We are interested in understanding the molecular basis for specificity in protein folding and protein-protein interactions.

Ligand binding by the LDL receptor.  The LDL receptor (LDLR) is the primary mechanism for uptake of cholesterol-carrying lipoprotein particles into cells.   We are studying the detailed molecular basis for recognition of lipoproteins by this receptor, and how mutations in the receptor give rise to the disease familial hypercholesterolemia (FH), which predisposes affected individuals to heart attacks at a young age.  Binding of lipoproteins is mediated by the amino-terminal domain of the receptor, which consists of seven tandemly repeated modules. Each module is ~40 amino-acid residues long and contains three disulfide bonds.  We are defining the minimum-length fragment of the receptor that is sufficient for binding to Apolipoprotein E and Apolipoprotein B-containing lipoproteins.  Subsequently, we plan to identify the specific contacts between the ligand and the receptor, evaluate the contributions of individual amino acids to ligand-binding, and understand how disease-causing point mutations within the LDL receptor give rise to FH.

Sequence alignment of over 300 LDL-A modules indicates that few residues within the 40-residue sequence are conserved, and that a wide variety of different residue types are tolerated at many of the positions within the repeat.  We are investigating both the kinetic pathway of folding and the thermodynamic determinants of formation of an LDL-A fold, using the fifth ligand-binding repeat of the LDLR as a model system.  Proper folding of native LDL-A modules requires calcium; we are attempting to repack the calcium coordination site with hydrophobic residues to eliminate the calcium dependence of module folding.  The results of these experiments will contribute to our understanding of how primary sequence determines topology in native proteins, and will be helpful in guiding future efforts at protein design.

Mechanism of ligand-dependent signal transduction by the human Notch1 receptor.  The human Notch1 (TAN-1) gene, originally discovered at the breakpoint of a recurrent chromosomal translocation found in human acute T-cell lymphoblastic leukemias, is a member of a conserved family of receptors that control differentiation in multicellular animals.  Although these receptors are large and complex, all Notch family members contain a conserved extracellular domain consisting of three LIN12 modules that are required for proper control of signaling.  We are currently determining the structure of a prototype LIN12 module of human Notch1 by NMR, and in collaboration with the laboratory of Dr. Jon Aster, studying the detailed role of these LIN12 modules in regulating Notch receptor function.

Selected Publications:
North C., and Blacklow, S.C.   Structural Independence of Ligand-Binding Modules Five and Six of the LDL Receptor.  (1999) Biochemistry, in press.

Aster, J.C., Simms, W.B., Zavala-Ruiz, Z., Patriub, V., North, C.L., and Blacklow, S.C.  (1999)  Folding and Structural Integrity of the First LIN12 Module of Human Notch1 Are Calcium Dependent.  Biochemistry,  in press.

Fass, D., Blacklow, S.C., Kim, P.S., and Berger, J.M.   Structure of LDL receptor module reveals molecular mechanisms of familial hypercholesterolemia.  (1997) Nature 388, 691-693.