Choline Acetyltransferase Mutations Causing Congenital Myasthenic Syndrome: Molecular Findings and Genotype-Phenotype Correlations

Abstract

Choline acetyltransferase catalyzes the synthesis of acetylcholine at cholinergic nerves. Mutations in human CHAT cause a congenital myasthenic syndrome due to impaired synthesis of ACh; this severe variant of the disease is frequently associated with unexpected episodes of potentially fatal apnea. The severity of this condition varies remarkably, and the molecular factors determining this variability are poorly understood. Furthermore, genotype-phenotype correlations have been difficult to establish in patients with biallelic mutations. We analyzed the protein expression of phosphorylated ChAT of seven CHAT mutations, p.Val136Met, p.Arg207His, p.Arg186Trp, p.Val194Leu, p.Pro211Ala, p.Arg566Cys, and p.Ser694Cys, in HEK-293 cells to phosphorylated ChAT, determined their enzyme kinetics and thermal stability, and examined their structural changes. Three mutations, p.Arg207His, p.Arg186Trp, and p.Arg566Cys, are novel, and p.Val136Met and p.Arg207His are homozygous in three families and associated with severe disease. The characterization of mutants showed a decrease in the overall catalytic efficiency of ChAT; in particular, those located near the active-site tunnel produced the most seriously disruptive phenotypic effects. On the other hand, p.Val136Met, which is located far from both active and substrate-binding sites, produced the most drastic reduction of ChAT expression. Overall, CHAT mutations producing low enzyme expression and severe kinetic effects are associated with the most severe phenotypes.

Keywords: ChAT; enzyme kinetics; genotype-phenotype correlations; phosphorylation.

Fig 1

Human ChAT structure. The tertiary structure of human ChAT is divided into two domains: a substrate binding domain comprising residues 118–207 and 510–733, and a catalytic domain with residues 208–509, based on the structure PDB 2FY2. This structure is missing the first 118 residues from the NH₂ terminal domain, and the crystal coordinates start at residue 8. The analyzed tertiary structure corresponds to the 83-kDa ChAT isoform 2. The catalytic histidine, His442 (red), is located at β-sheet 8 at the active-site tunnel, which runs through the enzyme and is located at the domain interface. The structure is represented in ribbon diagram; the α-helices are in blue, the β-sheets in green and the coils in orange. The mutated residues in magenta are located as follows: p.Val136Met at coil 1, p.Arg186Trp at α-helix 3, p.Val194Leu at α-helix 4, p.Arg207His at coil 5, p.Pro211Ala at coil 5, p.Arg566Cys at β-sheet 12 and p.Ser694Cys at β-sheet 16. When the mutations are introduced, the Swiss-PdbViewer algorithm selects side chain rotamer from a backbone-dependent rotamer library, consequently introducing minimal steric violations and with the most favorable hydrogen-bond interactions.

Fig 2

Protein expression of phosphorylated ChAT. ChAT expression of wild type and mutants in HEK293 cells. a Overexpression of ChAT proteins on HEK cells transfected with wild-type and mutant cDNA. Crude cell lysates were immunoblotted with ChAT antibody. Western blot variations of transfection efficiency were corrected by monitoring the expression of β-actin in the immunoblots (data not shown). The housekeeping β-actin protein was also used as a control to verify that all the samples had the same amount of loaded protein. b Western blot shows the affinity purified phosphorylated ChAT protein from HEK293 cells. c Bar graph showing the quantification of ChAT protein shown on Western blots (*p < 0.05).

Fig 3

Kinetic maps and position of p.Arg207His mutation at the active-site tunnel. The structure displays only hydrogen-bond interactions with neighbor residues that are close to Arg207 residue, and the hydrogen-bonding interactions are shown with green dotted lines. Hydrogen atoms are in blue, oxygen atoms in red, and Arg207 backbone in yellow. a Arg207 side chain (magenta) interacts with Gly284 backbone, Ser359, Asp206 and Asp288 to donate five H-bonds. Also, Arg207 backbone forms three hydrogen-bonds between Asn205, Arg358, and Asn206. b The mutated residue was best accommodated by rotamer #2. The mutation changes from the most hydrophilic polar residue (174 Da) to another, less hydrophilic polar, but smaller, His207 residue (155 Da) and changes all five H-bonds, but it forms three new H-bonds between its imidazole ring and Glu360 and Ser359. c 3D kinetic graph for the wild-type enzyme. d 3D kinetic graph for the mutant enzyme. This mutation produces the highest reduction in enzyme activity: the enzyme is basically inactive. Rotamer #2 has a score of −5 and p= 25%.

Fig 4

Mutation p.Arg566Cys close to His442 and p.Arg186Trp disrupts enzyme structure. The structure displays only hydrogen-bond interactions with neighbor residues that are close to Arg566 or Arg186. a Arg566 interacts with Asp446 backbone, His442 (red), Ser443, Arg566 backbone, Ile559 and Glu564, forming H-bonds. b The mutated residue was best accommodated by rotamer #1. The mutated residue changes from a large hydrophilic positive charge to Cys566, a smaller uncharged hydrophobic residue (121 Da). All four hydrogen-bonds interacting with Arg566 side chain are modified, and a hydrogen-bond is formed between Cys566 and Asp446. c Arg186, a polar very hydrophilic residue, changes to a Trp186 hydrophobic larger residue (205 Da). The Arg186 side chain donates hydrogen-bonds with Trp193 backbone, Thr190, Leu182, Leu183 and Lys189. d The mutated residue was best accommodated by rotamer #2. The Trp186 mutation changes the H-bond with Trp193, but forms many clash interactions (pink dotted lines) between the phenolic ring of Trp186 and Lys630, Lys183 and Tyr193. e 3D kinetic graph for p.Arg186Trp mutant enzyme. The mutation reduces the K_cat by 95% and the K_m for both substrates. f 3D kinetic graph for p.Arg566Cys mutant enzyme shows a decrease of K_cat by 48% and an increase in K_m for both substrates. Cys566 has rotamer #1 with a score of −3 and p = 45%. For Trp186 is rotamer #2 with a score of −3 and p = 32%

Fig 5

Mutations p.Ser694Cys and p.Val194Leu affect substrate binding site. The structure shows interactions with residues that are close to Ser694 or Val194. a Ser694 forms H-bonds with Ser694 backbone, Gly679 amino group, Gly679 and Tyr670 (red) backbone. b The mutated residue was best accommodated by rotamer #3. The Ser694 (105 Da) with a small polar hydroxyl side chain changes to Cys694 (121 Da), which has a larger hydrophobic side chain. The mutation forms a new H-bond between the Cys694 side chain NH₂ group and the Pro672 amino group. c The p.Val194Leu mutation is close to the acetyl-CoA binding site at α-helix 4. Val194 forms three H-bonds, with Ile559, Tyr197 and Asn192. The Val194 aliphatic side chain changes to Leu194 with a larger aliphatic side chain. d The mutated residue was best accommodated by rotamer #3. The Leu194 mutation creates clash interactions (pink dotted lines) with the Thr193 phenolic ring and with Asn192. e This mutation produces an increase of K_m for choline by 1.4-fold, for AcCoA by 3.8-fold, and for K_cat by 2-fold. f The 3D kinetic graph shows a decrease of 48% for K_cat and a 2-fold increase in K_m for choline. Cys694 has rotamer #3 with score of −4 and p= 22%. For Leu194 is rotamer #3 with a score of −4 and p = 40%

Fig 6

Mutations p.Val136Met and p.Pro211Ala produce small structural changes. The structure shows hydrogen-bonding interactions with residues that are close to Val136 and Pro211. a p.Val136Met mutation changes from a smaller branched-chain residue (117 Da) with an aliphatic side chain to a larger hydrophobic residue (149 Da) with long alkyl side chain. b The mutated residue was best accommodated by rotamer #4. The mutation Met136, which is a sulfur-containing residue, creates a clash (pink dotted lines) between its sulfur atoms with the aliphatic residue Leu184 side chain. Val136 and Met136 side chains do not produce any hydrogen-bonding with neighbor residues. c Pro211 cyclic amino residue creates one hydrogen-bond with Asn213. Pro211 side chain does not produce any H-bonds. d The Ala211 residue is very close to His442. Ala211 also does not produce any hydrogen-bonds. This mutation has 59% and 56% reduction of catalytic efficiency and overall catalytic efficiency respectively. Met136 has rotamer #4 with a score of −1 and p=10%.