Designing Molecules to Treat Neurologic and Ophthalmologic Diseases Caused by Sorbitol Excess: Engineering the Agrobacterium Vitis Protein

doi:10.21203/rs.3.rs-2231853/v1

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Designing Molecules to Treat Neurologic and Ophthalmologic Diseases Caused by Sorbitol Excess: Engineering the Agrobacterium Vitis Protein

https://doi.org/10.21203/rs.3.rs-2231853/v1

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This work presents the design of a new protein based on the adenosine triphosphate-binding cassette (ABC) transporter solute binding protein (SBP). The Protein Data Bank in Europe's dictionary of chemical components was utilized to identify sorbitol and D-allitol. Allitol bound to an ABC transporter SBP was identified in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB). Wizard Pair Fitting and Sculpting tools in PyMOL were used to replace bound allitol with sorbitol. PackMover Python code was used to induce mutations in the ABC transporter SBP’s binding pocket, and changes in free energy for each protein-sorbitol complex were identified. The results indicate that charged side chains form polar bonds with sorbitol in the binding pocket, thus increasing its stabilization. In theory, the novel protein can be used as a molecular sponge to remove sorbitol from tissue and therefore treat conditions affected by sorbitol dehydrogenase deficiency.

Biomedical Engineering

Computational Biology

Biotechnology and Bioengineering

Biopolymers

Biologic

Protein Engineering

PyRosetta

Cataract

Neuropathy

The polyol pathway is a biological mechanism utilized by tissues to convert glucose to fructose through converting glucose into an intermediary compound, sorbitol (Brownlee 2005). Sorbitol is the alcohol counterpart of glucose (Bhagavan 2002). Its structure effectively traps glucose within a cell, where it is then converted to fructose through the help of the enzyme sorbitol dehydrogenase (SORD) (Hodgson et al., 2017) (LeRoith et al., 2004) (Safi et al., 2014). Tissues with insufficient sorbitol dehydrogenase suffer from intracellular sorbitol accumulation, which subsequently causes osmotic damage. Excess sorbitol may deposit in the eyes, causing cataracts or retinopathy or manifest as peripheral neuropathy, similar to the effect of chronic hyperglycemia in diabetes (Jedziniak et al., 1981) (Burg and Kador, 1988).

Sorbitol dehydrogenase deficiencies currently affect one in 100,000 individuals and primarily manifest as the most common form of autosomal recessive peripheral neuropathy (Coretese et al., 2020). In patients with diabetes, excess glucose in the blood significantly increases the production of sorbitol and can lead to cell lesions. In the lens of the eye, diabetic patients can develop cataracts up to 20 years earlier (Vinson, 2006) because of osmotic imbalance from excess sorbitol and oxidative damage (Hashim and Zarina, 2012). These conditions can have drastic effects on quality of life and will require lifestyle changes and long-term management if not addressed in a timely manner.

To address the ophthalmic and neurological pathologies associated with sorbitol dehydrogenase deficiency, we propose a protein capable of binding sorbitol with high affinity and specificity. This protein, the adenosine triphosphate-binding cassette (ABC) transporter solute binding protein (SBP), is derived from the microbe Agrobacterium vitis (also known as Allorhizobium vitis), a Gram-negative, motile, plant pathogen that infects grapevines (Proteomes, 2021). The ABC transporter SBP itself has a ‘Venus flytrap’-like mechanism that allows for large conformational changes in its binding domains to allow for the encapsulation of ligands (Yadava et al. 2016). The structure of the ABC transporter SBP is specific for organic compounds such as allitol or the amino sugars glucosamine and galactosamine (Yadava et al. 2016) (Vetting et al. 2014).

Figure 1. Polyol Pathway. The metabolic pathway that converts glucose to fructose.

The small molecule allitol (synonymously referred to as D-allitol) has a molecular formula of ${C}_{6}{H}_{14}{O}_{6}$ and is a natural ligand for the ABC transporter SBP. Sorbitol is another small molecule and has the same molecular formula as allitol, ${C}_{6}{H}_{14}{O}_{6}$. Both molecules are six-carbon chains with each carbon having an attached hydroxyl ($-OH$) group. The difference between allitol and sorbitol is simply the stereochemistry of a single hydroxyl group at the third carbon position, as seen in Fig. 2. The similarity in the overall structures of allitol and sorbitol suggest that with minor modifications to the binding pocket of the ABC transporter SBP, the protein can be used as a molecular sponge that preferentially binds sorbitol over allitol for the purpose of removing sorbitol from tissue.

Figure 2. Ligands. A. Allitol is the naturally bound ligand of the ABC transporter SBP. B. Sorbitol, which differs in stereochemistry from allitol only at the third carbon hydroxyl group, is the ligand that the ABC transporter SBP can be engineered to preferentially bind to.

In this work we will design a new protein based on the adenosine triphosphate-binding cassette transporter solute binding protein to be used as a molecular sponge that can exchange its bound ligand allitol for a target ligand sorbitol. This new protein has the potential to be used as a drug for the treatment of cataracts, retinopathy, or peripheral neuropathy in individuals suffering from sorbitol dehydrogenase deficiency. Physiologically, the molecular sponge could decrease excess intracellular sorbitol and provide relief to individuals from the adverse effects associated with this condition.

2.1. Generating files for use in PyMOL

To identify the protein and ligands mentioned above, we used several online resources. Utilizing the Protein Data Bank (PDB) in Europe’s Chemical Components in the PDB, we identified that sorbitol, code name of SOR (Chemical Components in the PDB: SOR 2021), is a related compound of D-allitol, code name of X9X (Chemical Components in the PDB: X9X 2021). With this information, we searched the Research Collaboratory for Structural Bioinformatics (RCSB) (Research Collaboratory for Structural Bioinformatics Protein Data Bank 2021) PDB (‘X9X’ 2021, ‘SOR’ 2021) for a protein that contained allitol as a ligand in its structure, which we found in the protein with the code name 4WT7 (‘Crystal structure of an ABC transporter solute binding protein (IPR025997) from Agrobacterium Vitis (Avi_5165, Target EFI-511223) with bound allitol’) (‘4WT7’ 2021). We were able to download a ‘.pdb’ file directly for 4WT7, however this protein was a dimer of two identical monomers. We then deleted one monomer and saved the protein as a new ‘.pdb’ file. As X9X was contained within the structure of 4WT7, we isolated the ligand in the file and saved it as a separate ‘.pdb’ file. Finally, a ‘.sdf’ file for SOR was acquired which was run through an online web service integrated with OpenBabel to convert the file to a ‘.mdl’ file format (‘Open Babel’ 2021). This file was subsequently converted to a ‘.pdb’ file using the ‘molfile2params’ code.

The above mentioned ‘.pdb’ files were used in conjunction with PyMOL to calculate energy scores, run experiments, and generate images. A summary of these ‘.pdb’ files that will be referred to in subsequent sections is found in Table 1.

Table 1

**Summary of ‘.pdb’ Files Used.** Each ‘.pdb’ file with its corresponding nickname that we use in subsequent sections is listed in the table, along with a brief description of the protein and/or ligand the file contains.
Ligand/Protein/Complex Nickname	‘.pdb’ File	Details
Protein	protein.pdb	ABC transporter SBP (4WT7) which is downloaded from the PDB as a dimer
Allitol	allitol.pdb	Allitol (X9X) which is isolated from protein.pdb
Sorbitol	sorbitol.pdb	Sorbitol (SOR) which is generated via conversion through OpenBabel and ‘molfile2params’ code
Old Protein	proall.pdb	Protein-allitol complex which is the protein.pdb file but modified to be a monomer
New Protein	prosor.pdb	Protein-sorbitol complex which is essentially proall.pdb plus sorbitol.pdb minus allitol.pdb; the sorbitol molecule is docked in the binding pocket of the protein aligned to the carbon backbone of the allitol molecule before deleting allitol
New Protein after MinMover	minprosor.pdb	Protein-sorbitol complex after being run through the MinMover code
Mutated Protein	finprosor.pdb	Protein-sorbitol complex with selective mutation of 5 residues
New Protein after FastRelax	fastprosor.pdb	Protein-sorbitol complex after being run through the FastRelax code

2.2. Docking ligand to protein in PyMOL

For docking, we first opened protein.pdb in PyMOL and then imported sorbitol.pdb by dragging the file into the open PyMOL window (PyMOL 2021). Then we utilized a combination of the Wizard Pair Fitting and Wizard Sculpting tools in PyMOL to align the carbon backbone of the sorbitol molecule from the sorbitol.pdb file to the carbon backbone of the allitol molecule from the .pdb file (Fig. 3). The Wizard Pair Fitting tool allowed for movement and orientation of the sorbitol molecule while the Wizard Sculpting tool allowed for rotation of bonds to align the hydroxyl groups of sorbitol to those of allitol. This was an iterative process as sometimes using the Wizard Sculpting tool would cause unwanted movement of the sorbitol molecule, so the Wizard Pair Fitting tool was used again to re-align the carbon backbone, and once realigned, identified additional need for the Wizard Sculpting tool. Once the alignment of both the carbon backbone and hydroxyl groups (all but the hydroxyl group of the third carbon, as that differs in stereochemistry) was accomplished, the atoms of the allitol molecule were deleted from the complex. The subsequent protein-sorbitol complex was saved as a new molecule and ‘.pdb’ file.

Figure 3. Ligands in the Protein Binding Pocket. A-C. The ABC transporter SBP (in green) binds the ligand allitol (in red) in its binding pocket. The ligand sorbitol (in blue) can be docked in the same binding pocket aligned with the six-carbon backbone of allitol. D. The ligand sorbitol (solid) visualized inside the binding pocket of the protein (translucent mesh) with atoms specified by color (red = Oxygen, blue = Nitrogen, green = Carbon, white = Hydrogen).

2.3. Minimizing protein-ligand complex

For minimization, we used the MinMover code to find a local energy minimum of the new protein and the FastRelax code to find a structure with lower energy than the new protein. The results of each of these minimization methods are discussed in the Results section.

2.4. Mutating protein-ligand complex

Given that sorbitol fits in the binding pocket of the protein where allitol once was, we hypothesize that the amino acid side chains stabilizing bound allitol would similarly stabilize much of sorbitol; however, the difference in stereochemistry of the third carbon hydroxyl group between the two ligands suggest that modification to the protein residues in binding pocket would better stabilize sorbitol. To determine which residues to change, we performed an iterative experiment. We first used PyMOL to identify all residues of the protein close to (within 4 Angstroms of) the bound ligand sorbitol. The next step of our experimental method for mutating the new protein was to use the PackMover Python code to change all of these residues to ones better suited for residue-residue or residue-ligand interactions that could further stabilize sorbitol in the binding pocket. As we wanted more control over the design, we also generated a protein with only specific amino acids in the binding pocket that was mutated. We did this by running code to change the ‘resfiles’ values of these amino acids from NATRO (no mutation, no repacking) to ALLAA (all amino acids, all repacking) to subsequently change the amino acids. This described method generated a protein with all mutated residues in the binding pocket and selectively mutated residues in the binding pocket.

3.1. Selective mutation experiment

While in the binding pocket of the ABC transporter SBP, sorbitol has direct polar interactions with five residues in the binding pocket. There are a total of seven bonds created between the ligand and the five amino acid side chains. The details of these interactions are detailed in Table 2. The amino acids in the binding pocket of the ABC transporter SBP also include five side chains that appear to have the potential to participate in polar bonds, which were the target of the selective mutation experiment. Following the experiment, the polar bonds that were created among these residues are listed in Table 3.

Table 2

**Residues with Polar Bonds Before Mutation.** The five residues listed create a total of seven polar bonds with the ligand sorbitol when sorbitol is in the binding pocket of the protein prior to mutating any residues in the binding pocket of the protein.
	Before Mutation		After Mutation		After FastRelax
Residue Number	Residue	Number of Polar Bonds	Residue	Number of Polar Bonds	Residue	Number of Polar Bonds
61	HIS	1	HIS	1	HIS	1
89	ARG	1	ARG	1	ARG	2
187	ARG	3	ARG	3	ARG	2
268	ASP	1	ASP	1	ASP	0
290	GLN	1	GLN	1	GLN	1

Table 3

**Residues with Potential for Polar Bonds.** The five residues listed were within four Angstroms of the bound ligand sorbitol and did not create any polar bonds, though were oriented toward the ligand and appeared to have the potential to interact.
	Before Mutation		After Mutation		After FastRelax
Residue Number	Residue	Number of Polar Bonds	Residue	Number of Polar Bonds	Residue	Number of Polar Bonds
63	PHE	0	LYS	0	PHE	0
64	ASP	0	PRO	0	ASP	3
137	ASP	0	TYR	0	ASP	1
181	VAL	0	VAL	0	VAL	0
184	CYS	0	ILE	0	CYS	0

For the residues with polar bonds before mutation, once the mutation occurs the number of polar bonds does not change. Additionally, while after FastRelax the number of polar bonds decreases, it is imperative to note that there is an increase in other polar bonds formed among the residues that were identified as ones that had the potential for polar bonds, and thus an overall increase in the number of polar bonds formed. This result is visualized in Fig. 4. The change in overall protein structure is visualized in Fig. 5.

Figure 4. Sorbitol Polar Bonding in Protein Binding Pocket. Sorbitol (cyan) making polar bonds (yellow dashed line) with surrounding binding pocket residues. Highlighted atoms of sorbitol and residues are denoted by their color (Oxygen = red, Nitrogen = blue, Sulfur = yellow, Carbon = green).

Figure 5. Protein-Sorbitol Complex Structural Changes. A. Sorbitol (cyan) can be seen in the binding pocket of the new protein (green). B. Sorbitol (magenta) can be seen in the binding pocket of the new protein after MinMover (red). C. Sorbitol (magenta) can be seen in the binding pocket of the mutated protein (red). D. Sorbitol (magenta) can be seen in the binding pocket of the new protein after FastRelax (red).

3.2. Free energy changes associated with each complex

Different blocks of Python code were run at each step of the experiment to calculate energy scores for the new protein and subsequently generated structures. The changes in free energy are detailed in Table 4.

Table 4

**Energy Scores Resulting from Each Protein Structure.** Listed are the changes in free energy for each protein-sorbitol complex with corresponding descriptions of the complex and/or Python code run on the complex.
Complex	Energy Score	Details
New Protein	-140.67 (protein only: -171.51)	Protein-sorbitol complex
New Protein After RepeatMover	-143.10	Protein-sorbitol complex with iteration via RepeatMover code with variables $kT=1$and$n=20$
New Protein After MinMover	-214.97	Protein-sorbitol complex with backbone angles adjusted via MinMover code
Mutated Protein (All Binding Pocket Residues) After PackMover	-178.03	Mutated protein-sorbitol complex via PackMover (simulated annealing and Metropolis/Monte Carlo to find best residues and rotamers) for all 18 residues within four Angstroms of the binding pocket
Mutated Protein (Selected Binding Pocket Residues) After PackMover	-224.80 (protein only: -228.27)	Mutated protein-sorbitol complex via PackMover (simulated annealing and Metropolis/Monte Carlo to find best residues and rotamers) for a selected 5 residues within four Angstroms of the binding pocket
New Protein After FastRelax	-780.43	Protein-sorbitol complex with backbone angles adjusted and side chains repacked via FastRelax code

3.3. Thermodynamic energy cycle

Given the thermodynamic equation below, which estimates binding free energy

$${\varDelta G}_{bind}={G}_{C}-({G}_{P}+{G}_{L})$$

and

$$\varDelta {G}_{bind}^{*}={G}_{C*}-({G}_{P*}+{G}_{L})$$

we derive the following equation

$$\varDelta {\varDelta G}_{bind}=\varDelta {G}_{bind}^{*}-{\varDelta G}_{bind} ={G}_{C*}-{G}_{P*}-{G}_{C}+{G}_{P}$$

which results in a unitless number that describes the thermodynamic cycle of energy for our protein. Plugging in numbers, for our new protein and protein-sorbitol complex versus our selectively mutated protein and protein-sorbitol complex with five residues mutated, we find that

$$\varDelta {\varDelta G}_{bind}={G}_{C*}-{G}_{P*}-{G}_{C}+{G}_{P}$$

$$=\left(-224.80\right)-\left(-228.27\right)-\left(-140.67\right)+\left(-171.51\right)=-27.37$$

As $\varDelta {\varDelta G}_{bind}<0$, the mutated version of the complex is thermodynamically favorable, and thus we can conclude that the selective mutation allowed the protein to be better at binding the new ligand than the starting protein. It is important to note, however, that stabilizing the protein without the ligand in place such as with MinMover prior to mutation would not optimize for the protein when it is bound to the ligand. Thus, it is important that in the above calculation we used the energy score of the new protein prior to any minimization code (MinMover, Fast Relax, etc.) being run.

Based on the structure of sorbitol (six-carbon backbone with six hydroxyl groups), it is reasonable to assume that by changing non-charged side chains to charged side chains, the binding pocket can form new polar bonds with sorbitol and thus achieve greater stabilization (Ng and Henikoff, 2006). As the oxygen of the hydroxyl group has a partial negative charge and the hydrogen of the hydroxyl group has a partial positive charge, we can hypothetically predict a change for a particular binding pocket amino acid side chain given a different one may be more energetically favorable. Of the five selective changes summarized in Table 3, one residue that was hydrophobic changed to positive (PHE to LYS) which is a mutation that is consistent with what we should think would happen. Two residues that were negative changed to neutral (ASP to PRO, ASP to TYR), and the remaining mutation (CYS to ILE) perhaps occurred to better accommodate the other changes that were being made and further stabilize the structure, though indirectly. The last case (VAL to VAL) signified no mutation, which suggests that residue though close to the ligand does not participate in the stabilization as we had initially thought, or perhaps at all. Overall, the selective mutations were consistent with our predictions; however, importantly, the lack of new polar bonds in the mutated protein and the gain of polar bonds in the FastRelax protein suggest that while they are both effective strategies to decreasing free energy, mutating proteins is less effective than rotational conformation. The results of the free energy changes in Table 4 corroborated the assumption that each strategy yielded protein-sorbitol complexes that were more energetically favorable than the original.

The mutations and conformations discussed above may be beneficial as they stabilize the binding of the protein with sorbitol allowing for a strong complex between the ATP transporter SBP and its ligand. This selectively mutated protein isolated from Agrobacterium vitis, given a stronger binding affinity for sorbitol than allitol, could be used as a molecular sponge to preferentially bind and remove sorbitol from tissue, and thus potentially be utilized as a treatment for sorbitol dehydrogenase deficiency. Future development of this biologic and subsequent in vitro and in vivo experimentation can be utilized to study its efficacy.

Competing interests

The authors have no conflicts of interest to report.

Acknowledgements

We would like to thank the Massachusetts Institute of Technology teaching staff for 20.420 including Professor Alan Jasanoff, Professor Ernest Fraenkel, Christine Davis, Abigail McShane, Owen Porth for help during Office Hours and Python code support over Piazza.

Public Repository Link:

https://github.com/ShonitNS/Sorbitol

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Designing Molecules to Treat Neurologic and Ophthalmologic Diseases Caused by Sorbitol Excess: Engineering the Agrobacterium Vitis Protein

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Version 1

Abstract

Figures

1. Introduction

2. Methods

2.1. Generating files for use in PyMOL

2.2. Docking ligand to protein in PyMOL

2.3. Minimizing protein-ligand complex

2.4. Mutating protein-ligand complex

3. Results

3.1. Selective mutation experiment

3.2. Free energy changes associated with each complex

3.3. Thermodynamic energy cycle

4. Conclusion

Declarations

References

Supplementary Files

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