Insights into substrate recognition and specificity for IgG by Endoglycosidase S2

Antibodies bind foreign antigens with high affinity and specificity leading to their neutralization and/or clearance by the immune system. The conserved N-glycan on IgG has significant impact on antibody effector function, with the endoglycosidases of Streptococcus pyogenes deglycosylating the IgG to evade the immune system, a process catalyzed by the endoglycosidase EndoS2. Studies have shown that two of the four domains of EndoS2, the carbohydrate binding module (CBM) and the glycoside hydrolase (GH) domain are critical for catalytic activity. To yield structural insights into contributions of the CBM and the GH domains as well as the overall flexibility of EndoS2 to the proteins’ catalytic activity, models of EndoS2-Fc complexes were generated through enhanced-sampling molecular-dynamics (MD) simulations and site-identification by ligand competitive saturation (SILCS) docking followed by reconstruction and multi-microsecond MD simulations. Modeling results predict that EndoS2 initially interacts with the IgG through its CBM followed by interactions with the GH yielding catalytically competent states. These may involve the CBM and GH of EndoS2 simultaneously interacting with either the same Fc CH2/CH3 domain or individually with the two Fc CH2/CH3 domains, with EndoS2 predicted to assume closed conformations in the former case and open conformations in the latter. Apo EndoS2 is predicted to sample both the open and closed states, suggesting that either complex can directly form following initial IgG-EndoS2 encounter. Interactions of the CBM and GH domains with the IgG are predicted to occur through both its glycan and protein regions. Simulations also predict that the Fc glycan can directly transfer from the CBM to the GH, facilitating formation of catalytically competent complexes and how the 734 to 751 loop on the CBM can facilitate extraction of the glycan away from the Fc CH2/CH3 domain. The predicted models are compared and consistent with Hydrogen/Deuterium Exchange data. In addition, the complex models are consistent with the high specificity of EndoS2 for the glycans on IgG supporting the validity of the predicted models.

As the authors are aware, in isolated IgGs the two Fc-glycans are tightly packed within the Fc "horseshoe" structure, with each arm (considering complex N-glycans in human IgG1 for example) extending on either side of the Fc (see for example Harbison and Fadda, Glycobiology (2020) doi: https://doi.org/10.1093/glycob/cwz101). The crystal structure of the Endo-S2 in complex with the N-glycan (PDB 6MDS for one) was obtained with isolated N-glycans, i.e. not bound to the Fc. In view of this interactions, I believe, or as a general choice of strategy, molecular docking was used as the first step in making the models, by docking isolated Nglycans and then linking the Fc, if I understood correctly. Because the whole N-glycans do not extend at the sides of the Fc, so are not exposed, yet, as I mentioned earlier, extend across the Fc. Within this framework, I was wondering if the authors noticed in any of their simulations the interaction of only one of the arms on either glycans with the CBM, which in my opinion could potentially initiate extraction. More specifically, if the 1-6 on the CH2-CH3 side facing the domain interacts with the CBM, it could potentially trigger the opening/loosening of the Fc structure, increasing the accessibility to both glycans and promoting the binding of the whole glycan to the CBM and of the other glycan to the GH. This scenario would agree with model D, where the CBM acts as a 'grip' facilitating the removal of the opposite N-glycan by GH. The second deglycosylation event could occur according to model C, where the N-glycan bound to the CBM could be 'transferred' to the GH, which I found fascinating! I understand that the above is a mechanistic speculation, yet a plausible one based on the evidence presented in this work and published in the literature, in my opinion, unifying all the different scenarios the authors examined and could be presented in the discussion. In any case, I think it would be useful to comment on how the N-glycans are potentially extracted from within the Fc to bind the CBM and GH.
Reply: As suggested by the reviewer we have analyzed the simulation for potential extraction events of which 2 were identified in simulations based on Models A and C. Results from these simulations have been added to a new section at the end of the results as well as additional text added to the discussion.
As minor points, • I find that it would be really helpful to have Figures presenting the structures of the complexes in the main manuscript, indicating the positions/contacts of the glycans with CBM and GH in different models. Those could be integrated in Figure 1.
Reply: As requested images of the 4 models have been added to Figure 1 • Page 10 and throughout "long-time" MD simulations is probably not a specific term, consider multi-microsecond MD simulations or MD simulations in the low microsecond time range.

Reply: Updated
Reviewer #2: Let me first make one thing clear, I'm not a computational biologist, but very much interested in immunoglobulin glycosylation and bacterial modification of the functionally important Fc glycans. EndoS2 represents one such very specific strategy with hydrolysis of these glycans, and only when presented in the context of the CH2/CH3 domain of IgG. Some of the authors of the current study have successfully solved the crystal structure of EndoS2 and presented convincing data that both the glycoside hydrolase (GH)domain and the carbohydrate binding domain(CBM) are crucial for the activity on the Fc glycan. Further site directed mutagenesis the solvent exposed site chain of W712 in the CBM results in loss of activity.
If I understand the advanced modeling scheme, known crystal structures of EndoS and IgG Fc are used to investigate the following: 1. Do the CBM and GH interact with IgG in sequence or at the same time? 2. Do the CBM and GH simultaneously interact with the same IgG Fc and/or individually with the two Fc portions within the same IgG molecule? 3. Do the CBM and GH interact with the glycan and/or the protein backbone of CH2/CH3? 4. Can the glycan be transfered from the CBM to the GH and thereby form a catalytically active complex?
The modeling answers these question with that EndoS2 initially interacts with IgG through the CBM followed by interaction with GH to allow for hydrolysis in the chitiobiose core. Furthermore, it is suggested that EndoS2 can adopt both a closed and a more open conformation allowing the CBM and GH to either interact with the same heavy chain or with the two separate heavy chains within the same IgG molecule. Simulations also predict interaction with both the glycan and the protein backbone in the CH2/CH3 domain, and that the Fc glycan can transfer from the CBM within one EndoS2 and thereby facilitate enzymatic activity.
The results from the modeling is compared and consistent with previously presented hydrogen/deuterium exchange data, as well as with previous experimental data indicating the very high specificity of EndoS2 for IgG Fc glycans.
Taken together, the simulations beautifully presents a very plausible model for the detailed interactions between EndoS2 that also fits with earlier experimental findings. However, again I must reveal my somewhat poor understanding of the details of the modeling; is it possible to do some kind of negative control in the modeling (or is it already there?)? For instance, can you do in silico mutations of the solvent exposed side chains in the CBM, or test the know mutations in the GH that leads to loss of activity or a shift towards glycosyl transferase activity?
I have no criticism of the language, introduction to the field, the discussion, or appropriate acknowledgment of previous findings.