in buried regions results in low deuterium uptakes. The peptides in our dataset that include the Na⁺-binding site showed low HDX, likely due to poor solvent accessibility and structural stability. It is unclear what this reviewer meant by "increased dynamics at peptides covering the Na binding site on overall more dynamic helices." We do not observe increased dynamics in peptides covering Na⁺-binding sites.

(6) Previously stated HDX-MS results of MelB (Hariharan et al., 2024) state that the transmembrane helices are less dynamic than polypeptide termini and loops with similar distributions across all transmembrane bundles. The previous data was obtained in the presence of sodium. Does this remove the difference in dynamics in the sugar-binding helices and the cation-binding helices? Including this comparison would support the statement that the sodium-bound MelB is more stable than the Apo state, along with the lack of deprotection observed in the differential analysis.

Thanks for this suggestion. The previous datasets were collected in the presence of Na⁺. In the current study, we also have a Na-containing dataset. Both showed similar results: the multiple overlapping peptides covering the sugar-binding residues on helices I and V have higher HDX rates than those covering the Na⁺-binding residues, even when Na⁺ is present in both datasets.

(7) Have the authors considered carrying out an HDX-MS comparison between the WT and the D59C mutant? This may provide some further information on the WT structure (particularly a comparison with sugar-bound). This could be tied into a nice discussion of their structural data.

Thanks for this suggestion. Conducting the HDX-MS comparison between the WT and the D59C mutant is certainly interesting, especially given the growing amount of structural and biochemical/biophysical data available for this mutant. However, due to limited resources, we might consider doing it later.

(8) Have the authors considered utilising Li⁺ to infer how cation selectivity impacts the allostery? Do they expect similar stabilisation of a higher-affinity sugar binding state with all cations?

Thanks for this suggestion. We have demonstrated that Li⁺ also shows positive cooperativity with melibiose through ITC binding measurements. Li⁺ binds to MelB_St with higher affinity than Na⁺ but causes many different effects on MelB. It is worth investigating this thoroughly and individually. To address the second question, H⁺ is a poor coupling cation with minimal impact on melibiose binding. Since its pKa is around 6.5, only a small subpopulation of MelB_St is protonated at pH 7.5. The order of sugar-binding cooperativity is the highest with Na⁺, followed by Li⁺ and H⁺.

(9) MD of MelB suggests all transmembrane helices are reorientated during substrate translocation, yet substrate and cotransporter ligand binding only significantly impacts a small number of helices. Can the authors comment on the ensemble of states expected from each HDX experiment? The data presented here instead shows overall stabilisation of the transporter. This data can be compared to that of HDX on MFS sugar cation symporter XylE, where substrate binding induces a transition to OF state. There is no discussion of how this HDX data compares to previous MFS sugar transporter HDX. The manuscript could benefit from this comparison rather than a comparison to LacY. It is unlikely that there are universal mechanisms that can be inferred even from these model proteins. Highlighting differences instead between these transport systems provides broader insights into this protein class. Doi: 10.1021/jacs.2c06148 and 10.1038/s41467-018-06704-1.

The sugar translocation free-energy landscape simulations showed that both helix bundles move relative to the membrane plane. That analysis aimed to clarify a hypothesis in the field—that the MFS transporter can use an asymmetric mode to transition between inward- and outward-facing states. In the case of MelB, we clearly demonstrated that both domains move and each helix bundle moves as a unit, so the labeling changes were identified only in some extramembrane loops and a few highly flexible helices. Thanks for the suggestion about comparing with XylE. We will include a discussion on it.

(10) Additionally, the recent publication of SMFS data (by the authors: doi:10.1016/j.str.2022.11.011) states the following: "In the presence of either melibiose or a coupling Na⁺-cation, however, MelB increasingly populates the mechanically less stable state which shows a destabilized middle-loop C3." And "In the presence of both substrate and co-substrate, this mechanically less stable state of MelB is predominant.". It would benefit the authors to comment on these data in contrast to the HDX obtained here. Additionally, is the C3 loop covered, and does it show the destabilization suggested by these studies? HDX can provide a plethora of results that are missing from the current analysis on ligand allostery. The authors instead chose to reference CD and thermal denaturation methods as comparisons.

Thank this reviewer for reading the single-molecule force spectroscopy (SMFS) study on MelB_St. The C3 loop mentioned in this SMFS article is partially covered in the dataset Mel or Mel plus Na⁺ vs. Apo, and more coverage is in the Na⁺ vs. Apo. In either condition, no deprotection was detected. Two possible reasons the HDX data did not reflect the deprotection are: 1) The changes were too subtle and did not pass the statistical tests and 2) the longest labeling time point was still insufficient to detect the changes; much longer labeling times should be considered in future studies.

Reviewer #3:

Summary:

The melibiose permease from Salmonella enterica serovar Typhimurium (MelB_St) is a member of the Major Facilitator Superfamily (MFS). It catalyzes the symport of a galactopyranoside with Na⁺, H⁺, or Li⁺, and serves as a prototype model system for investigating cation-coupled transport mechanisms. In cation-coupled symporters, a coupling cation typically moves down its electrochemical gradient to drive the uphill transport of a primary substrate; however, the precise role and molecular contribution of the cation in substrate binding and translocation remain unclear. In a prior study, the authors showed that the binding affinity for melibiose is increased in the presence of Na⁺ by about 8-fold, but the molecular basis for the cooperative mechanism remains unclear. The objective of this study was to better understand the allosteric coupling between the Na⁺ and melibiose binding sites. To verify the sugar-recognition specific determinants, the authors solved the outward-facing crystal structures of a uniport mutant D59C with four sugar ligands containing different numbers of monosaccharide units (α-NPG, melibiose, raffinose, or α-MG). The structure with α-NPG bound has improved resolution (2.7 Å) compared to a previously published structure and to those with other sugars. These structures show that the specificity is clearly directed toward the galactosyl moiety. However, the increased affinity for α-NPG involves its hydrophobic phenyl group, positioned at 4 Å-distance from the phenyl group of Tyr26 forms a strong stacking interaction. Moreover, a water molecule bound to OH-4 in the structure with α-NPG was proposed to contribute to the sugar recognition and appears on the pathway between the two specificity-determining pockets. Next, the authors analyzed by hydrogen-to-deuterium exchange coupled to mass spectrometry (HDX-MS) the changes in structural dynamics of the transporter induced by melibiose, Na⁺, or both. The data support the conclusion that the binding of the coupling cation at a remote location stabilizes the sugar-binding residues to switch to a higher-affinity state. Therefore, the coupling cation in this symporter was proposed to be an allosteric activator.

Strengths:

(1) The manuscript is generally well written.

(2) This study builds on the authors' accumulated knowledge of the melibiose permease and integrates structural and HDX-MS analyses to better understand the communication between the sodium ion and sugar binding sites. A high sequence coverage was obtained for the HDX-MS data (86-87%), which is high for a membrane protein.

Thank this reviewer for your positive comments.

Weaknesses:

(1) I am not sure that the resolution of the structure (2.7 Å) is sufficiently high to unambiguously establish the presence of a water molecule bound to OH-4 of the α-NPG sugar. In Figure 2, the density for water 1 is not obvious to me, although it is indeed plausible that water mediates the interaction between OH4/OH6 and the residues Q372 and T373.

Thanks for your comments on the resolution. We will improve the density for the Water 1.

(2) Site-directed mutagenesis could help strengthen the conclusions of the authors. Would the mutation(s) of Q372 and/or T373 support the water hypothesis by decreasing the affinity for sugars? Mutations of Thr 121, Arg 295, combined with functional and/or HDX-MS analyses, may also help support some of the claims of the authors regarding the allosteric communication between the two substrate-binding sites.

The authors thank this reviewer for the thoughtful suggestions. MelB_St has been subjected to Cys-scanning mutagenesis (https://doi.org/10.1016/j.jbc.2021.101090). Placing a Cys residue on the hydrogen bond-donor Q372 significantly decreased the transport initial rate, accumulation, and melibiose fermentation, with little effect on protein expression, as shown in Figure 2 of this JBC paper. Although no binding data are available, the poor initial rate of transport with a similar amount of protein expressed suggested that the binding affinity is apparently decreased, supporting the role of water-1 in the binding pocket for better binding. The T373C mutant retained most activities of the WT. We will discuss the functional characterizations of these two mutants. Thanks.

(3) The main conclusion of the authors is that the binding of the coupling cation stabilizes those dynamic sidechains in the sugar-binding pocket, leading to a high-affinity state. This is visible when comparing panels c and a from Figure S5. However, there is both increased protection (blue, near the sugar) and decreased protection in other areas (red). The latter was less commented, could the increased flexibility in these red regions facilitate the transition between inward- and outward-facing conformations?

Thanks for this important question. We will discuss the deprotected data in the conformational transition between inward-facing and outward-facing states. The two regions, loop8-9 and loop1-2, are located in the gate area on both sides of the membrane and showed increased deuterium uptakes upon binding of melibiose plus Na⁺. They are likely involved in this process.

The HDX changes induced by the different ligands were compared to the apo form (see Figure S5). It might be worth it for data presentation to also analyze the deuterium uptake difference by comparing the conditions sodium ion+melibiose vs melibiose alone. It would make the effect of Na⁺ on the structural dynamics of the melibiose-bound transporter more visible. Similarly, the deuterium uptake difference between sodium ion+melibiose vs sodium ion alone could be analyzed too, in order to plot the effect of melibiose on the Na⁺-bound transporter.

We will analyze the data as suggested by this reviewer.

(4) For non-specialists, it would be beneficial to better introduce and explain the choice of using D59C for the structural analyses.

As response to the reviewer #1 at page 3, “Asp59 is the only site that responds to the binding of all coupling cations: Na⁺, Li⁺, or H⁺. Notably, this mutant selectively abolishes cation binding and cotransport. However, it still maintains intact sugar binding with slightly higher affinity and preserves the conformational transition, as demonstrated by an electroneutral transport reaction, the melibiose exchange, and fermentation assays with intact cells. Therefore, the structural data derived from this mutant are significant and offer important mechanistic insights into sugar transport. We will provide additional details during the revision.”.

(5) In Figure 5a, deuterium changes are plotted as a function of peptide ID number. It is hardly informative without making it clearer which regions it corresponds to. Only one peptide is indicated (213-226), I would recommend indicating more of them in areas where deuterium changes are substantial.

We appreciate this comment, which will make the plots more meaningful. In the previous article published in eLife (2024), we drew boxed to mark the transmembrane regions; however, it generated much confusion, such as why some helices are very short. The revised figure will label the full length of covered positions.

(6) From prior work of the authors, melibiose binding also substantially increases the affinity of the sodium ion. Can the authors interpret this observation based on the HDX data?

This is an intriguing mechanistic question. Based on current data, we believe that the bound melibiose physically prevents the release of Na⁺ or Li⁺ from the cation-binding pocket. The cation-binding pocket and surrounding regions, including the sugar-binding residue Asp124, show low HDX, supporting this idea. Since we lack a structure with both substrates bound, figuring out the details structurally is challenging. However, we have a hypothesis about the intracellular Na⁺ release as proposed in the 2024 JBC paper (https://doi.org/10.1016/j.jbc.2024.107427). After sugar release, the rotamer change of Asp55 will help Na⁺ exit the cation pocket to the sugar pocket, and the negative membrane potential will facilitate the further movement from MelB to the cytosol. We will discuss this during the revision.

E: bourgeois: (adj) of or characteristic of the middle class, typically with reference to its perceived materialistic values or conventional attitudes. (oxford languages dictionary)

What would we be considering "experimental" in terms of playwriting? Will we need to know more about playwriting terms or the history or will this be more based on what your argument is?

E: "Purport: appear or claim to be or do something, especially falsely; profess." (Oxford Languages)

I find it interesting that the author used "purport" to describe how poetry and prose fiction are always written in the past. Purport could also mean the gist of something like a document or speech, but this also implies that there are uncertainties with what is true or false in the events pointed out in these works of literature.

Some historians believe that Greek Theater was a method to show the population of what they would (or should) do in difficult situations (measuring dissatisfactions against characters - related to measuring your personal conviction or what you would do in their place)

https://www.physics.ucla.edu/~huffman/finddip.html

dweb.link

即使 temperature 设为 0，那么仍然结果是不确定性的。

A skill that somebody is good at.

A community.

something completed.

Something you need to do.

A high level course.

A lower level.

A some what demo for high school students that want to try college level courses.

A level made for college students.

ID given for your course or path.

A grade option.

A review in somebody information.

Class that gives no credits.

A record of grades.

A program that offers you work.

Money given.

Money given to a student for college student that has no source of income or has low income for him or she to get into a good college

A student loan.

Financial aid.

Money given to a student who can't pay for tuition.

A path that a student takes.

This is a paper showing you completed statues.

A degree is a certificate.

This is a class chosen by a student.

A level placement.

A fee for a class.

This refers to time in a year.

This is an exchange.

Something that involves grades.

Is the number the school provides you.

Means to have permission.

Means to sign up for program.

This means to sign up for something.

This means to start school from the begin.

A non mandatory class students have a choice in picking.

Financial aid

A non mandatory class students have a choice in picking.

Not being allowed to do something for not following the given rules.

A number given to a student for identification.

To be admitted is to be given permission to enter an activity, class, or program

Something a student academically specializes in

A group of people who collaborate to combine their knowledge, teaching others in the process

To enroll is to sign up for class of activity.

I have not heard of this

To sign up for something

A requirement that must be completed in order to take a college course.

The time during the year where students take their academic classes.

A course that is more difficult than most.

A course at a lower difficulty.

A course that is almost at or preparing you for a college level course

A course that is at the level of difficulty of a college course

A number for a specific course.

A multitude of ways a student can be graded

A review into someones information

College courses that don't contribute to achieving your degree, but still teaching you

A record of a students' grades

work that a school can give you.

Money given to someone.

Money given to a student by a college or institution to pay for their college education because the student has excelled in something like sports or academics.

Money the bank can give you that you must pay back, most of the time with interest, to help pay for college.

An application asking for financial aid.

Money to help students who may not be able to pay for tuition, pay for it.

The path a student takes to specialize in a certain field.

A paper showing official and proper documentation of status

An achievement granted to a college student for meeting an academic requirement.

A non-mandatory class a student may take due to interest.

The common level of education most people receive

The cost for a class in college.

A period of time in the academic year.

The amount of credits a student will receive in a unit.

Placed on temporary restrictions from certain things due to not meeting or following expectations.

A number or combination of numbers given to a student for identification.

To be accepted or granted access to something like a school you enrolled in.

Registering for something means to enroll or sign up for something

Formally signing up for something like a class or program.

The period of time in the year that school is open for people to attend.

when submitting these briefs.. outside voices can influence how courts interpret constitutional rights, including those protected by the first amendment - they ensure broader perspectives are considered in shaping our law.

it is interesting that the justice who writes the opinion needs to make sure it expresses all the views of the justices because it ties back into freedom of speech and the first amendment - the first amendment is very applicable here.

I find it so important and also commendatory that even people with different beliefs can still come together and shake hands out of a sign of respect for one another.

showing that once a decision is made it is almost instantaneous that the government starts to slowly process the case - using the word triggers ( a verb ) shows great effect.

Claim-> main point.

restate of claim

counter point

claim

limits

claim

evidence

I understand the article saying how widespread French is, but I wonder why they don't include how that came to be. French colonialism played a big part in the "galaxy of French speakers". Haiti, for example, has a large part of French speakers that had to fight for their independence. The galaxy of French speakers came about because of greed, and I believe it's important to highlight that.

I believe this graph is expressing the growth (or stagnation) of different languages throughout history with official counts along with estimates on the continued growth with dates going as far as 2040.

I believe that with the presence of graphs along with noticeable percentages and data points, this will be an informative article about the population of French speakers throughout the world.

Cognate, " Contexts, determines the elaboration of profiles of French speakers"

Cognate, "a percentage of the population"

Cognate, "evolution of the population of five different langues spoken with official population counts (i think)"

was this the beginning of color distinction between blue for democrats and red for republicans?

The idea of a new south was a mask or a blinded eye to a previous corruption that had taken place. Almost as if they were attempting to fool themselves.

This quote helps to show avenues of flourishment with the capitals transportation. avenues of these services came together to benefit the people but also created corruption with upperclassman and the owners capital.

This can create a challenge for teachers. If one kid does it and others find out, the whole idea can become obsolete.

There is a time and place for cell phones. In my opinion, it is not necessary to restrict phones for the whole day.

Students aren't purposely trying to pay attention. Taking phones out completely resists the urge

I think it is important for educators to note this, because phones are more than just a distraction.

Learning without thinking is unproductive. Thoughts and comments without knowledge of the topic are meaningless and misinformation.

a Man keep his promise and does not back track

This comment sticks out to me the most. I find this statement true and will say it throughout the time of human existence. To add new knowledge to the knowledge already gained.

Sees that he thinks he's the center of the universe or all-powerful.

Sounds like the perfect world. The rich show kindness, and the poor are happy.

Learn from the past, improve, and try again in the present and future.

Trust in his faith to guide him

Men should spread love and kindness everywhere they go, not hatred or suffering.

Tseng-tzu is challenging the Master on his accusations

This piques my interest. A true person has roots in something powerful or meaningful, like love, a family, or a moral compass.

classic community sales paradigm

If exposure alone doesn’t build competence, what kinds of structured training such as role-play, supervision, adapted CBT modules could actually prepare trainees? Creating a connecting bridge maybe useful in order for CBT to be effective in more regions of the world.

Second comment

I think this is something to note when taking care of the older adult population. Sadly, some older adults of other racial and ethnic groups are more "used to" the discrimination. While it may be a white older adult's first time being discriminated against. While they are developing more awareness of discrimination that they may have been blind to before, we should also acknowledge and stop our own biases and ageism towards these people. Whether they have experienced discrimination before or not, we should be offering them a safe space with the best care.

I thought this paragraph was very interesting because it explains how ageism directly affects older adults health. It stood out to me because it showed that as there is an increase in ageism, there is also an increase in the probability of pore physical health as well as poor mental health. I think part of this has to due with stereotypes of older adults having little independence and people not allowing them to complete tasks on their own.

I find it interesting that Haraway calls rivers, soil, and even technologies "collaborators" instead of just "resources". This idea could change how we think about environmental politics and responsibility by making the relationship more mutual. But at the same time, if everything becomes a "collaborator," the world might lose its meaning. So i wonder: is this idea really useful for political action, or does it risk becoming too vague..? It may risk stretching the very notion of collaboration beyond recognition.

Is it really the solution/best idea for us to call ourselves "compost"? Is there any risk of diminishing our sense of active responsibility? I mean it is true that all life eventually breaks down and circulates, but it is still unclear how it will lead to immediate political and social action.

We must make kin with all that lives on earth, sym-chthonically and sym-poietically, for we already share a common flesh---lateral, semiotic, genealogical.

While concepts like the Anthropocene or Capitalocene have already secured a place in academic and policy debates, Haraway introduces yet another term, which risk blurring rather than sharpening our understanding.

This is striking how the human refugee crisis and the refugee status of other species are interconnected. I believe that the world often think only of the human crisis, but Haraway makes us see other creatures as refugees as well...and reminds me about climate refugees, endangered animals, and habitat destruction.

This may sound radical at first but it's actually interesting in that it expands the human-centered concept of family and emphasizes relationships with non-human beings. Also, personally, it reminds me of the debate over the birth rate, and Haraway's proposal makes me imagine a new way of living together rather than simply population control.

Content must stay on topic/ try dot to de-rail topic of choice

Its vital to understand a target audience to achieve more awareness

Its vital to understand a target audience to achieve more awareness

insulin secretory granules.

phases. The first phase, which is the immediate post-meal response, begins within two minutes of nutrient ingestion. Insulin secretory granules docked to the beta cell plasma membrane are readily available to be released in this first phase.

In the second phase, the undocked insulin secretory granules are part of the reserve pool and are mobilized for exocytosis and release of their contents, while additional insulin is synthesized in the beta cell. This second phase has a lower rate of insulin release and can last for an hour or more.

It is a polypeptide synthesized in the beta cells of the pancreas. Proinsulin is folded in the rough endoplasmic reticulum (RER) where disulfide bonds form. Proinsulin is transported to the Golgi and then to immature secretory granules where most of the proinsulin is cleaved to form A and B chains held together by disulfide bonds, and the C-peptide is released. Mature "insulin secretory granules" fuse with the plasma membrane, and mature insulin, C-peptide and a small amount of proinsulin is released outside the cell.

I've read that schools are paid on their student attendance so there can be a bigger focus on getting them into classrooms. I understand that getting paid is important but students that miss class should have a support system that helps retain them so that their attendance isn't affected.

I understand that the Supreme Court gave teachers a degree protection for in-class curricular speech, but with the political climate today it feels not true. A teacher is also responsible for making their class a safe zone for their students.

One thing that worries me is that the code of ethics posted by a school might differ with my values a little too much. I know that working at a school is a hard job that not many can do. A teacher should do their best, and I believe that they should follow their heart when making decisions when there is no confliction with the law or a student's well being.

A teacher is an important member of a community because they work with the children who will one day be making choices about their surroundings. If you have a great teacher that really stimulates their students, then you'll see the difference in the community.

The teacher looks to better themselves because they want the respect from those that count or work with them. If a professional remains complacent then they won't advance in their careers. Their progress should be measured by a standard not their own personal goals though.

It is truly horrible that racism is still an ongoing issue, what even worse is that some teachers choose to ignore it and don't take action when they hear it in their environment.

It makes sense as to why a student would be disciplined. If a student is acting out it can disrupt the class and take away from those who are there to learn.

I see this and have experienced this in school as well, girls were typically the ones dress coded in my school. Specifically for wearing tank tops in the hot months and guys, who would also wear tanks, were never dress coded. It just felt very unfair and targeted.

This is a reflection of a student-teacher bond. If a teacher has a bond with their students, they feel more empathic, which makes scenarios like these hard.

Booker T. Washington believed Black people should go along with society and focus on work and money. He taught this during a time of big economic growth, but some felt he ignored bigger goals like freedom and education.

Black people were frustrated and tired of still being treated like slaves or servants, even after slavery was supposed to end. This frustration showed up in two major ways or movements.

When people are not just physically trapped (like in poverty or hard labor), but also surrounded by other people and their beliefs, the group that’s trapped can respond in three main ways: they may feel angry and want to fight back, they may try to fit in and do whatever the more powerful group wants, or, they may work hard to grow and become their true selves, even if others don’t support them.

People don’t usually say this criticism out loud. Even those who supported ending slavery don’t want to say that the schools started before Tuskegee were complete failures or should be laughed at, because those schools were started by good, unselfish people.

Democrats hold nothing but utter disrespect towards conservatives and all citizens on the right. Democrats' disrespect for them comes from a place of elitism.

in simpler words, let this be a lesson.

implying that no matter what you say to ChatGPT, the ai chatbot's goal is to make you feel fulfilled and satisfied. It will feed you affirmations and confirmations even if what you just said was completely wrong. You can test this yourself with questions that are open to discussions and debates with no "correct" answers.

Judge Castel was clearly showing signs of annoyance and irritation with all the signs (gesticulating, his voice rising, etc...)

Before the court hearing, he was cheerful and optimistic, but after the hearing he sat slumped, with his shoulders dropping and his head rising barely above the back of his chair, indicating that the hearing did not go well.

he was cheery and nervous at the same time, suggesting that he is trying to stay confident despite being anxious.

It’s crazy to think about how hard life would be if you couldn’t make new memories. Every situation would feel unfamiliar, which makes me realize how much we rely on memory without noticing.

It’s crazy to think about how hard life would be if you couldn’t make new memories. Every situation would feel unfamiliar, which makes me realize how much we rely on memory without noticing.

What stood out to me here is how schemas basically act like shortcuts for our brain. I can’t imagine trying to make sense of everything from scratch without them.

I like how the book compares The Thinker to controlled thinking because it makes the idea easier to picture. It’s interesting that most of our thinking actually happens automatically without us realizing it.

I like how the book compares The Thinker to controlled thinking because it makes the idea easier to picture. It’s interesting that most of our thinking actually happens automatically without us realizing it.

This part about quickly reading situations connects to the Detecting Deception slides from the Media Options. Both show how people make fast judgments based on little information, though those judgments aren’t always right.

This part stands out because it shows how naturally people process social information. It makes sense since we’re always judging situations and remembering details, even without thinking about it.

Reading about companies using technology to choose applicants reminds me of the Advertising-Priming Demo video. It shows how people’s decisions can be influenced without them realizing, which connects to how hiring tools might affect choices.

This part makes me think about how movies shape our fears of AI. It also connects to the Barnum Effect video since both show how easily people can believe dramatic ideas, even if they’re not fully based on reality.

I agree with this, the balancing of the support while developing capacity is difficult as they have a classroom as well. Systems within the school need to be in place to have a strong mentorship program for new teachers. The ideal will be to have a schedule for mentor teachers to support their mentee, the need to know their role and how does it look like and sound lilke. What is the difference between a site based coach and a mentor? Not all can rely on the mentor teacher.

The idea of scientific journalism is very interesting.

Surface charge density is the electric charge on a two-dimensional surface such as a metal plate. The units are coulombs per square meter (C/m^2). It is important to differentiate r and r^ in the formula (r is the distance from the charge element to the location of interest; r^ = vector)

I worked in a district where not everyone had consistent enforcement, and it often fell through the cracks.

When reading this, I think about how teachers, doctors, nurses, fast food workers, and restaurant employees are not using their phones while they are working and would likely get written up or fired for using them during times when they should be working. Thus, teaching it that way, such as not allowing them in the classroom and having them stored in the office or classroom basket, similar to a lounge or locker at a job, seems like a suitable response.

This takes away the much-needed time for teaching. If teachers have to consistently police phones and teach them to properly use them, they must stop their English, science, math, etc., and teach rules on phones. Whats more important here?

This section is essential in creating an environment similar to the times when phones were not allowed in classrooms.

Wow! I love all of this.

The problem with phones in general but especially in school. Students can not focus their attention long enough to make it through a class period of listening to a teacher. Thus, losing information and teaching time.

The amount of stress and negative self image that comes with social media and cell phones seems to be increasing significantly. Students are worried about who posted a picture with whom, what they look like in their "selfie", who liked or commented on their post, and how devastating it would be if that one person didn't. These are all things children never worried about before, but now is constantly on their minds. Especially when cell phones are allowed in schools and classrooms.

In other words, the colonials were disgusted.

This would be the Establishment Clause of the First Amendment.

Social because its said they are a "people" people, I am a very social person.

Significant for the professor to denote between his courses and assist in keeping the emails transparent. Good syllabus (: (Reposting it because of inappropriate username)

BIOmetaAnalysis

BIOmetaAnalysis

The highlighted text describes the "Tab' function, which is a navigational tool that allows disabled users to navigate the website using a keyboard alone. This website, "Audioeye.com", features this tool. The selected element is encased in a bright purple box, which could aid in making it clear what is being navigated. Citation : Rank, Sojin. "10 Accessible and ADA-Compliant Website Examples". Web. 7 August 2024. Accessed 10 Sept. 2025. https://www.audioeye.com/post/accessible-website-design-examples/

I believe this photo description does not go into precise enough detail about the contents of the screenshot and how it is relevant to the paragraph above it. I believe this lack of explanation could make it difficult for the visually impaired who rely on text-to-speech technology to understand why the image was relevant enough to include in the article. Citation : Rank, Sojin. "10 Accessible and ADA-Compliant Website Examples". Web. 7 August 2024. Accessed 10 Sept. 2025. https://www.audioeye.com/post/accessible-website-design-examples/ Federal Aviation Administration. Data And Research. Web. 12 Sept. 2025. Accessed 10 Sept. 2025. https://www.faa.gov/data_research

The use of subheadings helps create a logically structured layout, making the content easier to navigate and more digestible for readers.

This annotation applies broadly to the entire web article, as it demonstrates effective use of color contrast to make content distinguishable—especially beneficial for users with low vision.

: This text is a good example of effective alternative (alt) text. It briefly describes what is happening in the photo, providing context and enhancing the relevance of the visual content within the story.

I definitely agree. I live on the other side of the world from my family, so we use video calls all the time. It’s great to hear their voices and see their faces, but it still does not feel real. There’s no real sense of being together, it’s just a screen. No matter how clear the video is, it doesn’t feel like we’re actually together. It’s more like watching each other than being with each other. Video calls are interactive, but they’re not immersive, and that makes a big difference.