Structure of the human heparan-α-glucosaminide N-acetyltransferase (HGSNAT)

Reviewer #1 (Public Review):

This article by Navratna et al. reports the first structure of human HGSNAT in an acetyl-CoA-bound state. Through careful structural analysis, the authors propose potential reasons why certain human mutations lead to lysosomal storage disorders and outline a catalytic mechanism. The structural data are of good quality, and the manuscript is clearly written. This study represents an important step toward understanding the mechanism of HGSNAT and is valuable to the field. I have the following suggestions:

1. The authors should characterize whether the purified protein is active. Otherwise, how does one know if the detergent used maintains the protein in a biologically relevant state? The authors should at least attempt to do so. If these prove to be challenging, at the very least, the authors should try a cell-based assay to demonstrate that the GFP tag does not interfere with the function.

2. In Figure 5, the authors present a detailed schematic of the catalytic cycle, which I find to be too speculative. There is no evidence to suggest that this enzyme undergoes isomerization, similar to a transporter, between open-to-lumen and open-to-cytosol states. Could it not simply involve some movements of side chains to complete the acetyl transfer?

Reviewer #2 (Public Review):

Summary:
This work describes the structure of Heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), a lysosomal membrane protein that catalyzes the acetylation reaction of the terminal alpha-D-glucosamine group required for the degradation of heparan sulfate (HS). HS degradation takes place during the degradation of the extracellular matrix, a process required for restructuring tissue architecture, regulation of cellular function, and differentiation. During this process, HS is degraded into monosaccharides and free sulfate in lysosomes.

HGSNAT catalyzes the transfer of the acetyl group from acetyl-CoA to the terminal non-reducing amino group of alpha-D-glucosamine. The molecular mechanism by which this process occurs has not been described so far. One of the main reasons to study the mechanism of HGSNAT is that multiple mutations spanning the entire sequence of the protein, such as nonsense mutations, splice-site variants, and missense mutations lead to dysfunction that causes abnormal accumulation of HS within the lysosomes. This accumulation is a cause of mucopolysaccharidosis IIIC (MPS IIIC), an autosomal recessive neurodegenerative lysosomal storage disorder, for which there are no approved drugs or treatment strategies.

This paper provides a 3.26A structure of HGSNAT, determined by single-particle cryo-EM. The structure reveals that HGSNAT is a dimer in detergent micelles and a density assigned to acetyl-CoA. The authors speculate about the molecular mechanism of the acetylation reaction, map the mutations known to cause MPS IIIC on the structure and speculate about the nature of the HGSNAT disfunction caused by such mutations.

Strengths:
The description of the architecture of HGSNAT is the highlight of the paper since this corresponds to the first description of the structure of a member of the transmembrane acyl transferase (TmAT) superfamily. The high resolution of an HGSNAT bound to acetyl-CoA is an important leap in our understanding of the HGSNAT mechanism. The density map is of high quality, except for the luminal domain. The location of the acetyl-CoA allows speculation about the mechanistic role of multiple residues surrounding this molecule. The authors thoroughly describe the architecture of HGSNAT and map the mutations leading to MPS IIIC. The description of the dimeric interphase is a novel result, and future studies are left to confirm the importance of oligomerization for function.

Weaknesses:
Apart from the cryo-EM structure, the article does not provide any other experimental evidence to support or explain a molecular mechanism. Due to the complete absence of functional assays, mutagenesis analysis, or other structures such as a ternary complex or an acetylated enzyme intermediate, the mechanistic model depicted in Figure 5 should be taken with caution.

The authors discuss that H269 is an essential residue that participates in the acetylation reaction, possibly becoming acetylated during the process. However, there is no solid experimental evidence, e.g. mutagenesis analysis or structural analysis, in this or previous articles, that demonstrates this to be the case.

In the discussion part, the authors mention previous studies in which it was postulated that the catalytic reaction can be described by a random order mechanistic model or a Ping Pong Bi Bi model. However, the authors leave open the question of which of these mechanisms best describes the acetylation reaction. The structure presented here does not provide evidence that could support one mechanism or the other.

Although the authors map the mutations leading to MPS IIIC on the structure and use FoldX software to predict the impact of these mutations on folding and fold stability, there is no experimental evidence to support FoldX's predictions.

Reviewer #3 (Public Review):

Summary:
Navratna et al. have solved the first structure of a transmembrane N-acetyltransferase (TNAT), resolving the architecture of human heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) in the acetyl-CoA bound state using single particle cryo-electron microscopy (cryoEM). They show that the protein is a dimer, and define the architecture of the alpha- and beta- GSNAT fragments, as well as convincingly characterizing the binding site of acetyl-CoA.

Strengths:
This is the first structure of any member of the transmembrane acyl transferase superfamily, and as such it provides important insights into the architecture and acetyl-CoA binding site of this class of enzymes.

The structural data is of a high quality, with an isotropic cryoEM density map at 3.3Å facilitating the building of a high-confidence atomic model. Importantly, the density of the acetyl-CoA ligand is particularly well-defined, as are the contacting residues within the transmembrane domain.

The open-to-lumen structure of HSGNAT presented here will undoubtedly lay the groundwork for future structural and functional characterization of the reaction cycle of this class of enzymes.

Weaknesses:
While the structural data for the open-to-lumen state presented in this work is very convincing, and clearly defines the binding site of acetyl-CoA, to get a complete picture of the enzymatic mechanism of this family, additional structures of other states will be required.

A potentially significant weakness of the study is the lack of functional validation. The enzymatic activity of the enzyme characterized was not measured, and the enzyme lacks native proteolytic processing, so it is a little unclear whether the structure represents an active enzyme.

Author Response

eLife assessment

This important work describes the first high-resolution structure of HGSNAT, a lysosomal membrane protein required for the degradation of heparan sulfate (HS). Through careful structural analysis, this work proposes potential reasons why certain mutations in HGSNAT lead to lysosomal storage disorders and outlines the enzyme's catalytic mechanism. The experimental evidence presented provides incomplete support for the proposed molecular mechanism of the HS acetylation reaction and the impact of disease-causing mutations.

We thank the editors and reviewers for taking the time to provide a critical assessment of our manuscript. We appreciate the input and suggestions to improve the analysis. Included here are only our provisional responses. We will address the concerns raised in more detail and incorporate them in the revised version of the manuscript.

Reviewer #1 (Public Review):

This article by Navratna et al. reports the first structure of human HGSNAT in an acetyl-CoAbound state. Through careful structural analysis, the authors propose potential reasons why certain human mutations lead to lysosomal storage disorders and outline a catalytic mechanism. The structural data are of good quality, and the manuscript is clearly written. This study represents an important step toward understanding the mechanism of HGSNAT and is valuable to the field. I have the following suggestions:

We thank the reviewer for their encouraging and positive overall assessment of our work.

1. The authors should characterize whether the purified protein is active. Otherwise, how does one know if the detergent used maintains the protein in a biologically relevant state? The authors should at least attempt to do so. If these prove to be challenging, at the very least, the authors should try a cell-based assay to demonstrate that the GFP tag does not interfere with the function.

Thank you for highlighting this concern. The cryo-EM sample was prepared without the exogenous addition of ligand, as noted in the manuscript; the acetyl-CoA that we see in the structure was intrinsically bound to the protein, indicating the ability of GFP-tagged HGSNAT protein to bind the ligand. We purified the protein at a pH optimal for acetyl-CoA binding, as suggested by Bame, K. J. and Rome, L. H. (1985) and Meikle, P. J. et al., (1995). Because we see acetyl-CoA in a structure obtained using a GFP fusion, we argue that GFP does not interfere with protein stability and ability to bind to the co-substrate. As demonstrated by existing literature HGSNAT catalyzed reaction is compartmentalized spatially and conditionally. The binding of acetyl-CoA happens towards the cytosol and is optimal at pH 7-0.8.0, while the transfer of the acetyl group to heparan sulfate occurs towards the luminal side and is optimal at pH 5.0-6.0. We are working on establishing a robust assay to study this complicated and compartmentalized acetyl transfer assay.

1. In Figure 5, the authors present a detailed schematic of the catalytic cycle, which I find to be too speculative. There is no evidence to suggest that this enzyme undergoes isomerization, like a transporter, between open-to-lumen and open-to-cytosol states. Could it not simply involve some movements of side chains to complete the acetyl transfer?

The acetyl-CoA bound structure presented in the paper does not conclusively support a potential for isomerization and conformational dynamics. We agree with the reviewer that the reaction schematic presented in Figure 5 is speculative. We acknowledge in the discussion that our structure represents only a single step of the reaction, and defining the precise mechanism of acetyl transfer needs additional work. However, we will reword the discussion and change Figure 5 to address this concern raised by multiple reviewers.

Reviewer #2 (Public Review):

Summary:

This work describes the structure of Heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), a lysosomal membrane protein that catalyzes the acetylation reaction of the terminal alpha-D-glucosamine group required for the degradation of heparan sulfate (HS). HS degradation takes place during the degradation of the extracellular matrix, a process required for restructuring tissue architecture, regulation of cellular function, and differentiation. During this process, HS is degraded into monosaccharides and free sulfate in lysosomes.

HGSNAT catalyzes the transfer of the acetyl group from acetyl-CoA to the terminal non-reducing amino group of alpha-D-glucosamine. The molecular mechanism by which this process occurs has not been described so far. One of the main reasons to study the mechanism of HGSNAT is that multiple mutations spanning the entire sequence of the protein, such as nonsense mutations, splicesite variants, and missense mutations lead to dysfunction that causes abnormal accumulation of HS within the lysosomes. This accumulation is a cause of mucopolysaccharidosis IIIC (MPS IIIC), an autosomal recessive neurodegenerative lysosomal storage disorder, for which there are no approved drugs or treatment strategies.

This paper provides a 3.26A structure of HGSNAT, determined by single-particle cryo-EM. The structure reveals that HGSNAT is a dimer in detergent micelles and a density assigned to acetylCoA. The authors speculate about the molecular mechanism of the acetylation reaction, map the mutations known to cause MPS IIIC on the structure and speculate about the nature of the HGSNAT disfunction caused by such mutations.

Strengths:

The description of the architecture of HGSNAT is the highlight of the paper since this corresponds to the first description of the structure of a member of the transmembrane acyl transferase (TmAT) superfamily. The high resolution of an HGSNAT bound to acetyl-CoA is an important leap in our understanding of the HGSNAT mechanism. The density map is of high quality, except for the luminal domain. The location of the acetyl-CoA allows speculation about the mechanistic role of multiple residues surrounding this molecule. The authors thoroughly describe the architecture of HGSNAT and map the mutations leading to MPS IIIC. The description of the dimeric interphase is a novel result, and future studies are left to confirm the importance of oligomerization for function.

We thank the reviewer for their time and for highlighting both the quality and novelty of the structure presented in this work.

Weaknesses:

Apart from the cryo-EM structure, the article does not provide any other experimental evidence to support or explain a molecular mechanism. Due to the complete absence of functional assays, mutagenesis analysis, or other structures such as a ternary complex or an acetylated enzyme intermediate, the mechanistic model depicted in Figure 5 should be taken with caution.

Thank you for pointing out this concern. The proposed mechanistic model in Figure 5 is a hypothesis based on previously reported biochemical characterization of HGSNAT by Rome & Crain (1981), Rome et al, (1983), Miekle et al., (1995) and Fan et al., (2011). However, we agree with the reviewer that this schematic is not experimentally proven and is speculative at best. Especially because our structure presents only a single step of the reaction, which does not conclusively support either ping-pong or random-order bi-substrate reactions. We will rephrase this section of our discussion and edit Figure 5 to address this concern.

The authors discuss that H269 is an essential residue that participates in the acetylation reaction, possibly becoming acetylated during the process. However, there is no solid experimental evidence, e.g. mutagenesis analysis or structural analysis, in this or previous articles, that demonstrates this to be the case.

H269, as a crucial catalytic residue, was suggested by monitoring the effect of chemical modifications of amino acids on acetylation of HGSNAT membranes by Bame, K. J. and Rome, L. H. (1986). We agree that mutagenesis, catalysis, and structural evidence for the same are not currently available. We are pursuing a more thorough exploration of the role of both H269 (previous studies) and N258 (from this study) on the stability and function of HGSNAT.

In the discussion part, the authors mention previous studies in which it was postulated that the catalytic reaction can be described by a random order mechanistic model or a Ping Pong Bi Bi model. However, the authors leave open the question of which of these mechanisms best describes the acetylation reaction. The structure presented here does not provide evidence that could support one mechanism or the other.

We agree with the reviewer’s observation that the structure doesn’t indeed support one reaction mechanism or another. We are pursuing the structural and kinetic characterization of HGSNAT in the presence of other co-substrates and multiple pHs that are required to address this concern thoroughly.

Although the authors map the mutations leading to MPS IIIC on the structure and use FoldX software to predict the impact of these mutations on folding and fold stability, there is no experimental evidence to support FoldX's predictions.

We are working on assessing the impact of specific mutations on the stability of HGSNAT and will add them to the revised version of the manuscript. We thank the reviewer for this suggestion.

Reviewer #3 (Public Review):

Summary:

Navratna et al. have solved the first structure of a transmembrane N-acetyltransferase (TNAT), resolving the architecture of human heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) in the acetyl-CoA bound state using single particle cryo-electron microscopy (cryoEM). They show that the protein is a dimer and define the architecture of the alpha- and beta- GSNAT fragments, as well as convincingly characterizing the binding site of acetyl-CoA.

Strengths:

This is the first structure of any member of the transmembrane acyl transferase superfamily, and as such it provides important insights into the architecture and acetyl-CoA binding site of this class of enzymes.

The structural data is of a high quality, with an isotropic cryoEM density map at 3.3Å facilitating the building of a high-confidence atomic model. Importantly, the density of the acetyl-CoA ligand is particularly well-defined, as are the contacting residues within the transmembrane domain.

The open-to-lumen structure of HSGNAT presented here will undoubtedly lay the groundwork for future structural and functional characterization of the reaction cycle of this class of enzymes.

We thank the reviewer for their positive assessment of the data presented in this work. We really appreciate and agree with the reviewer's comment that the “structure of HSGNAT presented here will undoubtedly lay the groundwork for future structural and functional studies.”

Weaknesses:

While the structural data for the open-to-lumen state presented in this work is very convincing, and clearly defines the binding site of acetyl-CoA, to get a complete picture of the enzymatic mechanism of this family, additional structures of other states will be required.

We agree with the reviewers’ assessment and are heavily invested in pursuing the structures of all the steps of acetyl transfer by HGSNAT.

A potentially significant weakness of the study is the lack of functional validation. The enzymatic activity of the enzyme characterized was not measured, and the enzyme lacks native proteolytic processing, so it is a little unclear whether the structure represents an active enzyme.

We thank the reviewer for this comment. While the proteolytic cleavage of the protein remains debated, we find no evidence of such an event in our purification (SDS-PAGE and SEC). Studies like Durand et al., (2010) and Fan et al., (2011) suggest that even the ER retained monomeric HGSNAT is active. Because we see acetyl-CoA (co-substrate) bound to the protein in our structure, we surmise that proteolysis is not necessary for function, at least not for substrate binding. However, we are working towards the structural and kinetic characterization of recombinant α- and β-HGSNAT construct to explore the role of proteolysis on HGSNAT stability and function.