A 22-mer Segment in the Structurally Pliable Regulatory Domain of Metazoan CTP: Phosphocholine Cytidylyltransferase Facilitates Both Silencing and Activating Functions

Abstract

Background:The mechanism whereby CCT is auto-inhibited by its membrane-induced amphipathic helix (m-AH) is unknown. Results: m-AH regions sharing an amphipathic 22-mer element can be interchanged between CCTs with retention of catalytic silencing and activation by lipids. Conclusion:The 22-mer element is the principal auto-inhibitory motif. Significance: Multi-tasking and conformationally malleable motifs are widely used to regulate protein function; the CCT m-AH is a novel example of this.

Structural Basis for Autoinhibition of CTP:Phosphocholine Cytidylyltransferase (CCT), the Regulatory Enzyme in Phosphatidylcholine Synthesis, by Its Membrane-binding Amphipathic Helix

“…Protease accessibility, photocross-linking, and site-directed fluorescence anisotropy suggested a bipartite structure for domain M in the CCT soluble form: an N-terminal disordered segment (residues ϳ235-270) followed by a more structured segment (residues 272-301) (20,21). This bipartite structure is also predicted by many disorder prediction algorithms and is a feature of CCT M domains across phyla (16,22).…”

“…The K m for the other substrate, phosphocholine, is not regulated. However, membrane binding, which requires a functioning M domain, imparts an additional order of magnitude increase in catalytic efficiency above the increase observed upon domain M deletion (16). These data suggest that M has a dual role in CCT regulation: as a silencer in the soluble form and as an activator in its membrane-bound form.…”

“…All CCT variants were analyzed using a standard assay (23,25) in which substrate concentrations are optimal for WT CCT (16) in the absence or presence of egg PC/egg PG (1:1) sonicated vesicles (26). Lipid concentrations were 0.25-0.5 mM for maximal activation depending on the variant.…”

“…A recent analysis of chimeric enzymes revealed that the regulation of the catalytic domain of rat CCT␣ is tolerant of large variation in the sequence and length of the M domain, suggesting that physical features, such as hydrophobicity and amphipathy, are more important than sequence (16). However, deletion of a relatively conserved 22-mer from the C-terminal end generated a deregulated enzyme with catalytic properties similar to that of CCT-236, which is missing the entire M domain (16).…”

“…However, deletion of a relatively conserved 22-mer from the C-terminal end generated a deregulated enzyme with catalytic properties similar to that of CCT-236, which is missing the entire M domain (16). We refer to this 22-mer segment as the AI motif (see Fig.…”