MBB Faculty - Rosemary Cornell

Rosemary Cornell lab

Rosemary B. Cornell, Professor

Department of Molecular Biology and Biochemistry / Chemistry
Simon Fraser University

B.Sc., Houghton College
Ph.D., University of Pennsylvania

Office: SSB 6146
Phone: (778)-782-3709

Research Interests: Membrane biochemistry;

Regulation of peripheral membrane proteins by lipid interactions.

Regulatory enzymes that control metabolism are multi-domain structures whose catalytic sites are often suppressed by inter-domain interactions until some cellular signal binds and disrupts that inhibitory interaction. Many metabolic enzymes and proteins involved in signal transduction are amphitropic; i.e. they are regulated by reversible association with cell membranes, as illustrated on the right. One of these is CTP: phosphocholine cytidyltransferase (CCT), which catalyzes a key regulatory step in phosphatidyl-choline synthesis. We are investigating the interplay between CCT and membranes enriched in lipid mediators such as fatty acids and diacylglycerol. Membrane binding and activation of CCT is promoted by increases in membrane content of these minor lipid species, and by dephosphorylation of CCT’s C-terminal tail. (Cornell and Northwood 2000)

PROJECT OVERVIEWS:

How does CCT sense the lipid composition of the membrane? CCT’s lipid sensor is a membrane binding domain (domain M): a long amphipathic α-helix. with strips of positively charged amino acids at the interfaces between polar and non-polar faces. The helical structure is induced by membranes with lipid compositions and properties that promote adsorption (negative charge) and facilitate the insertion of the helical hydrophobic face into the bilayer (lipid packing stress). (Cornell & Taneva) CCT’s amphipathic helix bears a close resemblance in sequence and properties to that of α-synuclein, a synaptic vesicle protein, whose misfolding is linked to Parkinson’s Disease. We are comparing the binding of CCT and α- synuclein to lipid vesicles in an effort to determine how to prevent α-synuclein misfolding. We measure membrane partitioning using fluorescence, circular dichroism, direct vesicle or cell membrane binding assays, and light scattering.

How does membrane binding turn on the catalytic domain? Our hypothesis is that inter-domain interactions with the auto-inhibitory domain M turn the enzyme OFF in its soluble form. Membrane binding disrupts these to turn the enzyme ON. Using X-ray diffraction we (in collaboration with Dr. Mark Paetzel) have solved the structure to 2.2 A resolution of a soluble fragment lacking domain M to “see” the active form of the catalytic site, and hope to crystallize and solve the structure of a construct containing domain M to unravel the basis for the inactivation imposed by domain M. Recently some clues for the inactivation mechanism were obtained by site-specific cysteine engineering and photo-crosslinking. This approach has identified cross-bridges between several sites on domain M and peptides surrounding the active site cleft. These contacts are broken upon membrane insertion of domain M.

The CCT dimer; Residues 40-216 of rat CCTα provided clear electron density. Domain N (blue); Domain C (green). The reaction product, CDP-choline, is shown in stick representation, marking the active site (arrows). The catalytic domain is comprised of a 5-stranded beta sheet flanked by 6 helices.

CCT remodels membranes when it binds.

Proteins that insert partway into a lipid bilayer bend the membrane towards the water by deforming one leaflet with compensation by the opposite leaflet. In vitro this feature of CCT’s domain M causes tubulation of lipid vesicles, as shown below, and in cells CCT over-expression induces tubular invaginations of the nuclear envelope. Gehrig et. al.

The CCTa isoform can also promote tethering of two separate membranes in vitro. This property is due to its N-terminal nuclear localization signal (NLS) sequence, which doubles as a secondary membrane binding motif. (Teneva et. al) The beta isoform lacks an NLS, is non-tethering, and binds membranes more weakly than the alpha isoform. We are probing the differential regulation of these two non-redundant CCT isoforms by lipids and phosphorylation. The biological function of the membrane tethering and bending properties of CCT remain to be explored.

Last updated 09/08/2009