Structure of protein O-mannose kinase reveals a unique active site architecture

Thank you for submitting your article "Structure of protein O-mannose kinase reveals a unique active site architecture" for consideration by eLife. Your article has been favorably evaluated by Randy Schekman (Senior Editor) and three reviewers, one of whom, Reid Gilmore (Reviewer#1), is a member of our Board of Reviewing Editors. The following individuals involved in review of your submission have agreed to reveal their identity: Lance Wells (Reviewer #2); John D Scott (Reviewer #3).

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Summary:

The manuscript from Zhu et al. describes the structure of protein O-mannose kinase POMK), an enzyme required for synthesis of the glycan on α-dystroglycan. The authors present very strong evidence that POMK is an ER-localized glycan kinase, rather than a pseudokinase. The previous designation of POMK as a pseudokinase was based upon the lack of any of the active site residues found in protein kinases at any of the expected positions. The crystal structure shows that the residues necessary for ATP binding and catalysis are located at other positions indicating that POMK displays active site migration. One interesting feature of the structure is that the active site is linked to the activation loop via a disulfide that is conserved in POMK. The authors have done a nice job of analyzing a panel of POMK mutants, both those based upon the crystal structure, and two patient alleles. Analysis of the structure based point mutants confirms the importance of residues that contact ADP-AlF3 and GalNAc-β3-GlcNAc-β4-Man. This manuscript also serves the role as presenting data for how a pseudokinase can in fact be an actual functional kinase and thus suggests that other pseudokinases should be investigated for kinase enzymatic activity. The paper is well written, the experiments follow a logical order, and the findings are of significant moment. There are only a few minor questions/comments that are listed below and enthusiasm for this manuscript is extremely high. The three reviewers indicated that this manuscript needed very minor modifications before it would be appropriate for eLife.

1) While the authors reference the Di Costanzo manuscript, they don't discuss the impact of the Q109* mutation – given that the individual with this mutation displayed LGMD instead of the expected WWS, this seems worth at least mentioning any potential rationale for patient phenotype.

2) Could the authors comment on why they did not use the NMR titration method to determine whether the POMK point mutants in the substrate binding site have a reduced affinity for the trisaccharide. Were these not conducted due to the NMR method needing large amounts of purified mutant protein?

3) A considerable strength of this article is a comparative mechanistic analysis that maps disease-causing mutations onto Danio Rerio POMK ortholog. These are important observations that provide new mechanistic insight into how this important glycan kinase works. I suggest that the authors more clearly emphasize that this is a comparative mechanistic model

Reviewer #1:

The manuscript from Zhu et al. describes the structure of protein O-mannose kinase POMK), an enzyme required for synthesis of the α-dystroglycan. The authors present very strong evidence that POMK is an ER-localized glycan kinase, rather than a pseudokinase. The previous designation of POMK as a pseudokinase was based upon the lack of any of the active site residues found in protein kinases at any of the expected positions. The crystal structure shows that the residues necessary for ATP binding and catalysis are located at other positions indicating that POMK displays active site migration. One interesting feature of the structure is that the active site is linked to the activation loop by a disulfide that is conserved in POMK. The authors have done a nice job of analyzing a panel of POMK mutants, both those based upon the crystal structure, and two patient alleles. Analysis of the structure based point mutants confirms the importance of residues that contact ADP-AlF3 and GalNAc-β3-GlcNAc-β4-Man. Overall, this is an interesting and important manuscript that includes very high quality experimental evidence. The manuscript has been written so that it will be accessible to a broad audience. I do not have any major concerns with the current manuscript.

Reviewer #2:

This manuscript by Zhu et al. focuses on the structural elucidation of POMK. Several years ago, it was determined that mutations in POMK were causal for multiple forms of CMD ranging from LGMD to WWS. It had also been previously determined that POMK catalyzed the transfer of a phosphate to a trisaccharide structure on α-dystroglycan that served as the core structure that becomes functionally glycosylated with matriglycan. However, what was extremely puzzling is that POMK fits in the family of pseudokinases that presumably did not have actual kinase activity. This manuscript addresses at the structural level this quandary. Here they clearly define the key residues involved in catalysis and make comparisons to PKA, a typical known well-defined protein kinase. In addition, they present data that explains some of the naturally occurring mutations seen in individuals with CMD. This manuscript also serves the role as presenting data for how a pseudokinase can in fact be an actual functional kinase and thus suggests that other pseudokinases should be investigated for kinase enzymatic activity. The paper is well written, the experiments follow a logical order, and the findings are of significant moment. There are only a few minor questions/comments that are listed below and enthusiasm for this manuscript is extremely high.

1) While the authors determine the Kd of the trisaccharide for PrPOMK, they do not use their eloquent NMR approach to determine the Kd for several of the mutants they generate that would likely impact substrate binding. Could these be generated to further confirm their mechanism of binding and the impact of key mutants? Were these not conducted due to the NMR method needing large amounts of purified mutant protein?

2) While the authors reference the Di Costanzo manuscript, they don't discuss the impact of the Q109* mutation – given that the individual with this mutation displayed LGMD instead of the expected WWS, this seems worth at least mentioning any potential rationale for patient phenotype.

Reviewer #3:

This is a well-written and interesting article that provides important new structural insight into the mechanism of action of Danio Rerio POMK an O-mannose kinase. The significance of this work comes from evidence that the human ortholog is defective in diseases of the basement membrane where defects arise in the biosynthesis of functional α-distroglycan. This is an important and well-controlled study that represents a valuable contribution to our understanding of the burgeoning family of enzymes. Of particular note, this study helps to re-designate as a glycan kinase rather than a pseudokinase. I only have a few comments.

It is slightly confusing to open the Results section with a diagram of the human POMK otholog followed by the crystal structure of the Danio Rerio POMK ortholog. This needs to clearly defined in the text. One solution would be to have a comparison of the human and D. Rerio othologs in Figure 1A.

There is an element of redundancy in Figure 2A. I wonder if it would be better to superimpose the Danio Rerio POMK ortholog over the backbone of the PKA structure. This might be a convenient way to depict the similarities and differences between the two structures.

A considerable strength of this article is a comparative mechanistic analysis that maps disease-causing mutations onto Danio Rerio POMK ortholog. These are important observations that provide new mechanistic insight into how this important glycan kinase works. I suggest that the authors more clearly emphasize that this is a comparative mechanistic model.