Garman lab: Interconversion of lysosomal enzyme specificities

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Revision as of 15:20, 23 July 2018

1 How this page was created
2 Lysosomal storage disease
3 Immune Response
4 Enzymatic activity
5 Structures shown on this page

How this page was created

The goal of this page is to provide three-dimensional and interactive figures to explore the structures determined for the 2010 paper "Interconversion of the Specificities of Human Lysosomal Enzymes Associated with Fabry and Schindler Diseases" by Ivan B. Tomasic, Matthew C. Metcalf, Abigail I. Guce, Nathaniel E. Clark and Scott C. Garman [1]. The starting point are the figures found in this paper. Biochemistry students at Westfield State University recreated these figures in jmol, and revised them after getting feedback from the authors. A special thank you goes to Susan Al Mahrwuth, Samuel J. Butler, Susy Civil, Westin G. Cohen, Allison F. DeVivo, Tyler S. Fassett, Courtney M. Fisher, Kimberly Garcia, Stephanie L. Hardy, Maureen W. Kamau, Sienna R. Kardel, Allyson L. Kress, Julia M. Lahaie, Stephen A. Malerba, Brittany E. Ricci, Kimberly Rosario, Yelena Vynar, and Deanna N. Womack for creating the initial figures and captions. If you are interested to learn how these figures were made, take a look at the discussion page (2nd tab above).

Lysosomal storage disease

Lysosomal storage disorders are inherited metabolic diseases characterized by an accumulation of undigested various toxic materials. There are nearly 50 diseases and the two examples shown here are Fabry and Schindler disease. Fabry disease, which occurs between early childhood and adolescence, is characterized by the lack of the enzyme alpha galactosidase (α-Gal). Schindler disease can occur in infancy or in adulthood and is characterized by the lack on the enzyme alpha N-acetylgalactosaminidase (α-NaGal). There are currently no cures for lysosomal storage disorders however enzyme replacement therapy is a treatment option. The basic principle of enzyme replacement therapy is to over express the enzyme of interest heterologously, in this case α-Gal α-NaGal, in a cell line and to isolate and purify it from the culture. In enzyme replacement therapy, patients are injected with the enzymes that they lack in the hopes of restoring the enzymatic activity in their cells.

Immune Response

Individuals suffering from Fabry disease cannot produce the α-GAL protein that is necessary for breaking down Galactose. The usual treatment for this is giving the patient doses of the protein, but this poses a problem. Since the body does not produce the protein, an immune response ranging from severe anaphylaxis to mild discomfort can occur when the patient is given the protein. The body does however produce α-NAGAL, a protein with a similar active site and function as Alpha Gal. Altering the active site of α-NAGAL to match that of α-GAL allows doctors to administer a protein that serves the function of Alpha Gal but has the antigenicity of α-NAGAL, which means the body will recognize the protein and not elicit an immune response.

Enzymatic activity

α-Gal and α-NaGal have relatively identical active sites, which are conserved with the exception of alanine, serine, glutamate and leucine which are positioned differently. The two enzymes have the same folds and both function by cleaving glycosydic bonds however have different substrate specificities. The differences in substrate specificity occur because α-NaGal has a larger binding pocket thus interacting with larger molecules but smaller residues.

Galactose vs. N-acetyl-galactosamine

Structures shown on this page

3H54: the enyme α-NAGAL in complex with the sugar GalNAc

3HG5: the enyme α-GAL in complex with the sugar galactose

3LX9: the enyme α-GAL(SA) in complex with the sugar GalNAc

3LXA: the enyme α-GAL(SA) in complex with the sugar galactose

The initial shows the sugar N-acetyl galactosamine.

1 Overview of the research

Overview of the research

The research is about two related enzymes, GAL and NAGAL. They are found in the same location in the human body (in the lysosome, an acidic organelle responsible for breaking down molecules the cell no longer needs), their primary sequence is 50% identical, they catalyze the same reaction (hydrolysis of α-glycosidic bonds), and they have very similar active sites. However, they differ in substrate specificity (one cleaves the bond with galactose, and the other with N-acetyl glucosamine. In terms of structure, the backbone conformation is quite similar (compare and ). The active sites differ in only two residues, and the substrates bind in almost the same way (compare the active site of and ).

The researchers asked the following question: Is it possible to turn one into the other (in terms of reaction catalyzed)? Their hypothesis was that a simple swap of the two amino acids in the active site that are different would accomplish an interconverson of specificities.

To test this, they made variants (called GAL(SA) and NAGAL(EL) where one active site has the amino acids of the other active site and vice versa (by swapping the two residues that are different). The data obtained by enzyme kinetics supported their hypothesis; the preference for Galactose vs N-acetyl galactosamine is swapped (not shown here, but the data is in their paper). Crystal structures show how GAL(SA) is able to bind to either or . Comparing the structures of the NAGAL: N-acetyl galactosamine complex and teh GAL(SA) active sites bound to NAGAL shows that they bind the ligand in a very similar manner.

You can learn more about the structures by going through the figures below and clicking on the buttons.

Figure 1

Panel B (show )

Panel C (show )

Use these buttons to switch back and forth between the two enzymes or to animate the switching

If you click on pop-up on the bottom of the 3D browser window, maximize the pop-up window and turn on stereoview (right click on the model, select Style>Stereographic>...), the active site will really pop.

Figure X (bonus figure)

For the animation in Figure 1, the carbon alpha atoms of the shown active site residues were superimposed (RMSD = 0.3 Å). The following views of the active site differences shows a superposition of the six common carbon atoms (RMSD = 0.02 Å) in the bound sugar. It becomes obvious that the sugar is bound in a slightly different orientation with respect to the overall protein structure.

(use the buttons above to compare with α-NAGAL)

Figure Y (bonus figure)

The shape of the active site is often complementary to the molecule it binds to (lock-and-key concept). This figure shows the contacts between bound molecules and active site residues. Contacts are shown as the overlap of Van der Waals spheres around atoms. Slight overlap is shown in yellow while larger overlap is shown in red. When two functional groups form a hydrogen bond, atoms come closer than they would if they interact via Van der Waals interactions, so you expect to see red overlap for hydrogen bonds. Here are the contacts for and . These are observed structures, so the contacts seen explain why they bind to their ligands.

In contrast, here are the contacts for two hypothetical models, galactose bound to the α-NAGAL active site and N-acetyl galactosamine bound to the α-GAL active site. There are less contacts in the and severe clashes in the (clashes involve the acetyl group of the ligand and residues Glu 203 and Leu 206 of the active site). In fact, experiments show that α-NAGAL does bind galactose (though much more weakly than N-acetyl galactosamine) while α-GAL does not bind N-acetyl galactosamine.

Figure 2: Structure of GAL(SA)

GAL(SA) is derived from GAL by replacing actives site residues glutamate 203 with serine and leucine 206 with alanine. Having these smaller amino acids in the active site increases the substrate binding cavity, and makes the active site of αGAL(SA) very similar to that of αNAGAL. With these substitutions, the catalytic activity of GAL(SA) is more similar to NAGAL than to GAL (the data is not shown here, but can be found in the research paper).

: in complex with GalNAc

Crystal structure of α-GAL(SA) bound to GalNAc. α-GAL(SA) active site residues are shown in yellow and the product, GalNAc, is shown in gray. The blue mesh around GalNAc represents its electron density.

Use the buttons to hide the model of GalNAc in the figure. This is what a crystallographer would interpret to figure out what is bound in the active site.

: in complex with Gal

: Superposition with alpha-NAGAL bound to GalNAc

Superposition of structures: GAL(SA) bound to N-acetyl glucosamine, in yellow, and NAGAL bound to N-acetyl glucosamine, in dark blue. The sugar bound to GAL(SA) is shown in light brown and the one bound to NAGAL is shown in light blue.

For some reason, this figure breaks figure 1. To get back to figure one, press first.

Proteopedia Page Contributors and Editors (what is this?)

Karsten Theis

Retrieved from "http://52.214.119.220/wiki/index.php/Garman_lab:_Interconversion_of_lysosomal_enzyme_specificities"

@@ Line 26: / Line 26: @@
 LXA: the enyme α-GAL(SA) in complex with the sugar galactose
 <StructureSection load='' size='540' side='right' caption='' scene='78/786673/Galnac/2'>
 The initial <scene name='78/786673/Galnac/2'>scene on the right </scene> shows the sugar N-acetyl galactosamine.
+== Overview of the research ==
+The research is about two related enzymes, GAL and NAGAL. They are found in the same location in the human body (in the lysosome, an acidic organelle responsible for breaking down molecules the cell no longer needs), their primary sequence is 50% identical, they catalyze the same reaction (hydrolysis of α-glycosidic bonds), and they have very similar active sites. However, they differ in substrate specificity (one cleaves the bond with galactose, and the other with N-acetyl glucosamine. In terms of structure, the backbone conformation is quite similar (compare <scene name='78/786673/Gal_overall/5'>GAL</scene> and <scene name='78/786673/Nagal_overall/4'>NAGAL</scene>). The active sites differ in only two residues, and the substrates bind in almost the same way (compare the active site of <scene name='78/786673/Gal_active_site/5'>GAL</scene> and <scene name='78/786673/Nagal_active_site/3'>NAGAL</scene>).
+The researchers asked the following question: Is it possible to turn one into the other (in terms of reaction catalyzed)? Their hypothesis was that a simple swap of the two amino acids in the active site that are different would accomplish an interconverson of specificities.
+To test this, they made variants (called GAL(SA) and NAGAL(EL) where one active site has the amino acids of the other active site and vice versa (by swapping the two residues that are different). The data obtained by enzyme kinetics supported their hypothesis; the preference for Galactose vs N-acetyl galactosamine is swapped (not shown here, but the data is in their paper). Crystal structures show how GAL(SA) is able to bind to either <scene name='78/786673/Fig2a_galnac_complex/2'>N-acetyl galactosamine</scene> or <scene name='78/786673/Galsa_gal/21'>galactose</scene>. Comparing the structures of the NAGAL: N-acetyl galactosamine complex and teh <scene name='78/786673/Galsa_nagal/2'>GAL(SA): N-acetyl galactosamine complex</scene>GAL(SA) active sites bound to NAGAL shows that they bind the ligand in a very similar manner.
+You can learn more about the structures by going through the figures below and clicking on the buttons.
 ===Figure 1===
@@ Line 82: / Line 91: @@
 In contrast, here are the contacts for two hypothetical models, galactose bound to the α-NAGAL active site and N-acetyl galactosamine bound to the α-GAL active site. There are less contacts in the <scene name='78/786673/Nagal_swapped/1'>hypothetical α-NAGAL: galactose complex</scene> and severe clashes in the <scene name='78/786673/Gal_swapped/1'>hypothetical α-GAL: N-acetyl galactosamine complex</scene> (clashes involve the acetyl group of the ligand and residues Glu 203 and Leu 206 of the active site). In fact, experiments show that α-NAGAL does bind galactose (though much more weakly than N-acetyl galactosamine) while α-GAL does not bind N-acetyl galactosamine.
-===Figure 2: Structure of αGAL(SA)===
+===Figure 2: Structure of GAL(SA)===
-αGAL(SA) is derived from αGAL by replacing actives site residues glutamate 203 with serine and leucine 206 with alanine. Having these smaller amino acids in the active site increases the substrate binding cavity, and makes the active site of αGAL(SA) very similar to that of αNAGAL. With these substitutions, the catalytic activity of αGAL(SA) is more similar to αNAGAL than to αGAL (the data is not shown here, but can be found in the research paper).
+GAL(SA) is derived from GAL by replacing actives site residues glutamate 203 with serine and leucine 206 with alanine. Having these smaller amino acids in the active site increases the substrate binding cavity, and makes the active site of αGAL(SA) very similar to that of αNAGAL. With these substitutions, the catalytic activity of GAL(SA) is more similar to NAGAL than to GAL (the data is not shown here, but can be found in the research paper).
 <scene name='78/786673/Fig2a_galnac_complex/2'>Panel A</scene>: in complex with GalNAc
@@ Line 122: / Line 131: @@
 </jmol>
-<scene name='78/786673/From_scratch/1'>Panel D V2</scene>
 <scene name='78/786673/Galsa_nagal/2'>Panel D</scene>: Superposition with alpha-NAGAL bound to GalNAc
-Superposition of structures: alpha-GAL(SA) bound to GalNAc, in yellow, and alpha-NAGAL bound to GALNAc, in dark blue. GALNAc product of alpha-GAL(SA) is shown in light brown and GalNAc. product of alpha-NAGAL is shown in light blue. The two product structures are oriented slightly at different angles, but no difference in binding was found.
+Superposition of structures: GAL(SA) bound to N-acetyl glucosamine, in yellow, and NAGAL bound to  N-acetyl glucosamine, in dark blue. The sugar bound to GAL(SA) is shown in light brown and the one bound to NAGAL is shown in light blue.
 <jmol>
 <jmolButton>