Impact of pathological mutations on Alpha-Sarcoglycan Protein Structure

Name of Author: Tanish Rao

Name of Mentor- Dr. Simran Bawa

Affiliation: Junior High at Bellarmine College Preparatory, San Jose, California

1. ABSTRACT

Background: Sarcoglycans are transmembrane proteins associated with the Dystrophin-Glycoprotein components (DGC) that span the plasma membrane. The multimeric complex consists of four glycoproteins (alpha, beta, gamma, and delta), which assemble and form a sarcoglycan sub-complex (SGC) on the sarcolemma. The SGC proteins are tightly bound and are responsible for connecting the muscle fiber cytoskeleton with the extracellular matrix to prevent muscle damage during contraction. Mutations in the Sarcoglycan-alpha protein result in Limb-Girdle Muscular Dystrophy Type-2D (LGMD-2D), often termed sarcoglycanopathy. Limb-Girdle Muscular Dystrophy-2D belongs to the group of inherited autosomal recessive muscular dystrophies characterized by progressive weakness of the pelvic and shoulder muscles. It has been previously reported that R77C, the most prevalent point mutation in SGC-alpha, results in misfolded protein and causes aberrant protein degradation. However, the exact molecular mechanism behind the disease is not known. Therefore, to get more insight into the mechanism, we analyzed the effect of point mutations on the structure and the stability of the SGC-alpha protein.

Materials and methods: We performed computational analysis on R77C and other missense mutations (p.P73L, p.P73R, p.L31P, and p.D97G) and calculated the Gibbs free energy. We used Swiss model software to predict the structure of the alpha-sarcoglycan. The 3D model was predicted using homology modeling. Further, to assess the effect of point mutations on the structure of alpha-sarcoglycan, we manually changed the amino acid sequence and modeled the mutant structure using SWISS MODEL software. Pymol software was used to compare changes in the wild-type and mutant structure. Since mutations often have a debilitating effect on the protein structure. Therefore, we used Dynamut software to predict changes in the Gibbs free energy and the effect of mutations on protein stability and flexibility.

Results: Our findings demonstrate that all five pathological mutations are destabilizing and cause changes in the intramolecular amino acid interactions. Strikingly, both P73L and P73R mutations result in the shortening of the β-strand in one of the domains and alter the protein structure.

Conclusion: The amino acids mutated in the patients with LGMD-2D are located in the loops that connect the secondary structure and are solvent-exposed. Our analysis predicts that the mutations may inhibit fundamental protein-protein interactions essential for muscle function. We also think that loss in the intramolecular interactions between amino acids destabilizes the protein structure and renders it unstable, resulting in degradation.

Keywords: Alpha-sarcoglycan, point mutations, LGMD-2D, Gibbs free energy, protein structure

2. INTRODUCTION

Muscular Dystrophy is a term for a group of diseases characterized by progressive weakness and wasting of voluntary proximal muscles. In many instances, depending on the type and severity, muscular dystrophy can also cause cardiomyopathy, trouble breathing, and other respiratory issues. Onset of this MD varies from childhood all the way to early adulthood. Common symptoms of MD include trouble running, waddling gait, hypertrophied calf muscles, and muscle pain and stiffness.

Over 40 muscular dystrophy diseases exist, all classified into 9 main types. Table 1.1 shows the different types of MDs and their symptoms.

Table 1.1 Types of MDs and their Symptoms

3. RESULTS

Research has shown that the sarcoglycan complex can be considered not as a subunit, but at as its own complex.

Our analysis predicts that the mutations associated with LGMD-2D render the alpha-sarcoglycan protein unstable. These mutations affected the a-sarcoglycan subunit in different degrees, however. After analyzing the structure and calculating the Gibbs Free Energy for each mutation, it was determined that although some mutants presented no visible structural changes, they were shown to have a decrease in stability through a negative Gibbs Free Energy. The D97G mutation, in which aspartic acid changed to glycine at the 97th position produces no visible structural changes. However, the Gibbs Free Energy was -0.210 kcal, meaning that the mutation destabilized the protein. We can attribute this to the decrease in interatomic interactions, as seen in the interatomic analysis below. The L31P mutation, in which leucine changes to proline at the 31st position also shows no visible structural change, but a closer look at the interatomic analysis shows a great change in interatomic interactions, most likely due to the difference in structure between a branch-type leucine and ring-type proline. This change is not conducive to the stability of the alpha sarcoglycan subunit however, as the delta G of -1.086kcal shows. The next mutation, R77C, occurs when arginine changes to cysteine at the 77th position. This mutation also shows no major structural changes in the 3D model but the change in intramolecular interactions destabilizes the protein, as seen through a delta G value of -0.210kcal.

The next type of mutation is not only a destabilizing mutation, but also produces a visible structural change, mainly the shortening of a beta sheet in the protein complex. Out of all the 5 mutations that were analyzed, two produced this sort of an effect. The first mutation is the P73R mutation, which is when proline changes to arginine at the 73rd position. Leucine’s structure also greatly varies from proline’s structure, thus producing very different interatomic interactions that ultimately destabilize the protein, which produced a delta G value of -0.196kcal; this can be seen in the interatomic interaction analysis below. The other mutation that produced similar effects is the P73L mutation, which replaces a proline with a leucine at the 73rd position. Just like the arginine, leucine also has a branch-like structure which differs from proline’s ring-like structure. Given that the delta G value of this mutation is -0.276 kcal, it can be well inferred that this mutation is even more destabilizing than the P73R mutation.

Figure 1: Alpha-Sarcoglycan structure predicted using homology modeling. (A) SGCA protein prominently depicts anti parallel beta sheets and extended loops. (B) Replacing aspartic acid to glycine residue at the position 97 showed no effect on the overall structure of the protein.

Figure 2: Comparison between SGCA WT and D97G mutant protein intramolecular molecular interactions. D97G point mutation results in the loss of multiple side chain interactions with the neighboring amino acids.

Figure 3: Alpha-Sarcoglycan structure predicted using homology modeling. (A) SGCA protein prominently depicts anti parallel beta sheets and extended loops. (B) Replacing leucine to proline residue at the position 31 showed no effect on the overall structure of the protein.

Figure 4: Comparison between SGCA WT and L31P mutant protein intramolecular molecular interactions. L31P point mutation results in the loss of multiple side chain interactions with the neighboring amino acids and a formation of a ring structure as opposed to the original branch-like structure.

Figure 5: Alpha-Sarcoglycan structure predicted using homology modeling. (A) SGCA protein prominently depicts anti parallel beta sheets and extended loops. (B) Replacing cysteine to arginine residue at the position 77 showed no effect on the overall structure of the protein.

Figure 6: Comparison between SGCA WT and R77C mutant protein intramolecular molecular interactions. R77C point mutation results in the loss of multiple side chain interactions with the neighboring amino acids.

Figure 7: Alpha-Sarcoglycan structure predicted using homology modeling. (A) SGCA protein prominently depicts anti parallel beta sheets and extended loops. (B) Replacing arginine to proline residue at the position 73 resulted in shortening of one beta sheet strand.

Figure 8: Comparison between SGCA WT and P73R mutant protein intramolecular molecular interactions. P73R point mutation results in the loss of multiple side chain interactions with the neighboring amino acids and a change from a ring structure to a branch-like structure.

Figure 9: Alpha-Sarcoglycan structure predicted using homology modeling. (A) SGCA protein prominently depicts anti parallel beta sheets and extended loops. (B) Replacing leucine to proline residue at the position 73 resulted in shortening of one beta sheet strand.

Figure 10: Comparison between SGCA WT and P73L mutant protein intramolecular molecular interactions. P73L point mutation results in the loss of multiple side chain interactions with the neighboring amino acids and a change from a ring structure to a branch-like structure.