The folding and structural integrity of the first LIN-12 module of human Notch1 are calcium-dependent

Notch1 is a member of a conserved family of large modular type 1 transmembr ane receptors that control differentiation in multicellular animals. Notch function is mediated through a novel signal transduction pathway involving successive ligand-induced proteolytic cleavages that serve to release the i ntracellular domain of Notch, which then translocates to the nucleus and ac tivates downstream transcription factors, The extracellular domain of all N otch receptors have three iterated LIN-12 modules that appear to act as neg ative regulatory domains, possibly by Limiting proteolysis, Each LIN-12 mod ule contains three disulfide bonds and three conserved aspartate (D) or asp aragine (N) residues. To begin to understand the structural basis for LIN-1 2 function, the first LIN-12 module of human Notch1 (rLIN-12,1) has been ex pressed recombinantly in Escherichia coli and purified in a reduced form. I n redox buffers, rLIN-12.1 forms only one disulfide isomer in the presence of millimolar Ca2+ concentrations, whereas multiple disulfide isomers are o bserved in the presence of Mg2+ and EDTA, Further, mutation of conserved re sidues N1460, D1475, and D1478 to alanine abolishes Ca2+-dependent folding of this module. Mass spectrometric analysis of partially reduced rLIN-12,1 has been used to deduce that disulfide bonds are formed between the first a nd fifth (C1449-C1472), second and fourth (C1454-C1467), and third and sixt h (C1463-C1479) cysteines of this prototype module. This arrangement is dis tinct from that observed in other modules, such as EGF and LDL-A, that also contain three disulfide bonds. One-dimensional proton nuclear magnetic res onance shows that Ca2+ induces a dramatic increase in the extent of chemica l shift dispersion of the native rLIN-12.1 amide protons, as seen for the C a2+-binding LDL-A modules. We conclude that Ca2+ is required both for prope r folding and for the maintenance of the structural integrity of Notch/LIN- 12 modules.