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The biophysical characterisation and SAXS analysis of human NLRP1 uncover a new level of complexity of NLR proteins

DOI: 10.1371/journal.pone.0164662 DOI Help

Authors: Luigi Martino (Molecular Structure of Cell Signalling Laboratory, The Francis Crick Institute) , Louise Holland (Molecular Structure of Cell Signalling Laboratory, The Francis Crick Institute) , Evangelos Christodoulou (Structural Biology Science Technology Platform, The Francis Crick Institute) , Simone Kunzelmann (Structural Biology Science Technology Platform, The Francis Crick Institute) , Diego Esposito (Molecular Structure of Cell Signalling Laboratory, The Francis Crick Institute) , Katrin Rittinger (Molecular Structure of Cell Signalling Laboratory, The Francis Crick Institute)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Plos One , VOL 11

State: Published (Approved)
Published: October 2016
Diamond Proposal Number(s): 15579

Open Access Open Access

Abstract: NOD-like receptors represent an important class of germline-encoded pattern recognition receptors that play key roles in the regulation of inflammatory signalling pathways. They function as danger sensors and initiate inflammatory responses and the production of cytokines. Since NLR malfunction results in chronic inflammation and auto-immune diseases, there is a great interest in understanding how they work on a molecular level. To date, a lot of insight into the biological functions of NLRs is available but biophysical and structural studies have been hampered by the difficulty to produce soluble and stable recombinant NLR proteins. NLRP1 is an inflammasome forming NLR that is believed to be activated by binding to MDP and induces activation of caspase 1. Here, we report the identification of a soluble fragment of NLRP1 that contains the NACHT oligomerization domain and the putative MDP-sensing LRR domain. We describe the biophysical and biochemical characterization of this construct and a SEC-SAXS analysis that allowed the calculation of a low resolution molecular envelope. Our data indicate that the protein is constitutively bound to ATP with a negligible ability to hydrolyse the triphosphate nucleotide and that it adopts a monomeric extended conformation that is reminiscent of the structure adopted by NLRC4 in the inflammasome complex. Furthermore, we show that the presence of MDP is not sufficient to promote self-oligomerization of the NACHT-LRR fragment suggesting that MDP may either bind to regions outside the NACHT-LRR module or that it may not be the natural ligand of NLRP1. Taken together, our data suggest that the NLRP1 mechanism of action differs from that recently reported for other NLRs.

Journal Keywords: Insects; Nucleotides; Small-angle scattering; Immune receptor signaling; Toll-like receptors; Biophysics; Inflammasomes; Protein concentration assays

Subject Areas: Biology and Bio-materials


Instruments: B21-High Throughput SAXS

Added On: 10/11/2016 16:33

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Discipline Tags:

Structural biology Biophysics Life Sciences & Biotech

Technical Tags:

Scattering Small Angle X-ray Scattering (SAXS)