1. Structural insights into the p97/Ufd1/Npl4 complex via a new alanine methyl TROSY method Npl4 P97 N UBD Ufd C UfdN Ub N N N N N N D1 D2 The D1 and D2 domains form stacked hexameric rings. They bind and hydrolyse ATP to exert mechanical force on their substrates. The N-domains bind to adaptor proteins which recruit p97 for different cellular functions. Npl4 and Ufd1 associate 1:1 to form a heterodimeric complex of ~110 kDa which recruits the p97 hexamer to ubiquitin-related activities including Endoplasmic Reticulum- Associated Degradation and Regulated Ubiquitin Processing References 1) Isaacson, R.L., Simpson, P.J., Liu, M, Cota, E., Zhang, X., Freemont, P.S. & Matthews, S.J. (2007) J. Am. Chem. Soc. 129(50):15428-9 2) Isaacson, R.L., Pye, V.E., Simpson, P.J., Meyer, H.H., Zhang, X., Freemont, P.S. & Matthews S.J. (2007) J. Biol. Chem. 282(29):21361-9. 3) Tugarinov, V., Hwang, P. M., Ollerenshaw, J. E. & Kay, L. E. (2003) J. Am. Chem. Soc. 125(34):10420-10428 ABSTRACT TROSY of methyl-protonated, perdeuterated samples offers huge scope to extend the size limitations of protein NMR. Traditionally these experiments were performed on Ile/Leu/Val labelled samples. We present a new method of labelling alanine residues for such spectra and demonstrate the method on a large protein complex. We generate L-alanine-3- 13 C,2- 2 H in a tryptophan synthase-catalysed reaction and use it to label proteins in a perdeuterated rich media background 1. The homohexameric AAA ATPase, p97 achieves its versatility through binding to a wide range of cofactor proteins which adapt it to different cellular functions. The heterodimer UN (comprising Ufd1 and Npl4) is an adaptor complex that recruits p97 for numerous tasks, many of which involve the proteasome pathway. p97-UN is a ~650 KDa protein complex. We have elucidated structural information on this system by examining solution and interaction properties of individual domains 2. While informative, these studies have crucial limitations. Using our new technique we are now tackling the complex as a whole. p97 RUP ER p97-Npl4-Ufd1 ERAD Rivka Isaacson, Pete Simpson, Minhao Liu, Paul Freemont & Steve Matthews Why Alanine? Highly represented in proteins – 8.3% Widely distributed in both hydrophobic cores and protein surfaces Mutagenesis well tolerated Obstacles Overcome L-alanine-3- 13 C2- 2 H not commercially available – we make it using tryptophan synthase and L-alanine-3- 13 C2- 1 H Alanine scrambles into other amino acids – avoided by incorporating our precursor in a deuterated rich medium background Figure Legend (see right) TOP: 1 H- 13 C 2D Methyl TROSY spectrum 3 of 13 CH3-Ala,U- 2 H -labelled p97 ND1 – a 306 KDa hexamer. Residues from p97 N indicated in magenta rings BOTTOM: 1 H- 13 C 2D Methyl TROSY spectrum 3 of 13 CH3-Ile, 13 CH3-Ala,U- 2 H- labeled p97 ND1. This demonstrates comparable incorporation of labelled Ile (via precursor α-ketobutyrate and Ala (via our method) implemented simultaneously FAR LEFT: Expanded region of the 1 H- 13 C 2D Methyl TROSY spectrum 3 of 13 CH3-Ala,U- 2 H labelled hexameric p97- ND1 (306 KDa) in the absence (black) & presence (red) of U- 2 H Npl4 UBD LEFT: Structural model of the p97 ND1-Npl4 UBD complex with perturbed alanines indicated in red. Stoichiometry is known to be 1 Npl4 per p97 hexamer implying an asymmetry to the ring which we intend to probe further RIGHT: Reciprocal chemical shift perturbation studies yielded this HADDOCK- derived model for the Npl4 UBD (pink) – p97 N (blue) complex Salmonella typhimurium tryptophan synthase α2β2 complex was over-expressed, purified, crystallised in D 2 0 buffer and used to catalyse the above α-proton exchange reaction Reaction progress was followed by 1D NMR. Completed reaction mixture was used directly in perdeuterated rich media to label proteins with L-alanine- 3- 13 C,2- 2 H Synthesis of L-alanine-3- 13 C,2- 2 H