Mouse modeling and structural analysis of the p.G307S mutation in human cystathionine β-synthase (CBS) reveal effects on CBS activity but not stability

Abstract

Mutations in the cystathionine β-synthase (CBS) gene are the cause of classical homocystinuria, the most common inborn error in sulfur metabolism. The p.G307S mutation is the most frequent cause of CBS deficiency in Ireland, which has the highest prevalence of CBS deficiency in Europe. Individuals homozygous for this mutation tend to be severely affected and are pyridoxine nonresponsive, but the molecular basis for the strong effects of this mutation is unclear. Here, we characterized a transgenic mouse model lacking endogenous Cbs and expressing human p.G307S CBS protein from a zinc-inducible metallothionein promoter (Tg-G307S Cbs^-/-). Unlike mice expressing other mutant CBS alleles, the Tg-G307S transgene could not efficiently rescue neonatal lethality of Cbs^-/- in a C57BL/6J background. In a C3H/HeJ background, zinc-induced Tg-G307S Cbs^-/- mice expressed high levels of p.G307S CBS in the liver, and this protein variant forms multimers, similarly to mice expressing WT human CBS. However, the p.G307S enzyme had no detectable residual activity. Moreover, treating mice with proteasome inhibitors failed to significantly increase CBS-specific activity. These findings indicated that the G307S substitution likely affects catalytic function as opposed to causing a folding defect. Using molecular dynamics simulation techniques, we found that the G307S substitution likely impairs catalytic function by limiting the ability of the tyrosine at position 308 to assume the proper conformational state(s) required for the formation of the pyridoxal-cystathionine intermediate. These results indicate that the p.G307S CBS is stable but enzymatically inert and therefore unlikely to respond to chaperone-based therapy.

Keywords: bortezomib; cystathionine β-synthase; homocystinuria; inborn error of metabolism; methionine; missense mutation; mouse genetics; p.G307S; pyridoxal phosphate; pyridoxine; structural model; sulfur metabolism; transsulfuration pathway.

Figure 1.

G307S expression. A, immunoblot analysis of liver extracts from female mice from the indicated founder line on zinc-water. Blots were probed with a polyclonal serum that recognizes both human (hCBS) and mouse CBS (mCBS). A slight difference in migration is due to the presence of the hemagglutinin tag at the N terminus of hCBS. The same blot probed with actin is shown below. Lane C has extract from a control Tg-hCBS Cbs^+/− mouse. B, relative hCBS liver mRNA as determined by RT-quantitative PCR. Liver RNA was isolated from zinc-treated Tg-hCBS Cbs^−/− (n = 13) or Tg-G307S Cbs^−/− mice (n = 8). Error bar shows S.E. C, immunoblot analysis of p.G307S expression in Tg-G307S Cbs^−/− liver. Expression of transgenic CBS (hCBS) in zinc-treated male (M) and female (F) mice. Transgenic WT CBS (Tg-hCBS Cbs^−/−) extract is also shown. D, expression of transgenic CBS under native conditions.

Figure 2.

Comparison of Tg-G307S Cbs^−/− and Tg-G307S Cbs^+/− sex- and age-matched littermates. CBS genotype, age, and weight of each sibling pair are shown.

Figure 3.

Effect of proteostasis modulators on CBS. A, effect of bortezomib (B) at a dose of 0.49 mg/kg/day delivered constantly by subcutaneous pumps. Saline was used as vehicle (V). For each mouse genotype, treatment, tHcy, liver CBS activity, and liver CBS levels are shown. bd indicates that the activity was below our limit of detection (20 units). Note that samples in the two different blots with identical tHcy levels are the same. B, effect of oprozomib (O) at a dose of 40 mg/kg/day delivered in bolus by gavage. Carboxymethylcellulose (1% w/v in water) was used a vehicle (V).

Figure 4.

A, structural superposition of structures of the human CBS homodimer. PLP is shown as magenta sticks; heme is shown as red sticks, and the backbones of residues 307 and 308 adjacent to the active site are shown in green and yellow, respectively. B, close-up of the active site of CBS. Residues 304–310 are shown as sticks. PLP and its analogues are shown in magenta. Atoms are colored by element type (oxygen = red, nitrogen = blue, phosphorus = orange, and carbon = different colors for each residue). The most common backbone conformation for this segment places Tyr-308 into two different rotamers: gauche-minus in yellow and trans in cyan. A hydrogen bond (black dashed line) exists between the hydroxyl of Tyr-308 and the hydroxyl attached to the γ-carbon (OG) of serine-PLP in PDB entry 3PC4. An alternative backbone conformation of these residues from PDB entry 4PCU chain B is shown as gray sticks; this conformation places Tyr-308 in a different position from the other structures.

Figure 5.

Modeling of the external aldimine of cystathione in CBS enzyme. A, Tyr-308 in the gauche-minus conformation (yellow sticks) occupies volume required for the homocysteine moiety in the E-Cyst model. The empty volume in the absence of the external aldimine of cystathione is shown in a gray wire frame. The E-Cyst ligand does not fit into this volume when Tyr-308 is in the gauche-minus position. B, E-Cyst ligand fits into this volume when Tyr-308 is in the trans position (cyan sticks).

Figure 6.

Fluctuations of the main chain in molecular dynamics simulations along the sequence of CBS. Multiple structural alignments of 2,000 frames of chains A and B from each simulation were performed separately with the program Theseus. The values of RMSF represent root-mean-square deviations of C-α atoms from the mean structure calculated by Theseus.

Figure 7.

A, analysis of molecular dynamics simulations of the human CBS homodimer of the enzyme domain. Ramachandran plots for residues 305–310 for chains A and B of simulations of the wildtype (WT, magenta) and mutant (MUT, blue) homodimers. The A chain of mutant dimer exists predominantly in a flipped state at residues 307–308, whereas the WT protein is predominantly unflipped (similar to the starting conformation and most structures of human CBS). The B chain of mutant dimer exists predominantly in two flipped states at residues 306–307, whereas the WT protein is entirely in an unflipped conformation (similar to the starting conformation and most structures of human CBS). B, Kernel density estimates of the χ₁ dihedral of Tyr-308 from the WT and mutant simulations.

Figure 8.

Representative simulation structures from chain A of residues 306–308 and PLP attached to lysine 119. WT simulation is shown in cyan, and the G307S mutant is shown in magenta. Red shows oxygen atoms, and blue shows nitrogen. The green on PLP-K119 is the carbon where the substrate serine attacks to form the external aldimine.