Mutation-Structure-Function Relationship Based Integrated Strategy Reveals the Potential Impact of Deleterious Missense Mutations in Autophagy Related Proteins on Hepatocellular Carcinoma (HCC): A Comprehensive Informatics Approach

Autophagy, an evolutionary conserved multifaceted lysosome-mediated bulk degradation system, plays a vital role in liver pathologies including hepatocellular carcinoma (HCC). Post-translational modifications (PTMs) and genetic variations in autophagy components have emerged as significant determinants of autophagy related proteins. Identification of a comprehensive spectrum of genetic variations and PTMs of autophagy related proteins and their impact at molecular level will greatly expand our understanding of autophagy based regulation. In this study, we attempted to identify high risk missense mutations that are highly damaging to the structure as well as function of autophagy related proteins including LC3A, LC3B, BECN1 and SCD1. Number of putative structural and functional residues, including several sites that undergo PTMs were also identified. In total, 16 high-risk SNPs in LC3A, 18 in LC3B, 40 in BECN1 and 43 in SCD1 were prioritized. Out of these, 2 in LC3A (K49A, K51A), 1 in LC3B (S92C), 6 in BECN1 (S113R, R292C, R292H, Y338C, S346Y, Y352H) and 6 in SCD1 (Y41C, Y55D, R131W, R135Q, R135W, Y151C) coincide with potential PTM sites. Our integrated analysis found LC3B Y113C, BECN1 I403T, SCD1 R126S and SCD1 Y218C as highly deleterious HCC-associated mutations. This study is the first extensive in silico mutational analysis of the LC3A, LC3B, BECN1 and SCD1 proteins. We hope that the observed results will be a valuable resource for in-depth mechanistic insight into future investigations of pathological missense SNPs using an integrated computational platform.

The M residue is smaller and more hydrophobic than the WT residue. The WT residue was +vely charged, the M residue is neutral. The WT residue forms a salt bridge with: Asp at position 48. The difference in charge will disturb the ionic interactions.

Ala
The WT residue forms a hydrogen bond with Asp at position 4 and Thr at position 50. The size difference between WT and M residue makes that the new residue is not in the correct position to make the same hydrogen bond as the original WT residue did. The difference in hydrophobicity will affect hydrogen bond formation. The WT residue forms a salt bridge with Asp at position 4, Glu at position 19 and Glu at position 36. The difference in charge will disturb the ionic interactions.

Ala
The M residue is smaller than the WT residue. This will cause a probable loss of external interactions.

Leu
The M residue is bigger than the WT residue. The WT residue is a Pro. Pro is known to be very rigid and therefore induce a special backbone conformation which might be required at this position. The mutation can disturb this special conformation. The residue is located on the surface of the protein; mutation of this residue can disturb interactions with other molecules or other parts of the protein.

Ala
The M residue is smaller than the WT residue. The WT residue is a Pro. Pros are known to be very rigid and therefore induce a special backbone conformation which might be required at this position. The mutation can disturb this special conformation.

Cys
The M residue is smaller and more hydrophobic than the WT residue. The WT residue was +vely charged, the M residue is neutral. The WT residue forms a salt bridge with: Asp at position 48. The difference in charge will disturb the ionic interactions.

Arg
The WT residue was neutral, the M residue is +vely charged. The mutation introduces a charge at this position; this can cause repulsion between the M residue and neighboring residues. The residue is located on the surface of the protein; mutation of this residue can disturb interactions with other molecules or other parts of the protein.

Ala
The residue is buried in the core of a domain. The differences between the WT and M residue might disturb the core structure of this domain. The mutation will cause an empty space in the core of the protein.

Ser
The mutation will cause an empty space and loss of hydrophobic interactions in the core of the protein. The WT residue is predicted to be located in its preferred secondary structure, a β-strand. The M residue prefers to be in another secondary structure; therefore the local conformation will be slightly destabilized.

Lys
The WT residue forms a hydrogen bond with Glu at position 102. The size difference between WT and M residue makes that the new residue is not in the correct position to make the same hydrogen bond as the original WT residue did. The WT residue is involved in a multimer contact. The mutation introduces a bigger residue at this position; this can disturb the multimeric interactions. M residue will cause slight destabilization in local conformation. The mutation introduces a charge at this position; this can cause repulsion between the M residue and neighboring residues. The residue is located on the surface of the protein; mutation of this residue can disturb interactions with other molecules or other parts of the protein.

Pro
The WT residue is predicted (by the PHDsec DAS-server) to be located in an α-helix.
Pro disrupts an α-helix when not located at one of the first 3 positions of that helix. In case of the mutation at hand, the helix will be disturbed and this can have severe effects on the structure of the protein.

Asn
The WT residue forms a hydrogen bond with Asp at position 106, and a salt bridge with Lys at position 8 and Arg at position 16. The difference in charge will disturb the ionic interaction made by the original, WT residue.

Lys
The WT residue forms a salt bridge with Arg at position 16. The difference in charge will disturb the ionic interaction made by the original, WT residue. The residue is located on the surface of the protein; mutation of this residue can disturb interactions with other molecules or other parts of the protein.

Ala
The WT residue is a Gly, the most flexible of all residues. This flexibility might be necessary for the protein's function. Mutation of this Gly can abolish this function. Residues in the vicinity of the mutated residue are annotated in the UniProt database as being a binding site. The mutation could affect the local structure and as a consequence affect this binding site. Table S2. Evaluating the effect of the amino acid mutations in LC3B by HOPE sever. The distinct pattern and possible consequences of native and Mutant amino acid on protein structure based on size, charge, and hydrophobicity have been shown.

WT Amino Acid Position M amino Acid Characteristics and Possible Consequences
Arg 11

Cys
The M residue is smaller and more hydrophobic than the WT residue. The WT residue was +vely charged, the M residue is neutral. The WT residue forms a hydrogen bond and salt bridge with Asp at position 19. The difference in hydrophobicity will affect hydrogen bond formation. The difference in charge will disturb the ionic interaction made by the original, WT residue.

Gln
The M residue is smaller than the WT residue. The WT residue was +vely charged, the M residue is neutral. The WT residue forms a salt bridge with Glu at position 100. The difference in charge will disturb the ionic interaction made by the original, WT residue.

Leu
The M residue is bigger than the WT residue. The WT residue is a Pro. Pro is known to be very rigid and therefore induce a special backbone conformation which might be required at this position. The mutation can disturb this special conformation. The residue is buried in the core of a domain. The differences between the WT and M residue might disturb the core structure of this domain.

Ala
The M residue is smaller than the WT residue. The mutation will cause an empty space in the core of the protein.

Gln
The M residue is smaller and more hydrophobic than the WT residue. The WT residue was +vely charged, the M residue is neutral. The WT residue forms a hydrogen bond with Leu at position 44 and a salt bridge with Glu at position 41. The difference in charge will disturb the ionic interaction made by the original, WT residue.

Cys
The M residue is bigger and more hydrophobic than the WT residue. The WT residue is a Pro. The WT residue is a Gly, the most flexible of all residues. This flexibility might be necessary for the protein's function. Mutation of this Gly can abolish this function. The residue is located on the surface of the protein; mutation of this residue can disturb interactions with other molecules or other parts of the protein. The torsion angles for this residue are unusual. Only Gly is flexible enough to make these torsion angles, mutation into another residue will force the local backbone into an incorrect conformation and will disturb the local structure.

Ala
The M residue is smaller and more hydrophobic than the WT residue. The WT residue was +vely charged, the M residue is neutral. The charge of the WT residue is lost by this mutation. This can cause loss of interactions with other molecules.

Ala
The M residue is smaller and more hydrophobic than the WT residue. The WT residue was +vely charged, the M residue is neutral. The charge of the WT residue is lost by this mutation. This can cause loss of interactions with other molecules. Any hydrogen bond that could be made by the WT residue to other monomers will be lost now and affect the multimeric contacts.

His
The M residue is smaller than the WT residue. The WT residue was +vely charged, the M residue is neutral. The charge of the WT residue is lost by this mutation. This can cause loss of interactions with other molecules. Any hydrogen bond that could be made by the WT residue to other monomers will be lost now and affect the multimeric contacts.

Asp
The M residue is bigger and less hydrophobic than the WT residue. The WT residue was neutral, the M residue is -vely charged. The mutation introduces a charge at this position; this can cause repulsion between the M residue and neighboring residues.

Ser
The M residue is smaller and more hydrophobic than the WT residue. The WT residue was +vely charged, the M residue is neutral. The charge of the WT residue is lost by this mutation. The residue is buried in the core of a domain. The differences between the WT and M residue might disturb the core structure of this domain.

Cys
The M residue is more hydrophobic than the WT residue. The WT residue forms a hydrogen bond with: Ser at position 90. The difference in hydrophobicity will affect hydrogen bond formation.

Asn
The WT residue was -vely charged, the M residue is neutral. The WT residue forms a hydrogen bond with Lys at position 8 and Asp at position 106. The WT residue forms a salt bridge with Lys at position 8 and Arg at position 16. The difference in charge will disturb the ionic interaction made by the original, WT residue.

Gly
The M residue is smaller and more hydrophobic than the WT residue. The WT residue was -vely charged, the M residue is neutral. The mutation introduces a Gly at this position. Glys are very flexible and can disturb the required rigidity of the protein at this position. The WT residue forms a hydrogen bond with Arg at position 16, Phe at position 108 and Tyr at position 110. The difference in hydrophobicity will affect hydrogen bond formation. The WT residue forms a salt bridge with Lys at position 8 and Arg at position 16.

Cys
The M residue is smaller than the WT residue. The M residue is more hydrophobic than the WT residue. This will cause a possible loss of external interactions. The mutation will cause loss of hydrogen bonds in the core of the protein and as a result disturb correct folding.

Ser
The M residue is smaller than the WT residue. The mutation will cause an empty space in the core of the protein.

Val
The M residue is smaller and more hydrophobic than the WT residue. The WT residue was -vely charged, the M residue is neutral. The WT residue forms a salt bridge with Lys at position 42. The difference in charge will disturb the ionic interaction made by the original, WT residue. The mutation converts the WT residue in a residue that does not prefer α-helices as secondary structure.

Ala
The WT residue is a Gly, the most flexible of all residues. This flexibility might be necessary for the protein's function. Mutation of this Gly can abolish this function. The WT and M amino acids differ in size. The M residue is bigger than the WT residue. The residue is located on the surface of the protein; mutation of this residue can disturb interactions with other molecules or other parts of the protein. The torsion angles for this residue are unusual. Only Gly is flexible enough to make these torsion angles, mutation into another residue will force the local backbone into an incorrect conformation and will disturb the local structure. Table S3. Evaluating the effect of the amino acid mutations in BECN1 by HOPE sever. The distinct pattern and possible consequences of native and Mutant amino acid on protein structure based on size, charge, and hydrophobicity have been shown.

WT Amino Acid Position M Amino Acid Characteristics and Possible Consequences
Val 14

Glu
The M residue is bigger and less hydrophobic than the WT residue. The WT residue is predicted (by the PHDsec DAS-server) to be located in its preferred secondary structure, a β-strand. The M residue prefers to be in another secondary structure; therefore the local conformation will be slightly destabilized. The mutation introduces a charge; this can cause repulsion of ligands or other residues with the same charge.

Pro
The M residue is bigger than the WT residue. This might lead to bumps.

Cys
The mutation introduces a more hydrophobic residue at this position. This can result in loss of hydrogen bonds and/or disturb correct folding. The charge of the WT residue will be lost, this can cause loss of interactions with other molecules or residues.

Arg
In both the PDB-file and in the PISA-assembly, this residue was found to be involved in a multimer contact. The mutation introduces a bigger residue at this position, this can disturb the multimeric interactions. The mutation introduces a less hydrophobic residue. Sometimes, hydrophobicity is important for multimerisation and therefore this mutation could affect the multimer contacts. The residue is located on the surface of the protein; mutation of this residue can disturb interactions with other molecules or other parts of the protein. The mutation introduces a charge at this position, this can cause repulsion between the M residue and neighboring residues.

Arg
The WT residue forms a hydrogen bond with: Val at position 14, Val at position 14.
The size difference between WT and M residue makes that the new residue is not in the correct position to make the same hydrogen bond as the original WT residue did. The difference in hydrophobicity will affect hydrogen bond formation. In both the PDB-file and in the PISA-assembly, this residue was found to be involved in a multimer contact. The mutation introduces a bigger residue at this position, this can disturb the multimeric interactions. The mutation introduces a less hydrophobic residue. Sometimes, hydrophobicity is important for multimerisation and therefore this mutation could affect the multimer contacts.

Ala
In both the PDB-file and in the PISA-assembly, this residue was found to be involved in a multimer contact. The mutation introduces a smaller residue at this position. The new residue might be too small to make multimer contacts.

Leu
The WT residue forms a hydrogen bond with: Asp at position 124. The size difference between WT and M residue makes that the new residue is not in the correct position to make the same hydrogen bond as the original WT residue did. The difference in hydrophobicity will affect hydrogen bond formation. In both the PDB-file and in the PISA-assembly, this residue was found to be involved in a multimer contact. The mutation introduces a bigger residue at this position; this can disturb the multimeric interactions. A more hydrophobic residue is introduced here. Any hydrogen bond that could be made by the WT residue to other monomers will be lost now and affect the multimeric contacts.

Ala
The charge of the WT residue will be lost; this can cause loss of interactions with other molecules or residues. The mutation introduces a more hydrophobic residue at this position. This can result in loss of hydrogen bonds and/or disturb correct folding.

Phe
The WT residue is predicted (by the PHDsec DAS-server) to be located in an αhelix. The mutation converts the WT residue in a residue that does not prefer αhelices as secondary structure. The residue is located on the surface of the protein, mutation of this residue can disturb interactions with other molecules or other parts of the protein.

Ala
In the PDB file used for this analysis the mutated residue is involved in a multimer contact. The mutation introduces a smaller residue at this position. The new residue might be too small to make multimer contacts. A more hydrophobic residue is introduced here. Any hydrogen bond that could be made by the WT residue to other monomers will be lost now and affect the multimeric contacts.

Val
In the PDB file used for this analysis the mutated residue is involved in a multimer contact. The mutation introduces a smaller residue at this position. The new residue might be too small to make multimer contacts. A more hydrophobic residue is introduced here. Any hydrogen bond that could be made by the WT residue to other monomers will be lost now and affect the multimeric contacts.

Cys
The WT residue forms a salt bridge with: Glu at position 167. The difference in charge will disturb the ionic interaction made by the original, WT residue.

Pro
The WT residue is predicted (by the PHDsec DAS-server) to be located in an αhelix. Pro disrupts an α-helix when not located at one of the first 3 positions of that helix. In case of the mutation at hand, the helix will be disturbed and this can have severe effects on the structure of the protein.

Pro
The WT residue is predicted (by the PHDsec DAS-server) to be located in an αhelix. Pro disrupts an α-helix when not located at one of the first 3 positions of that helix. In case of the mutation at hand, the helix will be disturbed and this can have severe effects on the structure of the protein.

Cys
The WT residue forms a salt bridge with: Glu at position 202. The difference in charge will disturb the ionic interaction made by the original, WT residue. In both the PDB-file and in the PISA-assembly, this residue was found to be involved in a multimer contact. The mutation introduces a smaller residue at this position. The new residue might be too small to make multimer contacts. A more hydrophobic residue is introduced here. Any hydrogen bond that could be made by the WT residue to other monomers will be lost now and affect the multimeric contacts.

Pro
The WT residue is predicted (by the PHDsec DAS-server) to be located in an αhelix. Pro disrupts an α-helix when not located at one of the first 3 positions of that helix. In case of the mutation at hand, the helix will be disturbed and this can have severe effects on the structure of the protein.
According to the PISA-database, the mutated residue is involved in a multimer contact. The PISA-database contains protein assemblies that are highly likely to be biologically relevant. The mutation introduces a smaller residue at this position.
The new residue might be too small to make multimer contacts. A more hydrophobic residue is introduced here. Any hydrogen bond that could be made by the WT residue to other monomers will be lost now and affect the multimeric contacts.

Cys
The WT residue forms a salt bridge with: Asp at position 262. The difference in charge will disturb the ionic interaction made by the original, WT residue. The charge of the WT residue is lost by this mutation. This can cause loss of interactions with other molecules.

Ser
The mutation introduces a more hydrophobic residue at this position. This can result in loss of hydrogen bonds and/or disturb correct folding.

Ser
The residue is buried in the core of a domain. The differences between the WT and M residue might disturb the core structure of this domain. The mutation will cause an empty space in the core of the protein.

His
The WT residue forms a hydrogen bond with: Glu at position 422, Val at position 298. The size difference between WT and M residue makes that the new residue is not in the correct position to make the same hydrogen bond as the original WT residue did. The WT residue forms a salt bridge with: Glu at position 422. The difference in charge will disturb the ionic interaction made by the original, WT residue.

Cys
The WT residue forms a hydrogen bond with: Glu at position 422, Val at position 298. The size difference between WT and M residue makes that the new residue is not in the correct position to make the same hydrogen bond as the original WT residue did. The difference in hydrophobicity will affect hydrogen bond formation. The WT residue forms a salt bridge with: Glu at position 422. The difference in charge will disturb the ionic interaction made by the original, WT residue.

Ala
The residue is buried in the core of a domain. The differences between the WT and M residue might disturb the core structure of this domain. The mutation will cause an empty space in the core of the protein.

Met
The M residue is bigger than the WT residue. The WT residue was buried in the core of the protein. The M residue is bigger and probably will not fit.

Lys
The WT residue forms a hydrogen bond with: Arg at position 289, Arg at position 289. The size difference between WT and M residue makes that the new residue is not in the correct position to make the same hydrogen bond as the original WT residue did. The WT residue forms a salt bridge with: Arg at position 289. The difference in charge will disturb the ionic interaction made by the original, WT residue. The residue is located on the surface of the protein, mutation of this residue can disturb interactions with other molecules or other parts of the protein.

His
The mutation converts the WT residue in a residue that does not prefer α-helices as secondary structure. The residue is buried in the core of a domain. The differences between the WT and M residue might disturb the core structure of this domain.

Pro
The M residue is smaller than the WT residue. This will cause a possible loss of external interactions.

Cys
The M residue is smaller than the WT residue. The M residue is more hydrophobic than the WT residue. This will cause a possible loss of external interactions.

Tyr
The residue is located on the surface of the protein; mutation of this residue can disturb interactions with other molecules or other parts of the protein. The local conformation will be slightly destabilized.

Leu
The residue is buried in the core of a domain. The differences between the WT and M residue might disturb the core structure of this domain. The WT residue is a Pro.
Pros are known to be very rigid and therefore induce a special backbone conformation which might be required at this position. The mutation can disturb this special conformation.

Ala
The M residue is smaller and more hydrophobic than the WT residue. The WT residue is predicted (by the PHDsec DAS-server) to be located in its preferred secondary structure, a β-strand. The M residue prefers to be in another secondary structure; therefore the local conformation will be slightly destabilized.

His
The M residue is smaller and more hydrophobic than the WT residue. The WT residue is predicted (by the PHDsec DAS-server) to be located in its preferred secondary structure, a β-strand. The M residue prefers to be in another secondary structure; therefore the local conformation will be slightly destabilized.

Tyr
The WT residue was buried in the core of the protein. The M residue is bigger and probably will not fit. The hydrophobicity of the WT and M residue differs. The mutation will cause loss of hydrophobic interactions in the core of the protein.

Arg
The WT residue forms a hydrogen bond with Gly at position 355. The size difference between WT and M residue makes that the new residue is not in the correct position to make the same hydrogen bond as the original WT residue did. The difference in hydrophobicity will affect hydrogen bond formation. The mutation introduces a charge at this position, this can cause repulsion between the M residue and neighboring residues.

Arg
The M residue introduces a charge in a buried residue which can lead to protein folding problems. The WT residue was buried in the core of the protein. The M residue is bigger and probably will not fit.

Ser
The residue is buried in the core of a domain. The differences between the WT and M residue might disturb the core structure of this domain. The M residue is smaller and less hydrophobic than the WT residue.

Thr
The M residue is smaller and less hydrophobic than the WT residue. The residue is buried in the core of a domain. The differences between the WT and M residue might disturb the core structure of this domain. The mutation will cause an empty space in the core of the protein.

Arg
The WT residue is a Gly, the most flexible of all residues. This flexibility might be necessary for the protein's function. Mutation of this Gly can abolish this function. The torsion angles for this residue are unusual. Only Gly is flexible enough to make these torsion angles, mutation into another residue will force the local backbone into an incorrect conformation and will disturb the local structure.

Ala
The WT residue forms a hydrogen bond with: Ile at position 415. The size difference between WT and M residue makes that the new residue is not in the correct position to make the same hydrogen bond as the original WT residue did. The mutation will cause an empty space in the core of the protein.

Cys
The WT residue forms a hydrogen bond with: Ile at position 415. The size difference between WT and M residue makes that the new residue is not in the correct position to make the same hydrogen bond as the original WT residue did. The mutation will cause an empty space in the core of the protein.

Val
The M residue is smaller than the WT residue. The mutation will cause an empty space in the core of the protein. The residue is buried in the core of a domain. The differences between the WT and M residue might disturb the core structure of this domain. Table S4. Evaluating the effect of the amino acid mutations in SCD1 by HOPE sever. The distinct pattern and possible consequences of native and Mutant amino acid on protein structure based on size, charge, and hydrophobicity have been shown.

WT Amino Acid Position M Amino Acid Characteristics and Possible Consequences
Tyr 41

Cys
The M residue is smaller and more hydrophobic than the WT residue. This will cause a possible loss of external interactions. The mutation introduces a more hydrophobic residue at this position. This can result in loss of hydrogen bonds and/or disturb correct folding.

Arg
The M residue is bigger and less hydrophobic than the WT residue. The WT residue was neutral; the M residue is +vely charged. The WT residue was buried in the core of the protein. The M residue is bigger and probably will not fit. The WT residue is a Pro.
Pros are known to be very rigid and therefore induce a special backbone conformation which might be required at this position. The mutation can disturb this special conformation. Hydrophobic interactions, either in the core of the protein or on the surface, will be lost.

Asp
The M residue is smaller and less hydrophobic than the WT residue. The WT residue was neutral, the M residue is -vely charged. The mutation introduces a charge at this position; this can cause repulsion between the M residue and neighboring residues.

Gly
The M residue is smaller and more hydrophobic than the WT residue. The WT residue was -vely charged, the M residue is neutral. The mutated residue is not in direct contact with a ligand; however, the mutation could affect the local stability which in turn could affect the ligand-contacts made by one of the neighboring residues. The mutation introduces a Gly at this position. Glys are very flexible and can disturb the required rigidity of the protein at this position.

Cys
The M residue is smaller and more hydrophobic than the WT residue. This will cause a possible loss of external interactions. The mutated residue is located in a domain that is important for the activity of the protein and in contact with another domain that is also important for the activity. The interaction between these domains could be disturbed by the mutation, which might affect the function of the protein.

Arg
The M residue is bigger and less hydrophobic than the WT residue. The WT residue was neutral; the M residue is +vely charged. The WT residue is a Gly, the most flexible of all residues. This flexibility might be necessary for the protein's function. Mutation of this Gly can abolish this function. The torsion angles for this residue are unusual. Only Gly is flexible enough to make these torsion angles, mutation into another residue will force the local backbone into an incorrect conformation and will disturb the local structure.

Ile
The M residue is bigger and more hydrophobic than the WT residue. The mutation will cause loss of hydrogen bonds in the core of the protein and as a result disturb correct folding. The WT residue was buried in the core of the protein. The M residue is bigger and probably will not fit.

Pro
The M residue is smaller than the WT residue. The mutated residue is located in a domain that is important for the activity of the protein and in contact with another domain that is also important for the activity. The interaction between these domains could be disturbed by the mutation, which might affect the function of the protein.

Glu
The M residue is bigger and less hydrophobic than the WT residue. The WT residue was neutral; the M residue is -vely charged. The WT residue is a Gly, the most flexible of all residues. This flexibility might be necessary for the protein's function. Mutation of this Gly can abolish this function. The WT residue was buried in the core of the protein. The M residue is bigger and probably will not fit.

Cys
The M residue is smaller and more hydrophobic than the WT residue. The WT residue was +vely charged, the M residue is neutral. The mutated residue is not in contact with a metal; however, one of the neighboring residues does make a metal-contact that might be affected by the mutation in its vicinity. The mutated residue is not in direct contact with a ligand; however, the mutation could affect the local stability which in turn could affect the ligand-contacts made by one of the neighboring residues. The WT residue forms a hydrogen bond with: Glu at position 295, Thr at position 321, and Asn at position 318. The WT residue forms a salt bridge with: Glu at position 295, Asp at position 307 and Asp at position 325. The mutation will cause loss of hydrogen bonds in the core of the protein and as a result disturb correct folding.

Pro
The M residue is smaller and more hydrophobic the WT residue. The size differences between the WT and M residue disturb the interaction with the metal-ion: "ZN". The WT residue forms a hydrogen bond with His at position 120, His at position 157, His at position 157 and His at position 301. The difference in hydrophobicity will affect hydrogen bond formation.

Cys
The M residue is smaller and more hydrophobic the WT residue. The WT residue was +vely charged; the M residue is neutral. The WT residue forms a hydrogen bond with Asp at position 204, Glu at position 208 and Asp at position 204. The difference in hydrophobicity will affect hydrogen bond formation. The WT residue forms a salt bridge with Asp at position 204 and Glu at position 208. The difference in charge will disturb the ionic interaction made by the original, WT residue.

Ser
The M residue is smaller the WT residue. The WT residue was +vely charged; the M residue is neutral. The WT residue forms a hydrogen bond with Asp at position 204, Glu at position 208 and Asp at position 204. The difference in hydrophobicity will affect hydrogen bond formation. The WT residue forms a salt bridge with Asp at position 204 and Glu at position 208. The difference in charge will disturb the ionic interaction made by the original, WT residue.

Trp
The M residue is bigger and more hydrophobic than the WT residue. The WT residue was +vely; the M residue is neutral. The residue is located on the surface of the protein; mutation of this residue can disturb interactions with other molecules or other parts of the protein.

Gln
The M residue is smaller than the WT residue. The WT residue was +vely charged; the M residue is neutral. The WT residue forms a salt bridge with Glu at position 208. The difference in charge will disturb the ionic interaction made by the original, WT residue.

Trp
The M residue is bigger and more hydrophobic than the WT residue. The WT residue was +vely; the M residue is neutral. The residue is located on the surface of the protein; mutation of this residue can disturb interactions with other molecules or other parts of the protein.

Thr
The M residue is smaller and less hydrophobic than the WT residue. The mutation might cause loss of hydrophobic interactions with other molecules on the surface of the protein.

Cys
The M residue is smaller and more hydrophobic than the WT residue. The WT residue forms a hydrogen bond with Glu at position 206. The mutation will cause loss of hydrogen bonds in the core of the protein and as a result disturb correct folding.

Cys
The M residue is smaller and more hydrophobic the WT residue. The WT residue was +vely charged; the M residue is neutral. The WT residue forms a salt bridge with Glu at position 70. The difference in charge will disturb the ionic interaction made by the original, WT residue. The difference in hydrophobicity will affect hydrogen bond formation.

Tyr
The M residue is bigger and more hydrophobic the WT residue. The WT residue forms a hydrogen bond with Ala at position 192. The residue is buried in the core of a domain. The differences between the WT and M residue might disturb the core structure of this domain. The mutation will cause loss of hydrogen bonds in the core of the protein and as a result disturb correct folding.

213
Leu The M residue is smaller than the WT residue. This will cause a possible loss of external interactions.

Arg
The M residue is bigger than the WT residue. The WT residue was neutral; the M residue is +vely. The mutated residue is not in direct contact with a ligand; however, the mutation could affect the local stability which in turn could affect the ligandcontacts made by one of the neighboring residues. The WT residue forms a hydrogen bond with Val at position 150. The size difference between WT and M residue makes that the new residue is not in the correct position to make the same hydrogen bond as the original WT residue did. The WT residue was buried in the core of the protein.
The M residue is bigger and probably will not fit.

His
The M residue is smaller and less hydrophobic than the WT residue. The WT residue forms a hydrogen bond with Gln at position 147. The difference in hydrophobicity will affect hydrogen bond formation.

Cys
The M residue is smaller and more hydrophobic than the WT residue. The WT residue forms a hydrogen bond with Gln at position 147. The difference in hydrophobicity will affect hydrogen bond formation.

Thr
The M residue is smaller and less hydrophobic than the WT residue. The mutation might cause loss of hydrophobic interactions with other molecules on the surface of the protein.

Met
The M residue is bigger and more hydrophobic than the WT residue. The WT residue forms a hydrogen bond with Phe at position 227 and Thr at position 250. The mutation will cause loss of hydrogen bonds in the core of the protein and as a result disturb correct folding.

Gly
The M residue is smaller and more hydrophobic than the WT residue. The mutation introduces a Gly at this position. Glys are very flexible and can disturb the required rigidity of the protein at this position.

Arg
The M residue is smaller and more hydrophobic than the WT residue. The WT residue was neutral, the M residue is +vely charged. The mutation is located within a topological domain. The mutation might disrupt this topological domain.

Asp
The M residue is bigger and less hydrophobic than the WT residue. The WT residue was neutral, the M residue is -vely charged. The residue is buried in the core of a domain. The differences between the WT and M residue might disturb the core structure of this domain.

Arg
The M residue is bigger and less hydrophobic than the WT residue. The WT residue was neutral, the M residue is +vely charged. The WT residue is a Gly, the most flexible of all residues. This flexibility might be necessary for the protein's function. Mutation of this Gly can abolish this function. The residue is located on the surface of the protein; mutation of this residue can disturb interactions with other molecules or other parts of the protein. The torsion angles for this residue are unusual. Only Gly is flexible enough to make these torsion angles, mutation into another residue will force the local backbone into an incorrect conformation and will disturb the local structure.

Cys
The M residue is smaller and more hydrophobic than the WT residue. The WT residue is annotated to be located in its preferred secondary structure, a β-strand. The M residue prefers to be in another secondary structure; therefore the local conformation will be slightly destabilized.

Ile
The M residue is smaller and more hydrophobic than the WT residue.

Trp
The M residue is bigger and more hydrophobic than the WT residue. The WT residue was +vely charged, the M residue is neutral. The mutated residue is not in contact with a metal, however, one of the neighboring residues does make a metal-contact that might be affected by the mutation in its vicinity. The WT residue forms a salt bridge with Glu at position 165. The difference in charge will disturb the ionic interaction made by the original, WT residue. The residue is located on the surface of the protein; mutation of this residue can disturb interactions with other molecules or other parts of the protein.

Pro
The M residue is smaller than the WT residue. The WT residue is located in a region annotated to form an α-helix. Pro disrupts an α-helix when not located at one of the first 3 positions of that helix. In case of the mutation at hand, the helix will be disturbed and this can have severe effects on the structure of the protein.

Val
The M residue is smaller than the WT residue. This will cause a possible loss of external interactions.

Gly
The M residue is smaller and less hydrophobic than the WT residue. The mutation introduces a Gly at this position. Glys are very flexible and can disturb the required rigidity of the protein at this position.

326
Phe The M residue is bigger than the WT residue. The residue is located on the surface of the protein; mutation of this residue can disturb interactions with other molecules or other parts of the protein.

Val
The M residue is smaller than the WT residue. The WT residue is located in a region annotated to form an α-helix. The mutation converts the WT residue in a residue that does not prefer α-helices as secondary structure.

Ser
The M residue is bigger than the WT residue. The WT residue is a Gly, the most flexible of all residues. This flexibility might be necessary for the protein's function. Mutation of this Gly can abolish this function. The residue is located on the surface of the protein; mutation of this residue can disturb interactions with other molecules or other parts of the protein. The torsion angles for this residue are unusual. Only Gly is flexible enough to make these torsion angles, mutation into another residue will force the local backbone into an incorrect conformation and will disturb the local structure.

Thr
The M residue is bigger and more hydrophobic than the WT residue. The residue is buried in the core of a domain. The differences between the WT and M residue might disturb the core structure of this domain. The WT residue was buried in the core of the protein. The M residue is bigger and probably will not fit.

Gln
The M residue is smaller than the WT residue. The WT residue was +vely charged, the M residue is neutral. The WT residue forms a hydrogen bond with Asp at position 325. The size difference between WT and M residue makes that the new residue is not in the correct position to make the same hydrogen bond as the original WT residue did. The WT residue forms a salt bridge with Asp at position 325 and Asp at position 335. The charge of the WT residue is lost by this mutation. This can cause loss of interactions with other molecules.