Biochemical and structural studies of the LIN-2 and 7 (L27) dimerization domain: Linking proteins important to cell polarity.

Abstract

Asymmetric distribution of proteins and other molecules within cells allows for efficient completion of myriad complex tasks necessary for life. In mammalian epithelia, apical and basolateral proteins and lipids are prevented from mixing by tight junctions, a network of interacting transmembrane and peripheral membrane proteins comprising a physical barrier preventing passage of inorganic solutes and soluble growth factors between cells. Protein-protein interaction domains organize tight junctions and many other multimolecular complexes important for cell signaling and cellular polarity. One protein-protein interaction module of primary importance to these processes is the P&barbelow;ost-synaptic-density-95, D&barbelow;iscs Large, Z&barbelow;onula-occludens-1 (PDZ) domain. In <italic>C. elegans</italic>, the PDZ proteins LIN-2, LIN-7, and LIN-10 act in concert to localize LET-23 to the basolateral surface of body epithelia. A complex of the mammalian orthologs of these proteins, mLIN-2/CASK, mLIN-7/Veli and mLIN-10/X11alpha, was immunoprecipitated from brain lysate, but the moeities responsible for complex formation were unknown. Here, we chronicle the elucidation of the L&barbelow;IN-2&barbelow;, -7&barbelow; or L27 dimerization domain. Amino acids important to L27 domain integrity are described. It is also shown that the mLIN-7 L27 domain binds several membrane associated guanylate kinase proteins (MAGUKs) which themselves have two L27 domains, and the more carboxy terminal (L27C) domain mediates the binding. The mLIN-7 associated MAGUKs are mLIN-2, DLG2, DLG3, and two novel proteins, P&barbelow;roteins A&barbelow;ssociated with mL&barbelow;in-S&barbelow;even-1 and 2 (PALS1 and PALS2). We show that three mLIN-7 binding partners, mLIN-2, DLG3, and DLG2, specifically bind the mammalian ortholog of the <italic>Drosophila</italic> tumor suppressor Discs Large via L27 heterodimerization. The requirements for binding are analyzed and, surprisingly, two modes of interaction are uncovered. PALS1 was shown by others to have a partner for its more amino terminal L2 7N domain named P&barbelow;ALS1 a&barbelow;ssociated T&barbelow;ight J&barbelow;unction protein, or PATJ. This interaction was further determined to be essential for epithelial polarity. We determined the X-ray structure of the complex between PALS1(L27N) and PATJ(L27) domains. The structure reveals a novel fold with hydrophobic residues from three alpha helices of each monomer contributing to a compact binding interface. Taken together, this work elucidates a molecular basis for L27 domain heterodimerization between proteins important in cell polarity.