Author Response
eLife assessment
This study presents a useful comparison of the dynamic properties of two RNA-binding domains. The data collection and analysis are solid, making excellent use of a suite of NMR methods. However, evidence to support the proposed model linking dynamic behavior to RNA recognition and binding by the tandem domains remains incomplete. The work will be of interest to biophysicists working on RNA-binding proteins.
Response: We thank eLife for taking the time and effort to review our manuscript. Evidence from the literature and our study shows a great deal of parity between the dynamic behavior of dsRBDs and its dsRNA-recognition and -binding, which helped us culminate in proposing a fair model. As mentioned in the manuscript, we have been working on the suggested experiments to further support our proposed model.
Public Reviews:
Reviewer #1 (Public Review):
Summary:
In the manuscript entitled "Differential conformational dynamics in two type-A RNA-binding domains drive the double-stranded RNA recognition and binding," Chugh and co-workers utilize a suite of NMR relaxation methods to probe the dynamic landscape of the TAR RNA binding protein (TRBP) double-stranded RNA-binding domain 2 (dsRBD2) and compare these to their previously published results on TRBP dsRBD1. The authors show that, unlike dsRBD1, dsRBD2 is a rigid protein with minimal ps-ns or us-ms time scale dynamics in the absence of RNA. They then show that dsRBD2 binds to canonical A-form dsRNA with a higher affinity compared to dsRBD1 and does so without much alteration in protein dynamics. Using their previously published data, the authors propose a model whereby dsRBD2 recognizes dsRNA first and brings dsRBD1 into proximity to search for RNA bulge and internal loop structures.
Response: We thank the Reviewer for sending us an encouraging review. We have combined the findings reported in the literature with new ones, that led us to propose the dsRNA-binding model by tandem A-form dsRBDs.
We propose that dsRBD1 can first recognize a variety of sequential and structurally different dsRNAs. dsRBD2 assists the interaction with a higher affinity, thus fortifying the interaction between TRBP and a possible substrate. This may enable the other associated proteins like Dicer and Ago2 to perform critical biological functions.
However, the following statements made in the comment above are factually incorrect.
(1) They then show that dsRBD2 binds to canonical A-form dsRNA with a higher affinity compared to dsRBD1 and does so without much alteration in protein dynamics.
However, we have explicitly shown the perturbation in dsRBD2 dynamics upon RNA binding.
(2) Using their previously published data, the authors propose a model whereby dsRBD2 recognizes dsRNA first and brings dsRBD1 into proximity to search for RNA bulge and internal loop structures.
Our previously published data suggests that dsRBD1, owing to its high conformational dynamics in solution, is able to recognize a variety of structurally and sequentially different dsRNA (PMID: 35134335). dsRBDs preferably bind to the double-stranded region (minor-major-minor-groove) of an A-form RNA (PMID: 24801449; PMID: 27332119) and do not search for bulge and internal loop structures as a part of the binding event. Even though dsRBDs preferably bind to the double-stranded region, they can still accommodate perturbation in the A-form helix due to mismatch and bulges with decreased binding affinity (PMID 25608000). However, it is a matter of future research to identify how much of a deviation from the A-form structure can be accommodated by the dsRBDs. The diffusion event observed in the literature (PMID: 23251028) also does not show any direct implication to search for bulge and internal loop structures.
Strengths:
The authors expertly use a variety of NMR techniques to probe protein motions over six orders of magnitude in time. Other NMR titration experiments and ITC data support the RNA-binding model.
Weaknesses:
The data collection and analysis are sound. The only weakness in the manuscript is the lack of context with the much broader field of RNA-binding proteins. For example, many studies have shown that RNA recognition motif (RRM) domains have similar dynamic characteristics when binding diverse RNA substrates. Furthermore, there was no discussion about the entropy of binding derived from ITC. It might be interesting to compare with dynamics from NMR.
Response: We understand the reviewer’s point that this study is focused on a dsRNA-binding mechanism rather than addressing the much broader field of RNA-binding. There are multiple challenges in finding a single mechanism that works for all RNA-binding proteins. For instance, RRM is a single-stranded RNA binding domain that is able to read out the substrate base sequence. RRM behaves entirely differently than the dsRBD in terms of sequence specificity. Besides, several other RNA-binding domains like the KH-domain, Puf domains, Zinc finger domains, etc., showcase a unique RNA-binding behavior. Thus, it would be really difficult to draw a single rule of thumb for RNA-recognition behavior for all these diverse domains.
Thank you for pointing out the entropy of binding from ITC. We shall include the discussion about the entropy of binding in the revised manuscript.
Reviewer #2 (Public Review):
Summary:
Proteins that bind to double-stranded RNA regulate various cellular processes, including gene expression and viral recognition. Such proteins often contain multiple double-stranded RNA-binding domains (dsRBDs) that play an important role in target search and recognition. In this work, Chug and colleagues have characterized the backbone dynamics of one of the dsRBDs of a protein called TRBP2, which carries two tandem dsRBDs. Using solution NMR spectroscopy, the authors characterize the backbone motions of dsRBD2 in the absence and presence of dsRNA and compare these with their previously published results on dsRBD1. The authors show that dsRBD2 is comparatively more rigid than dsRBD1 and claim that these differences in backbone motions are important for target recognition.
Strengths:
The strengths of this study are multiple solution NMR measurements to characterize the backbone motions of dsRBD2. These include 15N-R1, R2, and HetNOE experiments in the absence and presence of RNA and the analysis of these data using an extended-model-free approach; HARD-15N-experiments and their analysis to characterize the kex. The authors also report differences in binding affinities of dsRBD1 and dsRBD2 using ITC and have performed MD simulations to probe the differential flexibility of these two domains.
Weaknesses:
While it may be true that dsRBD2 is more rigid than dsRBD1, the manuscript lacks conclusive and decisive proof that such changes in backbone dynamics are responsible for target search and recognition and the diffusion of TRBP2 along the RNA molecule. To conclusively prove the central claim of this manuscript, the authors could have considered a larger construct that carries both RBDs. With such a construct, authors can probe the characteristics of these two tandem domains (e.g., semi-independent tumbling) and their interactions with the RNA. Additionally, mutational experiments may be carried out where specific residues are altered to change the conformational dynamics of these two domains. The corresponding changes in interactions with RNA will provide additional evidence for the model presented in Figure 8 of the manuscript. Finally, there are inconsistencies in the reported data between different figures and tables.
Response: We thank the reviewer for the comprehensive and insightful review. A larger construct carrying both RBDs was not used because of the multiple challenges pertaining to dynamics study by NMR spectroscopy (intrinsic R2 rates of the dsRBD1-dsRBD2 construct would be high, resulting in broadened peaks) as per our previous experience (PMID: 35134335). There would be additional dynamics in that construct coming from domain-domain relative motions, difficult to deconvolute the dynamics information. Further, the dsRNA needed to bind to this construct will be longer, thereby causing further line broadening in NMR.
Coming to mutational studies, careful designing of domain mutants remains as a challenge because the conformational dynamics in both the domains are distributed all through the backbone rather than only in the RNA-binding residues. The mutational studies would need an exhaustive number of mutations in protein as well as RNA to draw a parallel between the binding and dynamics. Having said that, we are working on making such mutations in the protein (at several locations to freeze the dynamics site-specifically) and the RNA (to change the shape of the dsRNA) to systematically study this mechanism, which will be out of scope of this manuscript.
The reviewer has rightly pointed out some subtle superficial differences. These superficial differences are present because of the context in which we are describing the data. For example, in Figure S4 we are talking about the average relaxation rates and nOe values for only the common residues we were able to analyze between two magnetic field strengths 600 and 800 MHz. Whereas in Figure 6, we are comparing the averages of the core dsRBD residues at 600 MHz, in presence and absence of D12RNA. The differences however are minute falling well within the error range.
