Contribution of Each Membrane Binding Domain of the CTP:Phosphocholine Cytidylyltransferase-α Dimer to Its Activation, Membrane Binding, and Membrane Cross-bridging

Abstract

CTP:phosphocholine cytidylyltransferase (CCT), a rate-limiting enzyme in phosphatidylcholine synthesis, is regulated by reversible membrane interactions mediated by an amphipathic helical domain (M) that binds selectively to anionic lipids. CCT is a dimer; thus the functional unit has two M domains. To probe the functional contribution of each domain M we prepared a CCT heterodimer composed of one full-length subunit paired with a CCT subunit truncated before domain M that was also catalytically dead. We compared this heterodimer to the fulllength homodimer with respect to activation by anionic vesicles, vesicle binding affinities, and promotion of vesicle aggregation. Surprisingly for all three functions the dimer with just one domain M behaved similarly to the dimer with two M domains. Full activation of the wild-type subunit was not impaired by loss of one domain M in its partner. Membrane binding affinities were the same for dimers with one versus two M domains, suggesting that the two M domains of the dimer do not engage a single bilayer simultaneously. Vesicle cross-bridging was also unhindered by loss of one domain M, suggesting that another motif couples with domain M for cross-bridging anionic membranes. Mutagenesis revealed that the positively charged nuclear localization signal sequence constitutes that second motif for membrane cross-bridging. We propose that the two M domains of the CCT dimer engage a single bilayer via an alternating binding mechanism. The tethering function involves the cooperation of domain M and the nuclear localization signal sequence, each engaging separate membranes. Membrane binding of a single M domain is sufficient to fully activate the enzymatic activity of the CCT dimer while sustaining the low affinity, reversible membrane interaction required for regulation of CCT activity.Many amphitropic proteins have multiple membrane binding domains that serve to enhance binding affinity and to enable differential regulation by various lipid ligands. Multiple binding domains may in some cases enable a stepwise process leading to full activation of the enzyme. For example several protein kinase C isoforms have a C2 domain and two C1 domains that bind acidic lipids and diacylglycerol, respectively, with full activation dependent on engagement of both types of membrane binding modules (1). Other examples include phospholipase D 2 , which relies on both a PH 3 domain and a polybasic domain for targeting to phosphatidylinositol 4,5-bisphosphate-rich membranes (2) and phosphatidylinositol-phospholipase C ␤, ␥, and ␦, which contain C2 and PH domains for recognition of and regulation by Ca 2ϩ and phosphatidylinositol 4,5-bisphosphate, respectively (3).CCT, the rate-limiting enzyme in PC synthesis, is a well studied amphitropic enzyme whose weak, reversible binding is mediated by domain M, a long amphipathic helix (4). Domain M is situated just downstream of the catalytic domain in the linear sequence and is followed by a flexible, unstructured domain housing multiple phosphorylation sites....

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

“…The proteins were purified from the 20,000 ϫ g supernatant using nickel-agarose at 4°C (23). The peak elution fractions were dialyzed against 10 mM Tris, pH 7.4, 0.1 M NaCl, 0.15 mM Triton X-100, and 2 mM DTT and were frozen at Ϫ80°C.…”

“…pET14b-CCT Constructs-The preparation of pET14b CCT-236(K122A) was described in Taneva et al (23). The wild-type residue, Lys 122 , was restored by swapping a KpnI/SacI 650-bp fragment from pET14b-CCT-312.…”

“…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.…”

“…NaCl was added to 0.5 M, and the lysates were sedimented at 20,000 ϫ g for 10 min. HisCCT-367(8S) was purified from this supernatant using nickel-agarose (23). His-CCT-367(6A), -(6S), and -(10S) were mostly insoluble and were purified from the 20,000 ϫ g pellet after washing the pellet by brief sonication in 10 mM Tris, pH 7.4, 0.5 M NaCl, 0.25 mM Triton X-100, protease inhibitors, and 2 mM DTT; collecting the insoluble material; and solubilizing it in 8 M urea.…”

Crystal Structure of a Mammalian CTP: Phosphocholine Cytidylyltransferase Catalytic Domain Reveals Novel Active Site Residues within a Highly Conserved Nucleotidyltransferase Fold

“…An N-terminal domain (ϳ75 residues) housing its nuclear localization signal (NLS) sequence is followed by an ϳ150-residue catalytic domain, an ϳ60-residue membrane binding domain (domain M), and an unstructured phosphorylated tail (ϳ50 residues) (2,4). CCT functions as a homodimer (5).…”