An environment-dependent structural switch underlies the regulation of carnitine palmitoyltransferase 1A.

The enzyme carnitine palmitoyltransferase 1 (CPT1), which is anchored in the outer mitochondrial membrane (OMM), controls the rate-limiting step in fatty acid ß-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 Nß, 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 Nß to be noninhibitory, with the relative Nα/Nß 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/Nß ratio. For enzymes residing at the membrane-water interface, this constitutes an integrative regulatory mechanism of exceptional sophistication.

Structural studies on the RNA-recognition motif of NELF E, a cellular negative transcription elongation factor involved in the regulation of HIV transcription.

The elongation of transcription of HIV RNA at the TAR (transactivation-response element) is highly regulated by positive and negative factors. The cellular negative transcription elongation factor NELF (negative elongation factor) was suggested to be involved in transcriptional regulation of HIV-1 (HIV type 1) by binding to the stem of the viral TAR RNA which is synthesized by cellular RNA polymerase II at the viral long terminal repeat. NELF is a heterotetrameric protein consisting of NELF A, B, C or the splice variant D, and E. In the present study, we determined the solution structure of the RRM (RNA-recognition motif) of the RNA-binding subunit NELF E and studied its interaction with the viral TAR RNA. Our results show that the separately expressed recombinant NELF E RRM has alpha-helical and beta-strand elements adopting a betaalphabetabetaalphabeta fold and is able to bind to TAR RNA. Fluorescence equilibrium titrations with fluorescently labelled double- and single-stranded oligoribonucleotides representing the TAR RNA stem imply that NELF E RRM binds to the single-stranded TAR RNAs with K(d) values in the low-micromolar range.