Structural basis of EHEP-mediated offense against phlorotannin-induced defense from brown algae to protect akuBGL activity

Reviewer #1 (Public Review):

Sun and co-authors have determined the crystal structures of EHEP with/without phlorotannin analog, TNA, and akuBGL. Using the akuBGL apo structure, they also constructed model structures of akuBGL with phlorotannins (inhibitor) and laminarins (substrate) by docking calculation. They clearly showed the effects of TNA on akuBGL activity with/without EHEP and resolubilization of the EHEP-phlorotannin (eckol) precipitate under alkaline conditions (pH >8). Based on this knowledge, they propose the molecular mechanism of the akuBGL-phlorotannin/laminarin-EHEP system at the atomic level. Their proposed mechanism is useful for further understanding of the defensive-offensive association between algae and herbivores. However, there are several concerns, especially about structural information, that authors should address.

1. TNA binding to EHEP
The electron densities could not show the exact conformations of the five gallic acids of TNA, as the authors mentioned in the manuscript. On the other hand, the authors describe and discuss the detailed interaction between EHEP and TNA based on structural information. The above seems contradictory. In addition, the orientation of TNA, especially the core part, in Fig. 4 and PDB (8IN6) coordinates seem inconsistent. The authors should redraw Fig. 4 and revise the description accordingly to be slightly more qualitative.

2. Two domains of akuBGL
The authors concluded that only the GH1D2 domain affects its catalytic activity from a detailed structural comparison and the activity of recombinant GH1D1. That conclusion is probably reasonable. However, the recombinant GH1D2 (or GH1D1+GH1D2) and inactive mutants are essential to reliably substantiate conclusions. The authors failed to overexpress recombinant GH1D2 using the E. coli expression system. Have the authors tried GH1D1+GH1D2 expression and/or other expression systems?

3. Inhibitor binding of akuBGL
The authors constructed the docking structure of GH1D2 with TNA, phloroglucinol, and eckol because they could not determine complex structures by crystallography. The molecular weight of akuBGL would also allow structure determination by cryo-EM, but have the authors tried it? In addition, the authors describe and discuss the detailed interaction between GH1D2 and TNA/phloroglucinol/eckol based on docking structures. The authors should describe the accuracy of the docking structures in more detail, or in more qualitative terms if difficult.

Reviewer #2 (Public Review):

In this study the authors try to understand the interaction of a 110 kDa ß-glucosidase from the mollusk Aplysia kurodai, named akuBGL, with its substrate, laminarin, the main storage polysaccharide in brown algae. On the other hand, brown algae produce phlorotannin, a secondary metabolite that inhibits akuBGL. The authors study the interaction of phlorotannin with the protein EHEP, which protects akuBGL from phlorotannin by sequestering it in an insoluble complex.

The strongest aspect of this study is the outstanding crystallographic structures they obtained, including akuBGL (TNA soaked crystal) structure at 2.7 Å resolution, EHEP structure at 1.15 Å resolution, EHEP-TNA complex at 1.9 Å resolution, and phloroglucinol soaked EHEP structure at 1.4 Å resolution. EHEP structure is a new protein fold, constituting the major contribution of the study.

The drawback on EHEP structure is that protein purification, crystallization, phasing and initial model building were published somewhere else by the authors, so this structure is incremental research and not new.

Most of the conclusions are derived from the analysis of the crystallographic structures. Some of them are supported by other experimental data, but remain incomplete. The impossibility to obtain recombinant samples, implying that no mutants can be tested, makes it difficult to confirm some of the claims, especially about the substrate binding and the function of the two GH1Ds from akuBGL.

The authors hypothesize from their structure that the interaction of EHEP with phlorotannins might be pH dependent. Then they succeed to confirm their hypothesis, showing they can recover EHEP from precipitates at alkaline pH, and that the recovered EHEP can be reutilized.

A weakness in the model is raised by the fact that the stoichiometry of the complex EHEP:TNA is proposed to be 1:1, but in Figure 1 they show that 4 µM of EHEP protects akuBGL from 40 µM TNA, meaning EHEP sequesters more TNA than expected, this should be addressed in the manuscript.

The authors study the interaction of akuBGL with different ligands using docking. This technique is good for understanding the possible interaction between the two molecules but should not be used as evidence of binding affinity. This implies that the claims about the different binding affinities between laminarin and the inhibitors should be taken out of the preprint.

In the discussion section there is a mistake in the text that contradicts the results. It is written "EHEP-TNA could not dissolve in the buffer of pH > 8.0" but the result obtained is the opposite, the precipitate dissolved at alkaline pH.

Solving a new protein fold, as the authors report for EHEP, is relevant to the community because it contributes to the understanding of protein folding. The study is also relevant dew to the potential biotechnological application of the system in biofuel production. The understanding on how an enzyme as akuBGL can discriminate between substrates is important for the manipulation of such enzyme in terms of improving its activity or changing its specificity. The authors also provide with preliminary data that can be used by others to produce the proteins described or to design a strategy to recover EHEP from precipitates with phlorotannin at industrial scales.

In general methods are not carefully described, the section should be extended to improve the manuscript.

Reviewer #3 (Public Review):

The manuscript by Sun et al. reveals several crystal structures that help underpin the offensive-defensive relationship between the sea slug Aplysia kurodai and algae. These centre on TNA (a algal glycosyl hydrolase inhibitor), EHEP (a slug protein that protects against TNA and like compounds) and BGL (a glycosyl hydrolase that helps digest algae). The hypotheses generated from the crystal structures herein are supported by biochemical assays.

The crystal structures of apo and TNA-bound EHEP reveals the binding (and thus protection) mechanism. The authors then demonstrate that the precipitated EHEP-TNA complex can be resolubilised at an alkaline pH, potentially highlighting a mechanism for EHEP recycling in the A. kurodai midgut. The authors also present the crystal structures of akuBGL, a beta-glucosidase utilised by Aplysia kurodai to digest laminarin in algae into glucose. The structure revealed that akuBGL is composed of two GH1 domains, with only one GH1 domain having the necessary residue arrangement for catalytic activity, which was confirmed via hydrolytic activity assays. Docking was used to assess binding of the substrate laminaritetraose and the inhibitors TNA, eckol and phloroglucinol to akuBGL. The docking studies revealed that the inhibitors bound akuBGL at the glycone-binding suggesting a competitive inhibition mechanism. Overall, most of the claims made in this work are supported by the data presented.