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Ritter et al, 2003;

Blondeau et al, 2004). NECAP

1 expression levels are highest in the brain and dominant-negative NECAP

1 constructs disrupt synaptic vesicle endocytosis (Murshid et al, 2006). NECAP

1 is also expressed in non-neuronal tissues and cells and NECAP

2 is ubiquitously expressed. In contrast to most endocytic acces- sory proteins, both NECAP family members are enriched on CCVs together with AP-2 and clathrin (Mills et al, 2003;

Ritter et al, 2003;

Blondeau et al, 2004). The primary NECAP sequences are evolutionarily conserved but lack homology to other proteins. Our initial studies identi?ed and character- ized C-terminal peptide-binding motifs for AP-2 and the Golgi/endosome-associated clathrin adaptor proteins AP-1/ GGAs (Ritter et al, 2003, 2004;

Mattera et al, 2004). Here, we have solved the structure of the conserved N- terminal region in NECAP 1, revealing a new module in the pleckstrin homology (PH) domain superfamily. The PH do- main superfamily is one of the largest domain superfamilies and includes in addition to PH domains, phosphotyrosine- binding (PTB), Ena/VASP homology

1 (EVH1)/WASP homol- ogy

1 (WH1), and Ran-binding domains (RanBD) (Blomberg et al, 1999). The PH superfold is a seven-stranded b-barrel that is closed on one side by a C-terminal a-helix. PH domains were ?rst characterized as phospholipid-binding modules, but further studies revealed that only a few have high af?nity for phospholipids (Lemmon, 2004;

DiNitto and Lambright, 2006). In budding yeast, for example, only one of

33 PH domains in the genome strongly binds phospho- lipids (Yu et al, 2004). Many PH domains have now been Received:

11 April 2007;

accepted:

30 July 2007;

published online:

30 August

2007 *Corresponding authors. K Gehring, Department of Biochemistry, McGill University,

3655 Promenade Sir William Osler, Montreal, Canada H3G 1Y6. Tel.: ?

514 398 7287;

Fax: ?

514 847 0220;

E-mail: [email protected] or PS McPherson, Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University,

3801 University Street, Montreal, Canada H3A 2B4. Tel.: ?

514 398 7355;

Fax: ?

514 398 8106;

E-mail: [email protected]

3 These authors contributed equally to this work

4 Present address: De ?partement des Sciences Fondamentales, Universite ? du Que ?bec a ` Chicoutimi, Chicoutimi, G7H 2B1, Canada The EMBO Journal (2007) 26, 4066C4077 | &

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4066 recognized to bind proteins (Lemmon, 2004). Besides the common fold, the superfamily is characterized by low sequence similarity between family members and high varia- bility in the length of the loops connecting the core structural elements (DiNitto and Lambright, 2006). This ?exibility allows the stable PH superfold to morph into interfaces with the electrostatic potential and molecular surface neces- sary to adopt variable functions. Characterization of the PH-like domain of NECAPs identi?ed this module as a protein-binding interface that mimics the FxDxF motif bind- ing properties of the a-ear. We have thus named the module the PHear (PH fold with ear-like function) domain. As NECAPs themselves are stable components of CCVs, the PHear domain provides a means to regulate the access of FxDxF motif-bearing proteins to the assembling clathrin coat, thereby ?ne-tuning clathrin-mediated endocytosis. Results The PHear fold The N-terminal half of NECAPs is well conserved throughout evolution (Figure 1A and Supplementary Table I). Analysis of