Effects of natural mutations in lecithin:cholesterol acyltransferase on the enzyme structure and activity

Abstract

A molecular model was built for human lecithin:cholesterol acyltransferase (LCAT) based upon the structural homology between this enzyme and lipases (Peelman et al. 1998. Prot. Sci. 7: 585-597). We proposed that LCAT belongs to the ␣ / ␤ hydrolase fold family, and that the central domain of LCAT consists of a mixed seven-stranded ␤ -pleated sheet with four ␣ -helices and loops linking the ␤ -strands. The catalytic triad of LCAT was identified as Asp345 and His377, as well as Ser181. This model is used here for the interpretation of the structural defects linked to the point mutations identified in LCAT, which cause either familial LCAT deficiency (FLD) or fish-eye disease (FED). We show that these mutations occur in separate domains of the 3D structure of the enzyme. Most mutations causing familial LCAT deficiency are either clustered in the vicinity of the catalytic triad or affect conserved structural elements in LCAT. Most mutations causing fish-eye disease are localized on the outer hydrophilic surface of the amphipathic helical segments. These mutations affect only minimally the overall structure of the enzyme, but are likely to impair the interaction of the enzyme with its co-factor and/ or substrate.-

Relationship between structure and biochemical phenotype of lecithin:cholesterol acyltransferase (LCAT) mutants causing fish-eye disease

“…In summary, these experimental data strongly support the hypothesis previously proposed on the basis of molecular modeling (19), that one layer of helices in the ␣/␤ hydrolase fold of LCAT is involved in the lipoprotein substrate recognition, and that the conformation and orientation of these helices are critical for optimal activity. Measurements of LCAT activation by wild-type apoA-I and by natural or engineered mutants (38,39) suggest that residues 120-160 of apoA-I contribute to the LCAT activation properties of this apolipoprotein, and might represent the corresponding segment of apoA-I interacting with LCAT.…”

“…Because the three FED mutations T123I, N131D, and N391S have decreased activity on HDL, we previously proposed that the parallel helices ␣ 3-4 and ␣ His, which are on the same side of the central ␤ sheet of the LCAT fold, might specifically interact with HDL (19). To test this hypothesis, we mutated residues Q126, N127, and N130 on the hydrophilic face of helix ␣ 3-4 and residues E388 and H389 on the hydrophilic side of helix ␣ His to an alanine and tested the activity of these mutants on different substrates.…”

“…Class 4 mutants are residues contributing to the recognition of lipoprotein substrates by LCAT, through an optimal conformation of enzyme and substrate. In the classic FLD (Familial LCAT Deficiency) mutants, all enzymatic activities are severely impaired and we have previously shown that FLD mutations either perturb the architecture of the catalytic triad, impair the accessibility to the catalytic residues, or affect the fold of the enzyme (19).…”

“…As described above, the natural FED mutants T123I and N131D both belong to the amphipathic helix ␣ 3-4, spanning residues 116-134, while N391 belongs to the parallel C-terminal helix ␣ His (19). We mutated other residues, located in the vicinity of T123 and N131, in order to investigate their contribution to the enzymatic activity.…”

“…It is unclear how the two enzymatic activities are related and which residues, besides the catalytic triad, are critical for either activity or for both. The conformation of three of the natural FED mutants was examined in the 3D structure proposed for LCAT (19), showing that residues T123 and N131 belong to the amphipathic helix ␣ 3-4, while N391 is part of the C-terminal helix ␣ His. In the 3D model, the FED mutants T123I, N131D, N391S lie at about 12 Å of each other on the outer surface of the protein, thus creating a putative interface for enzyme-co-factor interactions (19).…”