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15 N-1 H HSQC spectra of an

15 N-enriched NECAP

1 construct encoding amino acids 1C178 revealed good signal dispersion for amino acids 1C132, while HSQC signals for amino acids 134C178 were located between 7.8C8.3 p.p.m. for

1 H nuclear magnetic resonance (NMR) and their heteronuclear

15 N{1 H}- NOEs were negative (data not shown), indicating that only residues 1C132 are folded. We thus focused on a shorter NECAP

1 construct encoding amino acids 1C133, which showed good signal dispersion (5A). The fold of amino acids 1C133 was determined using triple-resonance, multidimensional NMR spectroscopy on

15 N- and

13 C-,15 N-labeled samples. The structural statistics of the analysis are given in Table I and a ribbon presentation and backbone superposition for the deposited structure is shown in Figure 1B and C. The NECAP PHear domain possesses a b-barrel fold, with seven b-strands packed to- gether to form an antiparallel b-sandwich, closed by a long C- terminal a-helix (Figure 1B and C). A second, short helical structure is formed by amino acids 33C35 in the loop between b-strands

1 and

2 (Figure 1B and C). The hydrophobic core of the fold is formed primarily by residues V17, V19, L47, I49, L58, I60, V74, V82, I94, I96, and I106. A comparison of the Ca -atomic coordinates of the NECAP

1 PHear domain to known protein structures using the DALI program revealed

31 similar structures in the PDB database, with a Z-factor in the range of 5.0 to 6.6 (a total number of 84C110 equivalent residues;

Supplementary Table II), indicating a high statistical signi?cance for these structural domain alignments. The selected structures all share the common PH superfold (Blomberg et al, 1999). Within the PH domain superfamily, the NECAP fold best resembles PH domains. PH domains were initially characterized as phospholipid- binding modules. NMR titrations of the PHear domain with sodium phosphate and inositol-1,4,5-tris-phosphate revealed C AP-2 AP-1

2 2 VHS GGA

1 133

178 275 amino acids GAT GA E

1 1 Conserved region PHear domain A NECAP

1 2

1 C N B Figure

1 The NECAP fold. (A) Schematic representation of the organization of NECAP 1. The evolutionarily conserved N-terminal region (amino acids 1C178) is represented in green and the portion encoding the PHear fold (amino acids 1C133) is indicated by the purple line. The C-terminal binding motifs for AP-1/GGAs (blue) and AP-2 (brown) are assigned to the corresponding adaptor protein. (B) Ribbon representation of the solution structure of the NECAP

1 PHear domain with b-strands and a-helices indicated. (C) Stereoview of the backbone superposition of

10 low-energy structures. NECAPs recruit FxDxF motif proteins B Ritter et al &

2007 European Molecular Biology Organization The EMBO Journal VOL

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2007 4067 only low-af?nity interactions (Kd B10 mM) for residues in the spacial proximity of R90 and R113, which are outside the signature phospholipid binding motif (Lemmon and Ferguson, 2001) (data not shown). Structural alignment of the NECAP PHear domain with phospholipid-binding PH domains shows that most of the critical amino acids for phospholipid interaction are not conserved (Supplementary Figure 1A). Moreover, we failed to detect phospholipid bind- ing using PIP strips, despite binding of the epsin

1 ENTH domain (Supplementary Figure 1B), and lipid sedimentation assays failed to detect interaction of the PHear domain with puri?ed brain lipids (Supplementary Figure 1C). We did, however, detect a weak interaction with brain lipids supple- mented with 20% PtdIns(4,5)P2. Further studies will be needed to determine the biological signi?cance of this inter- action, but taken together, these studies indicate that phos- pholipids are unlikely the primary target of the NECAP PHear domain. Interaction with amphiphysin I To search for protein-binding partners in an unbiased screen, we performed af?nity selection experiments from rat brain extracts with puri?ed GST-NECAP