A new horizon in the phosphorylated sites of AGA: the structural impact of C163S mutation in aspartylglucosaminuria through molecular dynamics simulation

Abstract Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by insufficient aspartylglucosaminidase (AGA) activity leading to chronic neurodegeneration. We utilized the PhosphoSitePlus tool to identify the AGA protein’s phosphorylation sites. The phosphorylation was induced on the specific residue of the three-dimensional AGA protein, and the structural changes upon phosphorylation were studied via molecular dynamics simulation. Furthermore, the structural behaviour of C163S mutation and C163S mutation with adjacent phosphorylation was investigated. We have examined the structural impact of phosphorylated forms and C163S mutation in AGA. Molecular dynamics simulations (200 ns) exposed patterns of deviation, fluctuation, and change in compactness of Y178 phosphorylated AGA protein (Y178-p), T215 phosphorylated AGA protein (T215-p), T324 phosphorylated AGA protein (T324-p), C163S mutant AGA protein (C163S), and C163S mutation with Y178 phosphorylated AGA protein (C163S-Y178-p). Y178-p, T215-p, and C163S mutation demonstrated an increase in intramolecular hydrogen bonds, leading to greater compactness of the AGA forms. Principle component analysis (PCA) and Gibbs free energy of the phosphorylated/C163S mutation structures exhibit transition in motion/orientation than Wild type (WT). T215-p may be more dominant among these than the other studied phosphorylated forms. It might contribute to hydrolyzing L-asparagine functioning as an asparaginase, thereby regulating neurotransmitter activity. This study revealed structural insights into the phosphorylation of Y178, T215, and T324 in AGA protein. Additionally, it exposed the structural changes of the C163S mutation and C163S-Y178-p of AGA protein. This research will shed light on a better understanding of AGA’s phosphorylated mechanism. Communicated by Ramaswamy H. Sarma


Introduction
An inherited lysosomal storage disorder is known as AGU.It is brought on by losing the functional activity of the AGA enzymes (Arvio & Mononen, 2016).In cellular lysosomes, glycoproteins are broken down by the AGA enzyme.AGA substrate (aspartylglucosamine, GlcNAc-Asn) builds up in the lysosomes across all tissues and bodily fluids in people with mutant AGA genes.It usually occurs in modest amounts of faulty protein or perhaps an inactive precursor polypeptide (Arvio & Arvio, 2002;Arvio & Mononen, 2016).
AGU is a slow-moving, devastating neurodegenerative condition that causes early death, intellectual incapacity, and skeletal and motor deformities (Chen et al., 2021).Patients with AGU often live between 30 and 40 years.Patients rely highly on supportive care by the third decade of life because they have severe physical and intellectual disabilities.Lysosomal hypertrophy causes intellectual incapacity; and other symptoms, including skeletal and joint problems, result from substrate build-up.Due to gradual brain atrophy, AGU patients experience developmental difficulties, including delayed speech and reduced learning.
For AGU, no approved treatment addresses the diseases underlying causes.A few methods for treating AGU have been tried.Only a few bone marrow transplantation (BMT) instances have been successful (Arvio et al., 2001).Although peripheral delivery of enzyme replacement therapy (ERT) does not sufficiently treat the central nervous system, it has the potential to be a successful treatment (CNS).Additionally, producing the functional AGA with the proper post-translation modifications is extremely difficult, creating considerable obstacles to using ERT as a workable therapeutic therapy (Dunder et al., 2000(Dunder et al., , 2010)).One founder mutation (482 G > A) that results in an amino acid substitution (C163S) in AGA polypeptide are the source of the AGUFin primary mutation, which accounts for 98% of all Finnish disease alleles (Ikonen, Baumann, et al., 1991;Ikonen, Enomaa et al., 1991;Ikonen et al., 1993).
The AGA gene produces an inactive 346 amino acid preproprotein from which a co-translationally cleaved 23 amino acid proprotein signal sequence was formed in the endoplasmic reticulum (ER) (Saarela et al., 1998;Saito et al., 2008).It was hypothesized that the process indicating auto-proteolytic activation involves the dimerization of two inactive precursor molecules.Precursor molecules naturally cleave into N-terminal a-(�27 kDa) and C-terminal b-(�17 kDa) subunits when two precursor molecules dimerize (Ikonen et al., 1993).Dimerization occurs early in the folding process or when the polypeptides have nearly achieved their final folded state (Riikonen et al., 1996).Two a-and b-subunits make up the mature enzyme, a heterotetramer.The functional residue, a threonine, is accessible at the N-terminus of the a-subunits.At residues N38 and N308, each AGA subunit is N-linked to a high-mannose type of oligosaccharide chain.The mannose 6-phosphate pathway transports the tetrameric molecule to the lysosomes (Tikkanen et al., 1995).Neuronal cells also employ a similar mechanism (Kytt€ al€ a et al., 1998).Lysosomal proteases shorten the a-and b-subunits of the lysosomes Cterminal regions (Peltola et al., 1996).AGA can hydrolyze L- asparagine into L-aspartic acid and ammonia, acting as asparaginase (Liu et al., 1996;Noronkoski et al., 1997;Tanaka et al., 1973;Tarentino & Plummer, 1993).
The AGA polypeptide undergoes various mutations that prevent dimerization and consequent activation (Riikonen et al., 1996).In some cases, the activation does not occur, but the precursor is delivered to the lysosomes and cleaved due to some disease-causing mutations of AGA (Peltola et al., 1996;Tikkanen et al., 1996).Lysosomal proteins are recognized by UDP-N-acetylglucosamine phosphotransferase based on a surface structural determinant.On the surface of the human AGA molecule, the determinant comprises three separate and cooperative areas: oligosaccharide chains and lysine amino acids (Tikkanen et al., 1997).All recognition sites must be properly positioned for the cooperative interaction with phosphotransferase (Enomaa et al., 1995).The phosphotransferase cooperative interaction ensures that only appropriate folded molecules are phosphorylated and delivered to the lysosomes.(Enomaa et al., 1995;Tikkanen et al., 1995).
Molecular dynamics have produced temporal scales that are starting to match those of biological processes (Filipe & Loura, 2022).Currently, conformational changes can be successfully stimulated.The advancement in optimization of molecular dynamics, such as coarse-grained ones, the foundation of conventional molecular modeling, to the analysis of conformational ensembles.Conformational ensembles enable a better accurate depiction of actual macromolecules as they take flexibility and dynamic qualities (Almeida et al., 2022;de Almeida et al., 2023;Lima et al., 2022;Silva et al., 2023).
Here, molecular dynamics simulations studied the structural impact of the acquired phosphorylation sites in the AGA protein (Condic-Jurkic et al., 2018;Kato et al., 2017).We notably saw frequent C163S mutation close to the AGA protein's acquired phosphorylation site Y178.Hence, we further compared the structural changes of the C163S vs. C163S-Y178-p.

Data retrieval and pre-processing
The human AGA protein-related information was retrieved from the UniProt database (Apweiler et al., 2004) from accession id: P20933.The PDB structures of AGA were retrieved from the RCSB-PDB database with the accession id: 1APY.It belongs to the "AGA" protein and "Homo sapiens" species (Oinonen et al., 1995).Since the deposited AGA crystal had missing residues from 162 to 182 residual length, we adapted the use of missing residues construction using the SwissPDB viewer.The missing residue region was constructed & energy minimized, and validated using the SAVESv6.0 server (Laskowski et al., 1996).The validated structure was used for further processing.

Identification of phosphorylated site in AGA protein using PhosphositePlus
We utilized PhosphositePlus (https://www.phosphosite.org/homeAction.action)database to retrieve the Phosphorylated site in the AGA protein.PhosphositePlus is a free, extensive, manually maintained, and interactive resource for inspecting post-translational changes of human proteins that have been experimentally detected and reported.There are 130,000 non-redundant modification sites, mainly phosphorylation, ubiquitination, and acetylation (Hornbeck et al., 2012(Hornbeck et al., , 2019)).

System building using CHARMM-GUI
The validated AGA protein structure was utilized in the solution builder module in CHARMM-GUI to generate the protein system in the water solution (Jo et al., 2008).Then, PDB manipulation options enabled us to phosphorylate/mutate the specific residues of the AGA (Jo et al., 2014).Rectangle water box was created for each system with a 10 Å edge distance and solvated with TIP-3P water molecules.The solvent was neutralized using counter ions to allow the system to achieve electrostatic stability using the CHARMM-GUI module (Brooks et al., 2009).The parameters for further processing were built to run on the latest GROMACS software.

Molecular dynamics simulation and analysis
We generated six systems using CHARMM-GUI, namely; (i) WT -AGA protein, (ii) Y178-p, (iii) T215-p, (iv) T324-p, (v) C163S, and (vi) C163S-Y178-p.We applied the CHARMM36 force field for all the systems using CHARMM-GUI (Lee et al., 2016).The latest version of GROMACS utilized the CHARMM-GUI inputs for molecular dynamics simulations (Abraham et al., 2015).The steepest descent technique, implemented in GROMACS, was utilized to energy minimize the initial systems using a 10 kJ/mol/nm energy threshold.Following the minimization, 25 ns of NVT (constant particle number (N), volume (V), and temperature (T)) ensemble equilibration were run.The Berendsen algorithm applied temperature and pressure coupling.Constraints on proteins and side chains were applied in the equilibration run.Following that, production runs in an isothermal-isobaric ensemble (NPT-number of particles (N), pressure (P), and temperature (T)), in which all restrictions are removed.During the production run, the Semi-isotropic Parrinello-Rahman method was used for pressure coupling, and the Nose-Hoover temperature coupling method was employed for temperature coupling (Hoover, 1985).We used coupling constants of 1.0 ps for temperature and 5.0 ps for pressure.The pressure was held constant at 1 atm throughout the production run.Both equilibration and production run employed an integration timestep of 2 fs.A temperature of 303.15K was used for the simulations.With the verlet cutoff scheme, non-bonded interactions were truncated at 1.2 nm.The particle mesh Ewald (PME) approach represented long-range electrostatic interactions.Utilizing the LINCS78 algorithm, hydrogen bonds were restricted.In the semi-isotropic environment, simulations were subjected to periodic boundary conditions.We performed a total of 200 ns production for all the six systems each (Akhunzada et al., 2022).We performed the interim analysis from the final trajectories such as the root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent accessible surface area (SASA), the radius of gyration (Rg), intramolecular hydrogen bond, PCA, and Gibbs free energy calculation utilizing the in-built modules available in GROMACS.
The RMSD was studied to monitor the simulation trajectory effectiveness and convergence.The RMSF fluctuation was illustrated to provide an understanding of the flexibility of the amino acid residue for further insights into the structural modifications.The surface area in contact with the solvent surrounding a protein molecule is known as SASA.A protein's overall structure and folding depend heavily on its solvation capacity.The misfolded protein tends to have less efficacy than the WT protein.Therefore, it is crucial to investigate proteins SASA and packaging density and their folding behavior after structural modifications.Further, we determined the Rg for the AGA protein to demonstrate the protein compactness and provide a comprehensive overview of the distribution of molecules within proteins.Rg analysis provides detailed information about AGA protein dynamics.One of the crucial intramolecular interactions for maintaining the structural integrity of protein molecules is intramolecular hydrogen bonds.To better understand the structural integrity throughout the simulation, we observed the evolution of the number of intramolecular H-bonds in the WT, phosphorylated, and mutant forms.We carried out PCA to investigate the conformational changes and atomic motions of the AGA phosphorylation and C163S mutant.The phase space efficiency of a protein allows us to study its structural dynamics.

Phosphorylated sites and validation of AGA protein
A total of three phosphorylated sites of AGA protein were identified through the PhosphositePlus server: Y178-p, T215p, and T324-p (Hornbeck et al., 2012;Tikkanen et al., 1997), and the same has been depicted in Table 1.Fisher et.al.reported a predominant single nucleotide alteration in the AGA gene, resulting in cysteine substituted with serine (C163S) in the AGA enzyme protein leading to AGU (Fisher & Aronson, 1991).Hence, we studied the structural changes of the C163S-mutant along with phosphorylated sites of AGA via molecular dynamics simulation.Since the Y178 position is close to the most frequent mutation C163S among the three phosphorylated sites, we further determined structural changes in AGA with Y178-p and C163S mutation.Furthermore, the energy-minimized structure of AGA with the construction of missing residues was examined and evaluated using the SAVES v6.0 server.The verify3D server reports that 81.29% of the residues have an average 3D-1D score >0.2, and the score passed.The ERRAT server predicted a quality factor of 90.Ramachandran plot of the AGA protein model generated by the PROCHECK module demonstrated that >90% of the amino acid residues are located in the favoured and allowed regions (Supplementary Figure 1).

Molecular dynamics simulation and analysis
The resultant phosphorylated sites and reported mutant C163S were finalized and subjected to a 200 ns simulation using CHARMM-GUI.The simulation constructs, namely: (i) WT AGA protein (WT), (ii) Y178-p, (iii) T215-p, (iv) T324-p), (v) C163S, and (vi) C163S-Y178-p were simulated, and the trajectories acquired was carried out for analysis using the in-built available modules of GROMACS.A brief description of the analysis scores is depicted in Table 2.

Trajectory mean square deviation and fluctuation
The average RMSD value of WT vs. Phosphorylated forms of AGA, i.e., WT, Y178-p, T215-p, and T324-p, are 0.624, 0.531, 0.604, and 0.683 nm, respectively as (Figure 1A).There was a significant difference in the deviation and flexibility of the WT and phosphorylation forms.The average RMSD value for the C163S and C163S-Y178-p is 0.627 and 0.750 nm.We observed that the average RMSD value for C163S-Y178-p was higher than other structures, illustrating that C163S-Y178-p has highly deviated from the WT (Figure 1B).Subsequently, the average RMSF value for WT, Y178-p, T215-p, and T324-p is 0.373, 0.217, 0.162, and 0.295 nm, respectively (Figure 1C).The average RMSF value for C163S and C163S-Y178-p was 0.216 and 0.333 nm (Figure 1D).Based on the fluctuation score, the WT protein is more flexible than the phosphorylated and mutant AGA protein.Higher fluctuation values in the WT structure show that the C163S mutant caused constraints to be placed on the flexibility of the protein structure.We notably observed a lower fluctuation of three different phosphorylated AGA proteins in the switch I region than the WT protein.Additionally, we saw a higher fluctuation of the T324-p structure in the switch II region compared to other structures.

SASA, Rg, and intramolecular hydrogen bonds
The time evolution of the SASA of WT, phosphorylated and mutant forms was computed and plotted (Figure 2).The average SASA value of WT is 161.467nm 2 .We observed a lower SASA value in T215-p (155.436nm 2 ) than in other structures.
We found slightly higher SASA values in Y178-p (162.799nm 2 ) and T324-p (162.114nm 2 ) than in WT.T324-p showed higher SASA from 40 to 100 ns; after that gradually dropped to lower fluctuations from 120 to 200 ns than WT.T215-p observed lower SASA throughout the 200 ns simulation than WT.Likewise, we observed higher SASA in C163S (164.755nm 2 ) and C163S-Y178-p (166.566nm 2 ) compared to the WT.We observed higher SASA values after 170 ns in C163S and C163S-Y178-p compared to the WT, representing the greater extent of flexibility and instability of the structure.Subsequently, the variation of the Rg values over time was used to study the folding mechanism and conformational behavior of AGA WT, phosphorylated, and mutant structures.The average Rg value of WT, Y178-p, T215-p, and T324-p is 2.064, 2.062, 1.977, and 1.999 nm.The average value for C163S mutant and C163S-Y178-p is 2.187 nm and 2.067 nm (Figure 3).We noticed the lower average Rg values of T215-p and T324-p than the WT.The lower Rg value of T215-p and T324-p indicates higher compactness and signifies those structures have significantly deviated from the WT.The average Rg value of Y178-p was slightly lower than the WT.Whereas the C163S showed a higher average Rg, suggesting the looseness of the conformational state.Similarly, the C163S-Y178-p showed a slightly higher average Rg than the WT.
The WT-AGA protein forms an average of 207.296 intramolecular H-bonds, whereas the average intramolecular Hbonds for phosphorylated forms Y178-p, T215-p, and T324-p are 214.265, 214.722, and 203.384, respectively.The average number of intramolecular H-bonds for the C163S mutant and C163S-Y178-p structure are 208.609and 202.941, respectively (Figure 4).We observed higher intramolecular H-bonds in Y178-p, T215-p, and C163S compared to the WT.In T324-p and C163S-Y178-p, the observed lower numbers of intramolecular H-bonds indicate less compact than WT (Figure 4).We demonstrate that Y178-p, T215-p, and C163S of AGA protein were more compact than the WT.Hence, there may be a chance for fold induction brought by Y178p, T215p, and C163S forms in AGA protein.Intramolecular hydrogen bond formation significantly influences the structure and characteristics of molecules.We evaluated the hydrogen bonds formed within the 3 Å donor-acceptor and 20 degrees-angle cutoff distance in the average structures of the WT, Y178-p, T215-p, T324-p, C163S and C163S-Y178-p using VMD.We interpreted the total number of intramolecular hydrogen bonds formed in the AGA phosphorylated forms and C163S mutation against WT (Table 3).We chose the core residues based on the phosphorylated/mutated position (i.e) ± 3 residues from the phosphorylated/mutated position.We obtained the core interactions of each average structure, which is essential for structural compactness.Also, we compared the variations in residual co-operatively between the AGA phosphorylated forms and C163S mutation structures against WT (Table 4).Further, we provided detailed information on the core residues interactions, such as types of intramolecular hydrogen bonds, their position, and residues in Supplementary Table 1.

Principal component analysis (PCA) and Gibbs free energy
The PCA results exposed that well-defined cluster of motion direction in WT AGA PC1 (À 23 to 18 nm) and PC2 (À 13 to 18 nm) domain by covering the maximum region.However, the phosphorylated AGA such as Y178-p (PC1 À 11 to 11 nm and PC2 À 11 to 6 nm), T215-p (PC1 À 9 to 12 nm and PC2 À 7 to 6 nm), and T324-p (PC1 À 22 to 11 nm and PC2 À 15 to 11 nm) covered minimum region.Similarly, the mutant C163S (PC1 À 10 to 14 nm and PC2 À 10 to 14 nm) and C163S-Y178-p (PC1 À 16 to 21 nm and PC2 À 10 to 15 nm) covered minimum region compared to WT.The PCA plots have been illustrated in Figure 5. Further, we examined all atoms in the PC1 and PC2 systems of WT, Y178-p, T215-p, T324-p, C163S, and C163S-Y178-p structures by the Gibbs free energy utilizing trajectories to determine the direction of the fluctuation (Figure 5).The DG values for WT, Y178-p, T215-p, T324-p, C163S, and C163S-Y178-p were 11.5, 9.88, 10.3, 14.2, 8.55, and 11.4 kJ/mol, respectively (Figure 6).The plot represents the lower energy states with the most stable protein conformations.In contrast, the blue region represents the high-energy states.The plot's more minor concentrated red lower energy area indicates a highly stable structure.We can observe the changes of lower energy on the Phosphorylated and C163S mutant AGA structures compared to WT.A widespread conserved energy orientation in the WT underwent a transition in orientation in the phosphorylated forms of AGA (Figure 6A-D).Also, a paradigm shift in the energy plane was observed in the AGA-mutant and mutant phosphorylated form (Figure 6E, F).

Discussion
The most prevalent condition of glycoprotein degradation, AGU, is a recessively inherited lysosomal storage disease with a high frequency in the Finnish population (Ikonen et al., 1991).It is a chronic illness that long-term impacts the patient's appearance, intellect, adaptive abilities, physical development, personality, body structure, and health (Arvio et al., 1997(Arvio et al., , 1999;;Arvio & Arvio, 2002).AGU was caused by a mutation in the AGA gene, which has nine exons on the 4q34.3.The AGA sequence is an evolutionarily well-conserved region (Liu et al., 1996).AGA cleaves the N-glycosidic link between the L-asparagine and N-acetylglucosamine subunits of GlcNAc-Asn.AGA enzyme deficiency causes a buildup of undegraded AGA and several other glycoasparaginase, such as glycoconjugates having an L-asparagine moiety attached to the carbohydrate chain, in the affected person's tissues and bodily fluids (Mononen et al., 1993).Earlier researchers used transient expression of polypeptides with specific amino acid changes to characterize the phosphotransferase recognition signals of human AGA.They discovered three lysine residues (Lys177 or Tyr178, Lys183 and Lys214), and a tyrosine located in three spatially different areas of the AGA polypeptide is required for oligosaccharide phosphorylation.Two of the lysines (Lys183 and Lys214) are particularly critical for AGAs lysosomal targeting effectiveness, which appears to be determined mainly by the degree of phosphorylation of the subunit oligosaccharide (Tikkanen et al., 1997).However, the mechanism and structural changes of the AGA phosphorylations and AGA mutation causing AGU were not clearly understood.In this study, we used the molecular dynamics simulation analysis and compared the structural impacts of three different residue-specific phosphorylation of AGA (Y178-p, T215-p, and T324-p) and C163S mutation.
The flexibility differences between residues are analyzed using RMSF with the periodic molecular dynamic simulation conformation.A high RMSF value indicates a mobile region, whereas a low RMSF value indicates a restricted number of movements during molecular dynamic simulation relative to its average (Ardalan et al., 2018;Parra-Cruz et al., 2018).We observed lower RMSF values for the phosphorylated, C163S, and C163S-Y178-p of AGA compared to the WT, which indicates reduced flexibility.According to the average RMSF value, we found that three phosphorylation of AGA makes the structural constraints and reduce the flexibility.Similarly, we discovered that C163S and C163S-Y178-p had the lowest RMSF value in the switch I region, suggesting that C163S and C163S-Y178-p flexibility was reduced even more than our studied phosphorylated AGA around the mutation region.
The average H-bonds of C163S were slightly higher than the WT, and it states that mutation of AGA increased the intramolecular H-bond interaction and decreased the flexibility.The average H-bonds of T324-p and C163S-Y178-p were lower than the WT, indicating that structures are more flexible.In contrast, the average H-bonds of Y178-p and T215-p were higher than the C163S, indicating that phosphorylation of AGA structures increased the rigidity and reduced the flexibility of the protein.Due to the tighter chain distance brought on by the increased density, the system has more H-bonds (Li et al., 2021).Based on the total number of intermolecular hydrogen bonds formed within the 3 Å donoracceptor and 20 degrees-angle cutoff distance, we observed reduced main chain-main chain interactions upon AGA phosphorylation and mutations.But while including the main chain-side chain and side chain-side chain interactions, we observed almost similar interactions in T324-p and WT, the least number of interactions seen in other phosphorylated AGA (Y178-p and T215-p) and C163S mutation (Table 3).The C163S-Y178-p formed more intramolecular interactions than WT but with varied occupancy (Table 3).The effect of conformational changes related to protein-protein recognition may be predicted using SASA.Additionally, it has been demonstrated that bound conformations exhibit greater SASA than their unbound forms (Mukherjee & Bahadur, 2018).We found a higher SASA value in the C163S compared to WT and an even higher value in Y178 phosphorylated AGA with C163S mutation, suggesting that the C163S-Y178-p structure gained solvent accessibility or folded structure.We noticed the most negligible SASA value in the T215-p, thus indicating a loss of solvent accessibility or unfolded structure.
Still, the Y178-p AGA structure was compact, rigid, and resembled the WT.Whereas T215-p, and T324-p observed higher Rg values, indicating more rigid than the WT.PCA and Gibbs free energy landscape was a well-known approach for obtaining large-scale coordinated motions (Ali et al., 2019;Valente et al., 2020).The Gibbs free energy plot revealed that C163S has a lower value than WT, signifying a more stable structure than WT.However, the phosphorylation of Y178 with the mutant C163S has no significant changes, retaining the same energy and showing a similar structure to the WT.Moreover, the phosphorylated form of 215 AGA might be more dominant than the other phosphorylated forms, which can hydrolyze L-Aspargine into Laspartic acid and ammonia acting as an asparaginase, thereby regulating neurotransmitter activity (Sriroopreddy et al., 2021).Furthermore, we looked at the structural insights of AGA C163S mutated with Y178 phosphorylated protein.Figure 7 shows the structures of AGA protein for WT, Y178-p, T215-p, and T324-p, C163S, and C163S-Y178-p after 200 ns simulations.
In WT AGA secondary structure, the helix formed from Thr149 to Ala160, the coil formed from Arg161 to Lys196, the helix formed from Glu197 to Glu199, coil formed in  Asp200 and Asp205.We observed sheet formation from Thr206 to 213 and turns from Lys214 to Gly216 (Figure 7A).In Y178-p secondary structures are altered by induction of turns (Arg161 to Asn162 and Lys177 to Cys179), and the helix shifted from (Glu197-Glu199) to (Ile194 -Thr198) due to the phosphorylation of the residue 178 (Figure 7B).In the T215-p structure, helix reduction (Thr149 to Leu159) and induction of turns (Ala160 to Arg161, Pro175 to Lys177, Pro181 to Tyr182, Ile194 to 195, and Ser238 to Pro239) were observed after phosphorylation of Thr215 (Figure 7C).In T324-p, the helix was reduced (Ser151 to Trp158), followed by the helix being replaced by the coil in Leu159, induction of turns (Arg161 to Cys163, Asn166 to Asn170, Pro175 to Ser176, and Pro193 to Ile194), Sheet reduction (Thr206 to Ile212), induction of coil from His213 to His217, and other minor changes were noticed in the structure (Figure 7D).In contrast, in C163S AGA mutant secondary structure transformed the WT state by helix reduction (Thr149 to Trp158), replacing the helix in the Leu159 -Ala160 into the coil, induction of turns Trp168 -Asn170, Pro185 -Gly186, and Ile194 -His195.The coil length extends till Asp205, and turn reduction in the Lys214 -Thr215 (Figure 7E).In C163S-Y178p, helix reduction Ser151 to Ala160, induction of turns (Thr149 -Ala150, Arg161, Pro175 -Ser 176) followed by helix replacing the coil and extended till Thr206 in the structure (Figure 7F).The superimposed structure of C163S and C163S- Y178-p were illustrated in Figure 7G to understand the structural changes upon the AGA phosphorylation of the mutant C163S.As mentioned above, a transitional shift in the orientation took place in the phosphorylated forms compared to the WT, leading us to assume that the structural modification due to phosphorylation within the protein structure plays a vital role in the man-nose-6-phosphate pathway.Some of the reports which support our findings have been elucidated.The AGA mutations influence the AGA molecules folding, dimerization, activation, or transport and further help to expose the residues essential for any of these required processes.Riikonen et al., reported a significant mutation R161Q þ C163S of the AGUFin, causing improper folding and early degradation (Riikonen et al., 1996).The glycosylasparaginase enzyme protein undergoes two nucleotide changes, the first substitution causes glutamine to replace arginine (R161Q), and the second one causes cysteine to serine substitution (C163S), resulting in a lack of glycosylasparaginase (AGA) activity (Fisher & Aronson, 1991).The mutation prevents the inactive precursor enzyme protein from autocleavage into subunits and an active enzyme protein by destroying a disulfide link and altering its shape (McCormack et al., 1995;Sui et al., 2014).The disulfide bridge between C163 and C179 cannot form because of C163S, leading to structural issues and the instability of the loop structure that connects the two dimers of AGA.However, C163 lies close to K177 and Y178, two of the three phosphotransferase binding sites.Thus, the alteration also inhibits phosphorylation via changing local folding (Tikkanen et al., 1997).Banning et al., demonstrated that T122K or AGU-Fin (C163S mutation plus R161Q polymorphism) mutation in AGA causes it to be produced normally and localized in lysosomes.Still, it also reduces AGA activity because the precursor molecule cannot be processed properly into subunits.The precursor is processed into subunits when T122K or AGU-Fin is co-expressed with wild-type AGA, suggesting that the mutation produces a local misfolding that prevents the precursor from being processed (Banning et al., 2016).
Our structural analysis of AGA phosphorylation and C163S mutation that causes AGU will shed light for us to understand the AGA phosphorylation process.We found that AGA C163S mutation altered the structure by induction of turns and helix reduction.This alteration may inhibit phosphorylation and cause early degradation (Figure 7).Further, we also elucidated that transitional shift in the orientation of the AGA phosphorylated structure as compared to the WT.This structural modification in AGA phosphorylation plays a significant role in the auto cleavage and mannose-6-phosphate pathway.

Conclusions
A systematic structural insight was demonstrated on the three-residue-specific phosphorylation, Y178, T215, and T324 and its impact on AGA.Molecular dynamics simulations enabled us to analyze the orientation and structural changes over the 200 ns simulation run.Our research found that phosphorylation enables the AGA activity to perform cleavage.Also, the mutant showed a distinct impact on the specific phosphorylated site, rendering the AGA enzyme's structure-function activity.Hence, this study revealed the structural insights of the three phosphorylation sites, C163S mutation, and its effect on phosphorylation in AGA protein through molecular dynamics simulation.Our findings deliver new fundamental insights into structural changes of the C163S mutation, which may lead to phosphorylation inhibition, cause early degradation of AGA protein, and cause AGU.This structural analysis and the phosphorylation process of AGA will lay a foundation for a better understanding of AGA phosphorylation for further research.

Figure 3 .
Figure 3.The scatter radius of the gyration plot of the AGA protein WT, Y178-p, T215-p, T234-p, C163S mutation, and C163S-Y178-p.(A) The black color indicates the WT AGA.(B) The orange color indicates Y178-p AGA.(C) The yellow color indicates the T215-p AGA.(D) The purple color indicates the T234-p AGA (E).The red color indicates C163S AGA.(F) The blue color indicates the C163S-Y178-p AGA.

Figure 4 .
Figure 4.The number of intramolecular hydrogen bonds formed in the AGA protein WT, Y178-p, T215-p, T234-p, C163S mutation, and C163S-Y178-p.The X-axis depicts time (ns), whereas the Y-axis depicts the number of intramolecular hydrogen bonds.(A) The black color indicates the WT AGA.(B) The orange color indicates Y178-p AGA.(C) The yellow color indicates the T215-p AGA.(D) The purple color indicates the T234-p AGA (E).The red color indicates C163S AGA.(F) The blue color indicates the C163S-Y178-p AGA.

Figure 6 .
Figure 6.Gibbs free energy plot of the AGA protein (A) WT, (B) Y178-p, (C) T215-p, (D) T234-p, (E) C163S mutation, and (F) C163S-Y178-p.The X-axis depicts PC1, while Y-axis depicts PC2 of the system.The color of Gibbs free energy (KJ/mol) ranges from blue to red.Blue indicates lower Gibbs free energy, while red indicates higher Gibbs free energy.

Table 1 .
The list of phosphorylated sites in the AGA gene attained from the PhosphoSitePlus server.

Table 2 .
The RMSD, RMSF, Rg, SASA, and hydrogen bonds generated in phosphorylated and mutant AGA were compared on an average basis.

Table 3 .
Total number of intra-molecular hydrogen bonds formed in the AGA phosphorylated forms and C163S mutation from the average structures.

Table 4 .
The number and type of intra-molecular hydrogen bonds formed on the core residues of AGA phosphorylated forms and C163S mutation compared to WT.