An Environment-dependent Structural Switch Underlies the Regulation of Carnitine Palmitoyltransferase 1A

Jampani N Rao,Gemma Z.L Warren,Sara Estolt-Povedano,Victor A Zammit,Tobias S Ulmer

doi:10.1074/jbc.m111.306951

Abstract

The enzyme carnitine palmitoyltransferase 1 (CPT1), which is anchored in the outer mitochondrial membrane (OMM), controls the rate-limiting step in fatty acid β-oxidation in mammalian tissues. It is inhibited by malonyl-CoA, the first intermediate of fatty acid synthesis, and it responds to OMM curvature and lipid characteristics, which reflect long term nutrient/hormone availability. Here, we show that the N-terminal regulatory domain (N) of CPT1A can adopt two complex amphiphilic structural states, termed Nα and Nβ, that interchange in a switch-like manner in response to offered binding surface curvature. Structure-based site-directed mutageneses of native CPT1A suggest Nα to be inhibitory and Nβ to be noninhibitory, with the relative Nα/Nβ ratio setting the prevalent malonyl-CoA sensitivity of the enzyme. Based on the amphiphilic nature of N and molecular modeling, we propose malonyl-CoA sensitivity to be coupled to the properties of the OMM by Nα-OMM associations that alter the Nα/Nβ ratio. For enzymes residing at the membrane-water interface, this constitutes an integrative regulatory mechanism of exceptional sophistication.

Highlights

The enzyme carnitine palmitoyltransferase 1 regulates the rate of fatty acid oxidation
The enzyme carnitine palmitoyltransferase 1 (CPT1), which is anchored in the outer mitochondrial membrane (OMM), controls the rate-limiting step in fatty acid ␤-oxidation in mammalian tissues
N-terminal CPT1A Domain Can Adopt Two Structural States—Based on the functional properties of CPT1, we hypothesized that N might associate with the catalytic domain (CD), with the OMM or exist “free” within the aqueous environment (Fig. 1A)

Summary

Background

The enzyme carnitine palmitoyltransferase 1 regulates the rate of fatty acid oxidation. The enzyme carnitine palmitoyltransferase 1 (CPT1), which is anchored in the outer mitochondrial membrane (OMM), controls the rate-limiting step in fatty acid ␤-oxidation in mammalian tissues. It is inhibited by malonyl-CoA, the first intermediate of fatty acid synthesis, and it responds to OMM curvature and lipid characteristics, which reflect long term nutrient/hormone availability. Structure-based site-directed mutageneses in conjunction with functional assays of the fulllength protein ascribe physiological relevance to both structures and suggest that their environment-dependent population ratio is used to integrate MCoA concentration, membrane composition, and curvature into one regulatory signal. These results provide fundamental insight into the regulation of enzymes residing at the membrane-water interface

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

Deviations from idealized covalent geometry Bonds Angles Impropers