Multi-barrier unfolding of the double-knotted protein, TrmD–Tm1570, revealed by single-molecule force spectroscopy and molecular dynamics

Reviewer #1 (Public review):

Summary:

This paper investigates the thermal and mechanical unfolding pathways of the doubly knotted protein TrmD-Tm1570 using molecular simulations, optical tweezers experiments, and other methods. In particular, the detailed analysis of the four major unfolding pathways using a well-established simulation method is an interesting and valuable result.

Strengths:

A key finding that lends credibility to the simulation results is that the molecular simulations at least qualitatively reproduce the characteristic force-extension distance profiles obtained from optical tweezers experiments during mechanical unfolding. Furthermore, a major strength is that the authors have consistently studied the folding and unfolding processes of knotted proteins, and this paper represents a careful advancement building upon that foundation.

Weaknesses:

While optical tweezers experiments offer valuable insights, the knowledge gained from them is limited, as the experiments are restricted to this single technique.

The paper mentions that the high aggregation propensity of the TrmD-Tm1570 protein appears to hinder other types of experiments. This is likely the reason why a key aspect, such as whether a ribosome or molecular chaperones are essential for the folding of TrmD-Tm1570, has not been experimentally clarified, even though it should be possible in principle.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors combined coarse-grained structure-based model simulation, optical tweezer experiments, and AI-based analysis to assess the knotting behavior of the TrmD-Tm1570 protein. Interestingly, they found that while the structure-based model can fold the single knot from TrmD and Tm1570, the double-knot protein TrmD-Tm1570 cannot form a knot itself, suggesting the need for chaperone proteins to facilitate this knotting process. This study has strong potential to understand the molecular mechanism of knotted proteins, supported by many experimental and simulation evidence. However, there are a few places that appear to lack sufficient details, and more clarification in the presentation is needed.

Strengths:

A combination of both experimental and computational studies.

Weaknesses:

There is a lack of detail to support some statements.

(1) The use of the AI-based method, SOM, can be emphasized further, especially in its analysis of the simulated unfolding trajectories and discovery of the four unfolding/folding pathways. This will strengthen the statistical robustness of the discovery.

(2) The manuscript would benefit from a clearer description of the correlation between the simulation and experimental results. The current correlation, presented in the paragraph starting from Line 250, focuses on measured distances. The authors could consider providing additional evidence on the order of events observed experimentally and computationally. More statistical analyses on the experimental curves presented in Figure 4 supplement would be helpful.

(3) How did the authors calibrate the timescale between simulation and experiment? Specifically, what is the value \tau used in Line 270, and how was it calculated? Relevant information would strengthen the connection between simulation and experiment.

(4) In Line 342, the authors comment that whether using native contacts or not, they cannot fold double-knotted TrmD-Tm1570. Could the authors provide more details on how non-native interactions were analyzed?

(5) It appears that the manuscript lacks simulation or experimental evidence to support the statement at Line 343: While each domain can self-tie into its native knot, this process inhibits the knotting of the other domain. Specifically, more clarification on this inhibition is needed.