Structural insights into the p97/Ufd1/Npl4 complex via a new alanine methyl TROSY method Npl4 P97 N...
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Transcript of Structural insights into the p97/Ufd1/Npl4 complex via a new alanine methyl TROSY method Npl4 P97 N...
Structural insights into the p97/Ufd1/Npl4 complex via a new alanine methyl TROSY method
Npl4P97 NUBD
Ufd C
UfdN
Ub
N
N
N
N
N
ND1
D1
D1D1
D1
D1
D2
D2D2
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
ABSTRACTTROSY 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-3-13C,2-2H in a tryptophan synthase-catalysed reaction and use it to label proteins in a perdeuterated rich media background1.
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 domains2. While informative, these studies have crucial limitations. Using our new technique we are now tackling the complex as a whole.
p97p97
RUP
ERERp97-Npl4-Ufd1p97-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-3-13C2-2H not commercially available – we make it using tryptophan synthase and L-alanine-3-13C2-1H
● Alanine scrambles into other amino acids – avoided by incorporating our precursor in a deuterated rich medium background
Figure Legend (see right)
● TOP: 1H-13C 2D Methyl TROSY spectrum3 of 13CH3-Ala,U-2H -labelled p97 ND1 – a 306 KDa hexamer. Residues from p97 N indicated in magenta rings
● BOTTOM: 1H-13C 2D Methyl TROSY spectrum3 of 13CH3-Ile,13CH3-Ala,U-2H- 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 the1H-13C 2D Methyl TROSY
spectrum3 of 13CH3-Ala,U-2H labelled hexameric p97- ND1 (306 KDa) in the
absence (black) & presence (red)
of U-2H Npl4UBD
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 D20 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-3-13C,2-2H
Synthesis of L-alanine-3-13C,2-2H