Structure of human lysosomal acid α-glucosidase–a guide for the treatment of Pompe disease

Pompe disease, a rare lysosomal storage disease caused by deficiency of the lysosomal acid α-glucosidase (GAA), is characterized by glycogen accumulation, triggering severe secondary cellular damage and resulting in progressive motor handicap and premature death. Numerous disease-causing mutations in the gaa gene have been reported, but the structural effects of the pathological variants were unknown. Here we present the high-resolution crystal structures of recombinant human GAA (rhGAA), the standard care of Pompe disease. These structures portray the unbound form of rhGAA and complexes thereof with active site-directed inhibitors, providing insight into substrate recognition and the molecular framework for the rationalization of the deleterious effects of disease-causing mutations. Furthermore, we report the structure of rhGAA in complex with the allosteric pharmacological chaperone N-acetylcysteine, which reveals the stabilizing function of this chaperone at the structural level.

The authors also make the interesting observation that a trefoil type -P domain appears to have a binding site that might anchor the enzyme on glycogen particles. I was initially surprised and slightly disappointed that the authors have not provided an annotated list of all known disease-associated mutations or an image showing their locations on the structure. On reflection, I think that the combination of structure determination, several crystallographic binding studies, and investigations of thermal stability is probably enough for one paper. However, I look forward to seeing a detailed structural analysis of the effects of diseaseassociated mutations.
In a revised manuscript, the authors should mention that, shortly after submission of their manuscript, another group released two isomorphous structures of the same enzyme at the PDB, though there is no peer-reviewed publication accompanying those structures yet. The PDB entries 5kzw and 5kzx were released on 26 July 2017, but the only associated publication so far is an abstract from the WORLDSymposium 2016 meeting, published last year in Molecular Genetics and Metabolism.
There are a few minor corrections that would improve the manuscript.
In the introduction (e.g p.2 line 46 and p.3 line 56, the term "pharmaceutical chaperone" should be replaced by "pharmacological chaperone" for consistency with standard usage and with the rest of the manuscript. In the discussion of N-acetylcysteine binding (p.8), it would help to have some idea of the concentration of N-acetylcysteine, so that the occupancies of the two sites can be understood in the context of the concentration dependence of the chaperone activity. Were the volume of the crystallization drop and the amount of powder added to it (p. 15) measured at least approximately?
On p.9, the term "thermal agitation factor" is appealing but it would be better to use something more standard, like "thermal displacement parameter" or "thermal motion factor".
The figures use attractive colours, but more saturated versions of the lighter colours should be used for labels, some of which are almost invisible. In particular, it is very difficult to see the light blue label for the isomaltose in Figure 4a, and even the molecule is difficult to see in this colour.
In the supplementary material, the pairs of images in Supp Figs 2 and 7 don't appear to be stereos. Either the right and left images are identical, or the pinch angle is so small that there is no visible stereo effect.
Supplementary Figure 5 has not reproduced in the PDF version of the supplement, although what is in the small box is visible in the Word version of this document.
Randy J Read C ambridge Institute for Medical Research University of C ambridge

Reviewer #2 (Remarks to the Author):
The authors are congratulated on an interesting and clear story providing structural evidence for the activity of NAC as a stabilizer for hGAA. The following comments/suggestions should not delay publication significantly but would be helpful to consider. 1. Pages are not numbered but on Page 6 of the MS, how was the superposition onto the B. obeum enzyme carried out? What atoms were used and is there an rms deviation? 2. Acarvosine in the supplemental binding site: is this presumed to be acarbose where part is not visible? If not, where did it come from? 3. Figure 4 -bound compounds: The authors are commended for showing the 'unbiased' difference map. However, the density is not terribly convincing at 3-sigma. How about adding a lower contour level in another color? 4. B-factor stabilization: Fig S7: I don't follow the argument for 'correcting' the B -factors as described in the caption to Fig S7. Is this the same in effect as comparing the differences between the atoms in question and the average B -factor over all atoms for each structure? Also, if the compound is stabilizing the surrounding residues, should it not have a B -factor roughly matching those residues? Was the occupancy constrained to 1.0? 5. The goal is to stabilize the structure for activity in the lysosome. The stability measurements were done at pH7.4 (p. 16). Is the compound equally stabilizing at low pH? Similarly -may be quibbling -but the enzyme activity comparison between wild-type and mutant (Pg 4) was at what pH? pH 4.0 as in Methods?
Minor / typos: Suggest the GH family (GH31) is stated explicitly in the intro, rather than implied later. Pg 6, line 5 -"by virtue of" Pg 15, line 10 should be "scaled" SI Fig 4 caption, halfway down, should be "sucrase"

Reviewer #1 (Remarks to the Author):
This manuscript describes the long-awaited structure of human lysosomal acid-alphaglucosidase (GAA), the enzyme mutated in Pompe disease. Despite availability of large quantities of pure enzyme produced for enzyme replacement therapy, a number of previous attempts to obtain good crystals had failed. The structure will be extremely valuable in helping to inform the development of new therapies for this rare but devastating lysosomal storage disease.
In addition to providing a high-resolution structure of excellent quality for this enzyme, the authors have contributed ligand-binding studies that will be of great value to those who wish to develop new therapies. Complexes with substrate analogues and with active-site directed inhibitors (deoxynojirimycin and its N-hydroxyethyl derivative) that stabilise the fold will contribute to the further development of typical pharmacological chaperones, i.e. those that improve the trafficking of active enzyme to the lysosome but at the cost of at least temporary inhibition of enzyme activity. A favourable alternative is to produce allosteric pharmacological chaperones that stabilise the enzyme without inhibiting it, and the structure of a complex with N-acetylcysteine demonstrates the mode of interaction with promising binding sites outside of the active site cleft.
The authors also make the interesting observation that a trefoil type-P domain appears to have a binding site that might anchor the enzyme on glycogen particles. I was initially surprised and slightly disappointed that the authors have not provided an annotated list of all known disease-associated mutations or an image showing their locations on the structure. On reflection, I think that the combination of structure determination, several crystallographic binding studies, and investigations of thermal stability is probably enough for one paper. However, I look forward to seeing a detailed structural analysis of the effects of disease-associated mutations.
In a revised manuscript, the authors should mention that, shortly after submission of their manuscript, another group released two isomorphous structures of the same enzyme at the PDB, though there is no peer-reviewed publication accompanying those structures yet. The PDB entries 5kzw and 5kzx were released on 26 July 2017, but the only associated publication so far is an abstract from the WORLDSymposium 2016 meeting, published last year in Molecular Genetics and Metabolism.
There are a few minor corrections that would improve the manuscript.
In the introduction (e.g p.2 line 46 and p.3 line 56, the term "pharmaceutical chaperone" should be replaced by "pharmacological chaperone" for consistency with standard usage and with the rest of the manuscript. In the discussion of N-acetylcysteine binding (p.8), it would help to have some idea of the concentration of N-acetylcysteine, so that the occupancies of the two sites can be understood in the context of the concentration dependence of the chaperone activity. Were the volume of the crystallization drop and the amount of powder added to it (p. 15) measured at least approximately?
On p.9, the term "thermal agitation factor" is appealing but it would be better to use something more standard, like "thermal displacement parameter" or "thermal motion factor".
The figures use attractive colours, but more saturated versions of the lighter colours should be used for labels, some of which are almost invisible. In particular, it is very difficult to see the light blue label for the isomaltose in Figure 4a, and even the molecule is difficult to see in this colour.
In the supplementary material, the pairs of images in Supp Figs 2 and 7 don't appear to be stereos. Either the right and left images are identical, or the pinch angle is so small that there is no visible stereo effect.
Supplementary Figure 5 has not reproduced in the PDF version of the supplement, although what is in the small box is visible in the Word version of this document.

Reviewer #2 (Remarks to the Author):
The authors are congratulated on an interesting and clear story providing structural evidence for the activity of NAC as a stabilizer for hGAA. The following comments/suggestions should not delay publication significantly but would be helpful to consider. 1. Pages are not numbered but on Page 6 of the MS, how was the superposition onto the B. obeum enzyme carried out? What atoms were used and is there an rms deviation? 2. Acarvosine in the supplemental binding site: is this presumed to be acarbose where part is not visible? If not, where did it come from? 3. Figure 4 -bound compounds: The authors are commended for showing the 'unbiased' difference map. However, the density is not terribly convincing at 3-sigma. How about adding a lower contour level in another color? 4. B-factor stabilization: Fig S7: I don't follow the argument for 'correcting' the B-factors as described in the caption to Fig S7. Is this the same in effect as comparing the differences between the atoms in question and the average B-factor over all atoms for each structure? Also, if the compound is stabilizing the surrounding residues, should it not have a B-factor roughly matching those residues? Was the occupancy constrained to 1.0? 5. The goal is to stabilize the structure for activity in the lysosome. The stability measurements were done at pH7.4 (p. 16). Is the compound equally stabilizing at low pH? Similarly -may be quibbling -but the enzyme activity comparison between wild-type and mutant (Pg 4) was at what pH? pH 4.0 as in Methods?
Minor / typos: Suggest the GH family (GH31) is stated explicitly in the intro, rather than implied later. Pg 6, line 5 -"by virtue of" Pg 15, line 10 should be "scaled" SI Fig 4 caption, halfway down, should be "sucrase"

Reviewer #1 (Remarks to the Author):
This manuscript describes the long-awaited structure of human lysosomal acid-alphaglucosidase (GAA), the enzyme mutated in Pompe disease. Despite availability of large quantities of pure enzyme produced for enzyme replacement therapy, a number of previous attempts to obtain good crystals had failed. The structure will be extremely valuable in helping to inform the development of new therapies for this rare but devastating lysosomal storage disease.
In addition to providing a high-resolution structure of excellent quality for this enzyme, the authors have contributed ligand-binding studies that will be of great value to those who wish to develop new therapies. Complexes with substrate analogues and with active-site directed inhibitors (deoxynojirimycin and its N-hydroxyethyl derivative) that stabilise the fold will contribute to the further development of typical pharmacological chaperones, i.e. those that improve the trafficking of active enzyme to the lysosome but at the cost of at least temporary inhibition of enzyme activity. A favourable alternative is to produce allosteric pharmacological chaperones that stabilise the enzyme without inhibiting it, and the structure of a complex with N-acetylcysteine demonstrates the mode of interaction with promising binding sites outside of the active site cleft.
The authors also make the interesting observation that a trefoil type-P domain appears to have a binding site that might anchor the enzyme on glycogen particles.

Response:
We thank the reviewer for the positive and constructive feedback. In the revised manuscript we have taken into account the reviewer's comments and suggestions and our responses to the points raised are listed below: I was initially surprised and slightly disappointed that the authors have not provided an annotated list of all known disease-associated mutations or an image showing their locations on the structure. On reflection, I think that the combination of structure determination, several crystallographic binding studies, and investigations of thermal stability is probably enough for one paper. However, I look forward to seeing a detailed structural analysis of the effects of disease-associated mutations.
Response: We thank the reviewer for this suggestion and have added to Supplementary a Table describing the structural consequences of missense mutations associated with Pompe disease.
In a revised manuscript, the authors should mention that, shortly after submission of their manuscript, another group released two isomorphous structures of the same enzyme at the PDB, though there is no peer-reviewed publication accompanying those structures yet. The PDB entries 5kzw and 5kzx were released on 26 July 2017, but the only associated publication so far is an abstract from the WORLDSymposium 2016 meeting, published last year in Molecular Genetics and Metabolism.

Response:
We have added a sentence to the Discussion section mentioning that the Garman group has released two isomorphous structures of rhGAA at the Protein Data Bank shortly after submission of our manuscript. An overlap of these structures onto the unbound form of rhGAA described in our manuscript reveals that the structures are almost identical, with an rmsd of 0.19 Å for 844 aligned Cα positions. Given this significant similarity we feel that at structural comparison would be obsolete in the context of this manuscript.
There are a few minor corrections that would improve the manuscript.
In the introduction (e.g p.2 line 46 and p.3 line 56, the term "pharmaceutical chaperone" should be replaced by "pharmacological chaperone" for consistency with standard usage and with the rest of the manuscript.

Response:
We thank the referee for pointing out this language abuse and have corrected the mistake.
In the discussion of N-acetylcysteine binding (p.8), it would help to have some idea of the concentration of N-acetylcysteine, so that the occupancies of the two sites can be understood in the context of the concentration dependence of the chaperone activity. Were the volume of the crystallization drop and the amount of powder added to it (p. 15) measured at least approximately? Response: We thank the referee for the suggestion and have added to the methods section that the amount of powder added to crystallizations droplets of ~1.5 μl corresponded to "1-2 snowflake-like crystals of approximate dimensions of 0.5 mm". We refrain from going any further in quantification of "real" concentration of NAC within crystal solvent channels, as we feel that this would be too much of a speculation on density of snowflake-like crystals, diffusion rate, solvent accessibility within crystal and competition with small molecules present in the crystallization medium.
The figures use attractive colours, but more saturated versions of the lighter colours should be used for labels, some of which are almost invisible. In particular, it is very difficult to see the light blue label for the isomaltose in Figure 4a, and even the molecule is difficult to see in this colour.

Response:
We thank the reviewer for the appreciation of the colouring scheme used to illustrate the diverse structural features of rhGAA and are grateful for the constructive suggestion. We have redrawn Figures 2, 3 and 4 and Supplementary Figures 3 and 9 (corresponding to numbering of revised version of Supplementary) using more saturated colours for labels and depiction of the isomaltose molecule in Figure 4a. We sincerely hope that in this new version the figures will be intelligible to the readers.
In the supplementary material, the pairs of images in Supp Figs 2 and 7 don't appear to be stereos. Either the right and left images are identical, or the pinch angle is so small that there is no visible stereo effect.

Response:
We are grateful to the reviewer for pointing out our regrettable mistake made by reducing pinch angles when scaling images to A4 format. We have redrawn all stereo images (Supplementary Figures 2, 4 and 8) and verified the stereo effect on printed paper.