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

Sapna Gupta,Simon Kelow,Liqun Wang,Mark D Andrake,Roland L Dunbrack,Warren D Kruger

doi:10.1074/jbc.ra118.002164

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.

Highlights

Mutations in the cystathionine ␤-synthase (CBS) gene are the cause of classical homocystinuria, the most common inborn error in sulfur metabolism
We describe the creation and characterization of a mouse model for the p.G307S mutation and used computational modeling to explain its behavior
Our key findings are as follows: 1) p.G307S expression is unable to rescue the neonatal lethality associated with being homozygous null for Cbs in the C57BL6 background; 2) p.G307S CBS appears to be stable and forms multimers but has minimal residual enzyme activity; 3) p.G307S cannot be rescued by treatment with proteasome inhibitors; and 4) a molecular dynamics simulation of p.G307S suggests that this mutation affects the position and orientation of a key tyrosine at position 308 hindering the CBS enzyme reaction

Summary

Introduction

Mutations in the cystathionine ␤-synthase (CBS) gene are the cause of classical homocystinuria, the most common inborn error in sulfur metabolism. 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 unlikely to respond to chaperone-based therapy. Mammalian CBS is a 63-kDa protein that forms homotetramers and is expressed at high levels in liver and kidney It has three functional domains: an N-terminal heme-binding domain, a middle catalytic domain, and a C-terminal regulatory domain [10]. In fluctuation; SAM, S-adenosylmethionine; RMSD, root mean squared deviation; tHcy, total homocysteine; E-Cyst, external aldimine of cystathionine

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