The tetrameric L27 domain complex as an organization platform for supramolecular assemblies

Abstract

Clustering and asymmetric distribution of receptors, ion channels and associated protein complexes are essential to the polarity of neurons and epithelial cells. Such asymmetric targeting of large molecular assemblies is thought to be governed in part by modular scaffold proteins. L27 domain, initially identified in the C. elegans Lin-2 and Lin-7 proteins, is a protein interaction module that exists in a large family of scaffold proteins 1 . Formation of the trimeric Lin-2-Lin-7-Lin-10 complex requires L27 domains 2,3 and has a central role in targeting receptor tyrosine kinase Let-23 signalsome to the basolateral surface of vulval precursor cells 4,5 . Mutations in Lin-2, Lin-7 or Lin-10 lead to a vulvaless phenotype, presumably by mistargeting of 5). The mammalian orthologs of Lin-2, Lin-7 and Lin-10 are known as mammalian Lin-2 (mLin-2)/CASK, mLin-7/Velis/Mals and mLin-10/X11α/Mint1, respectively 2,6-9 . The mLin-2-mLin-7-mLin-10 tripartite complex has also been observed in the brain 2,6 , and this evolutionarily conserved protein complex has been implicated in the targeting of NMDA receptors and β-catenin assemblies to membrane subdomains in epithelia and neurons 10,11 .A bioinformatics survey reveals that L27 domain-containing proteins are invariably scaffold proteins containing multiple proteinprotein interaction domains without intrinsic enzyme activities 12 . For example, SAP97 and mLin-2/CASK, which are members of a subset of membrane-associated guanylate kinases (MAGUKs), contain one and two L27 domains, respectively, in addition to their PDZ, SH3 and guanylate kinase-like domains. The N-terminal L27 domain of SAP97 can form specific heteromeric complexes with the N-terminal L27 domain of mLin-2, Dlg2 and Dlg3, respectively 3,13,14 . The C-terminal L27 domain of mLin-2 specifically binds to the L27 domain of 15). The discovery of L27 domains as specific protein-protein interaction modules capable of forming heteromeric complexes suggests that L27 domains can integrate multiple scaffold proteins into supramolecular assemblies 1,3,13,15,16 . However, the molecular basis of the L27 domain-mediated protein assembly formation remains unclear. The structures of isolated L27 domains and their cognate heteromeric complexes are not known.Here we show that the L27 domain of SAP97 and the N-terminal L27 domain of mLin-2 form a heterotetrameric complex. The structure of the SAP97-mLin-2 L27 complex was solved by NMR spectroscopy. The structure of the complex, together with data derived from various biochemical studies, establishes the roles of specific residues in the formation of the L27 heterotetrameric complex. The structure of the SAP97-mLin-2 L27 complex further suggests a mechanistic model for polymerization of L27 domain scaffold proteins. RESULTS L27 domains form specific heterotetrameric complexesWe first characterized the structural properties of the isolated L27 domains of SAP97 (referred to as L27S below), mLin-7, the N-terminal L27 domain (referred to as L27N below), and the C-terminal L27 domain (ref...

A unified assembly mode revealed by the structures of tetrameric L27 domain complexes formed by mLin-2/mLin-7 and Patj/Pals1 scaffold proteins

“…The overall structure of the L27 Lin7 ͞L27 Lin2C tetramer is similar to the tetramer structure of the L27 domain complex formed by the SAP97 (L27 SAP97 ) and mLin-2 (L27 Lin2N ) (17). Other than a few residues from the two termini, the structure of the L27 Lin7 ͞L27 Lin2C tetrameric complex is well defined (Fig.…”

“…Distance restraints were generated as described in ref. 17. Backbone dihedral angle restraints were derived from the secondary structure of the protein and backbone chemical shift analysis program TALOS (23).…”

“…The two N-terminal helices (␣A and ␣B) of each L27 domain pack together to form a tight, four-helix bundle in the heterodimer. The third helix (␣C) of each L27 domain forms another four-helix bundle that assembles the two units of the heterodimer into a tetramer (17). Formation of heteromeric L27 domain complexes are highly specific (15,17,18).…”

“…Despite the availability of the two pairs of L27 domain structures, the molecular basis for the formation of the cognate L27 domain complexes is still poorly defined. In addition, the mode of assembly of L27 domain heterodimers into higher-order structures is controversial (17,18). Elucidation of the higher-order assembly mode of cognate pairs of L27 domains is critical for understanding the roles of L27 domain scaffold proteins in organizing supramolecular protein complexes.…”

“…The third helix (␣C) of each L27 domain forms another four-helix bundle that assembles the two units of the heterodimer into a tetramer (17). Formation of heteromeric L27 domain complexes are highly specific (15,17,18). A subsequent structural study of the L27 domain complex formed by the L27 domain of Patj and the N-terminal L27 domain of Pals1 also showed that the packing of the two N-terminal helices from each L27 domain into a four-helix bundle is a general mechanism for L27 heterodimer formation (18).…”