Cohesin-dockerin code in cellulosomal dual binding modes and its allosteric regulation by proline isomerization
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
Article activity feed
-
-
###Reviewer #3:
The manuscript by Vera et al. reports on cohesin-dockerin interaction studies of cellulosomal subunits using mainly single-molecule FRET, but also molecular dynamics simulations and NMR measurements. The authors study a range of cohesin-dockerin pairs and discover a varying distribution of two alternative binding modes that apparently follows a built-in cohesin-dockerin code. Finally, the authors show that prolyl isomerase activity can regulate kinetics towards equilibrium/steady state as well as distribution of the binding modes. The results are important for understanding the mechanistic basis cellulosome function.
In my opinion, this is an important paper, which provides new interesting insight into cellulosome function. The single-molecule FRET and molecular dynamics parts of the study are well designed, the …
###Reviewer #3:
The manuscript by Vera et al. reports on cohesin-dockerin interaction studies of cellulosomal subunits using mainly single-molecule FRET, but also molecular dynamics simulations and NMR measurements. The authors study a range of cohesin-dockerin pairs and discover a varying distribution of two alternative binding modes that apparently follows a built-in cohesin-dockerin code. Finally, the authors show that prolyl isomerase activity can regulate kinetics towards equilibrium/steady state as well as distribution of the binding modes. The results are important for understanding the mechanistic basis cellulosome function.
In my opinion, this is an important paper, which provides new interesting insight into cellulosome function. The single-molecule FRET and molecular dynamics parts of the study are well designed, the corresponding experiments are thoroughly performed, and data are carefully analysed. The manuscript is also very well written. However, there are several issues that need to be addressed:
The authors claim to have uncovered a built-in cohesin-dockerin code. However, the principles of the code remain elusive. For example, what is the relationship between the Pro66 cis/trans conformation and the binding mode? What needs to be known to predict the dockerin binding mode? This point should be elaborated in the manuscript.
The conclusion that prolyl isomerase activity is able to change the distribution of binding modes requires more consideration and/or research. First, it seems from Figure 6A that the expected steady-state B1 fraction of c1C-CcCel5A and c1C-CcCel5A+prolyl isomerase could be the same within error ranges. Second, it is unlikely that the enzyme will change the equilibrium ratio of Pro66 cis/trans conformation that is controlled by thermodynamics. Therefore, the prolyl isomerase activity may only be relevant in case of slow re-equilibration kinetics.
NMR measurements were performed in order to check if the dockerin ́s Leu65 - Pro66 peptide bond is in the cis conformation in the cohesin-dockerin complex. The authors found very similar dockerin chemical shifts in the absence or presence of 1.3 equivalents of cohesin suggesting that the binding does not significantly alter the conformation. However, this is an indirect measurement, although NMR also allows direct determination of Pro cis/trans conformation (based on 13C chemical shifts and NOE patterns, e.g. see https://doi.org/10.1107/S1744309110005890 ). The authors should check if direct determination of the cis conformation is possible in their case. Also, peak doubling in the 15N-1H HSQC spectrum should be checked, which is an indication of Pro cis/trans equilibria.
Furthermore, a direct measurement of the Pro66 cis/trans ratio for two cohesin-dockerin pairs that show distinct B1/B2 preferences would be useful to clarify the role of Pro66.
-
###Reviewer #2:
By analyzing the formation of a series of dockerin-cohesin complexes from the cellulosome of two species of the Clostridium bacteria using smFRET experiments and other techniques, the authors conclude that the overall equilibrium between the two binding modes of the complex can be allosterically regulated by the enzymatic isomerization of dockerin's proline 66, which is part of a structural clasp between the N and C terminus of the protein. They speculate that a mechanism of enzymatically or environmentally driven clasp de/stabilization may be present in other dockerin-cohesin complexes, as well, and may provide the cellulosome with the required plasticity to carry out its function.
In large part the work is clearly written and the claims seems to be supported by the data provided, however there are few issues that the …
###Reviewer #2:
By analyzing the formation of a series of dockerin-cohesin complexes from the cellulosome of two species of the Clostridium bacteria using smFRET experiments and other techniques, the authors conclude that the overall equilibrium between the two binding modes of the complex can be allosterically regulated by the enzymatic isomerization of dockerin's proline 66, which is part of a structural clasp between the N and C terminus of the protein. They speculate that a mechanism of enzymatically or environmentally driven clasp de/stabilization may be present in other dockerin-cohesin complexes, as well, and may provide the cellulosome with the required plasticity to carry out its function.
In large part the work is clearly written and the claims seems to be supported by the data provided, however there are few issues that the authors should address:
The computer simulations presented in the manuscript are not described very clearly. For example on page 19 regarding the foldX MC method: the author identifies two variables to describe the binding: an "axis" Z and a rotation angle phi. An axis, however, is defined by three coordinates, while the authors always associate a single number to Z. The reader has to guess that the axis is the axis of symmetry of the two binding modes and Z is only an offset along the axis. Similarly in eq. (4) the authors associate the sum over the conformations indexed by i to an average (first line page 20) but in reality that sum and the others that appear in the argument of the logarithm of equation 4 are an estimate of the partition function of the system.
The computer simulations of the complex do not seem to add significant information to the overall message of the manuscript: the rigid-body coarse grained approach does not allow to distinguish allosteric effects as the authors already admit, while the FoldX approach provides only very large errors. Most probably, given the presence of well defined crystallographic structures for some of the complexes, simple free-energy estimation techniques (i.e. metadynamics, steered MD etc.) based on classical atomistic molecular dynamics simulations (with limited homology modelling for the mutants) would have provided more accurate results. The authors should explain why they did not consider this approach.
The data about the time dependency of the FRET signal in C. cellulolyticum are a bit worrying. The authors should dissect them more carefully, possibly adding additional control experiments to exclude artifacts (whose possible presence is also admitted by the authors in the caption of Fig.6 figure supplement 3). Then, if the process is confirmed, they should really try and identify the underlying process in a more precise way.
Fig 5C and Fig 5F show two different curves for the same data. Similarly Figure 6 figure supplement 4 C shows two different histograms for the same complex. If this is the result of repeated experiments, the authors should make an effort and report histograms with error bars. Visual comparison of histograms which have a large intrinsic variability may be misleading.
A picture showing a model of the molecular structure of the dyes attached to the molecular structure of the proteins would be very useful to to understand the relative size of the objects.
-
###Reviewer #1:
Vera et al. report the detection of binding and quantification of populations of two different orientations of assembly of dockerin and cohesin, which define structural organization and plasticity of bacterial cellulosome multi-enzyme complexes. The authors apply smFRET spectroscopy in in-vitro experiments carried out on isolated, modified domains. Vera et al. find uneven distributions of populations of the protein in the two modes of binding. Vera et al. investigate the molecular origins of the observed bias by studying homologous sequences obtained from various organisms, by mutagenesis and by domain-swap experiments. The authors complement experimental studies by Monte Carlo and molecular dynamics simulations. The authors arrive at the conclusion to having identified a cohesion-dockerin "code" of binding and a novel …
###Reviewer #1:
Vera et al. report the detection of binding and quantification of populations of two different orientations of assembly of dockerin and cohesin, which define structural organization and plasticity of bacterial cellulosome multi-enzyme complexes. The authors apply smFRET spectroscopy in in-vitro experiments carried out on isolated, modified domains. Vera et al. find uneven distributions of populations of the protein in the two modes of binding. Vera et al. investigate the molecular origins of the observed bias by studying homologous sequences obtained from various organisms, by mutagenesis and by domain-swap experiments. The authors complement experimental studies by Monte Carlo and molecular dynamics simulations. The authors arrive at the conclusion to having identified a cohesion-dockerin "code" of binding and a novel allosteric control mechanism involving cis/trans isomerization of a C-terminal proline residue in dockerin.
Structural plasticity of the cellulosome induced by variable assembly of the cohesion-dockerin adapter, facilitated by rotational symmetry of the two-helical binding interface, is an interesting biological phenomenon. The dual binding mode is already reported in the literature (refs. 23, 24, Wojciechowsky et al. Sci Rep 2018, 8:5051), somewhat limiting the novelty of findings. But forces and mechanisms that drive the orientations are not yet understood. The authors successfully developed a smFRET assay that can distinguish the two binding modes of the cohesion-dockerin interaction and that can measure the respective populations in vitro. Their homology, mutagenesis and domain-swap experiments show that specific interactions within the binding interface are not responsible for modulation of orientation. Instead, they show that interactions of a C-terminal proline can modulate binding. However, the relevance of findings for the in-vivo situation appear unclear.
I have the following concerns:
The authors' smFRET assay clearly distinguishes the two binding modes B1 and B2. A key element of their work, which goes beyond state of the art, is the quantification of populations estimated from integrals of smFRET histograms and PDA. Their FRET analysis presumes that photophysics or quantum yields of donor/acceptor fluorophores are independent on orientation of binding. But the protein micro-environment at the positions of the labels close to the binding interface may change in B1 and B2 orientation, which may modulate photophysics and thus FRET. This would, in turn, lead to errors in estimation of populations. The authors could test for such effects by measuring fluorescence of donor-only and acceptor-only constructs in B1 and B2 orientations.
From their study of homologous sequences, mutagenesis experiments and swap of helix 1 and 2 of dockerin, the authors provide a solid body of data that shows that specific interactions within the binding interface are not responsible for the swap of binding mode. Instead, their results show that interactions of a C-terminal proline can modulate binding through an elusive mechanism. Proline mutagenesis experiments and enzymatic cis/trans isomerization show significant effects. But the relevance of a prolyl isomerase for the modulation of the dockerin-cohesin interaction in vivo remains speculation. The conclusion calls for additional experiments where, e.g., changes in catalytic activity of cellulosomes are measured upon application of a prolyl isomerase. Alternatively, the packing of enzyme subunits in the dense cellulosome may be responsible for alternate binding. Such protein-protein interactions may also modulate a proline interaction.
An allosteric mechanism of the proline interaction in modulating binding, as proposed in this work, is not sufficiently supported by the data presented. The flexibility of the C-terminal tail of dockerin, which hosts the proline, and its close proximity to the cohesin binding interface, evident in structures (please provide PDB IDs in Fig. 1), may allow a direct interaction of the proline with cohesin.
The impact of the intrachain proline/tyrosine interaction on binding, however, identified by the authors, is very interesting. This finding calls for further investigations on mechanistic details. Here high-resolution techniques, like NMR, which can provide atomic details of protein structure and dynamics, are desirable. Such experiments could help to identify potential allosteric effects on the conformation and thus on binding.
Having said that, the authors state (in the abstract and introduction) to have performed NMR experiments in their study. But no NMR data are shown or discussed in this manuscript.
If the C-terminal proline was a biologically relevant switch that modulates binding, this residue should be conserved. Have the authors checked conservation of the C-terminal proline in homologous sequences?
The authors conclude to have identified a cohesion-dockerin "code". The word "code" in this context is unclear to me. What do the authors mean by "code"?
The authors conducted and analysed a set of kinetic experiments. But these experiments are not described at sufficient detail in the results and methods sections.
-
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript.
###Summary:
The reviewers find your work of interest and acknowledge your development of an elegant smFRET assay that can detect and quantify populations of cohesion-dockerin binding orientations. They further acknowledge your interesting finding of a role of the molecular clasp in modulating binding orientation involving a terminal proline. The reviewers find, however, that your conclusions of an enzymatic and allosteric control mechanism present in the cellulosome is not sufficiently supported by the data presented. …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript.
###Summary:
The reviewers find your work of interest and acknowledge your development of an elegant smFRET assay that can detect and quantify populations of cohesion-dockerin binding orientations. They further acknowledge your interesting finding of a role of the molecular clasp in modulating binding orientation involving a terminal proline. The reviewers find, however, that your conclusions of an enzymatic and allosteric control mechanism present in the cellulosome is not sufficiently supported by the data presented. The study lacks molecular-level information required to identify allosteric effects, which could, for example, be obtained using NMR spectroscopy that falls short in the present work. The proposed Monte Carlo approach and coarse-grained computer simulation does not provide sufficient molecular details and dynamic information to obtain mechanistic insight. There are further issues with the kinetic experiments. Some reported quantities are within error and controls are required to exclude artefacts.
-
-