title: The Biophysical Characterisation and SAXS Analysis of Human NLRP1 Uncover a New Level of Complexity of NLR Proteins.
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.
authors.affiliation: Molecular Structure of Cell Signalling Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, United Kingdom.
authors.forename: Luigi
authors.initials: L
authors.lastname: Martino
authors.affiliation: Molecular Structure of Cell Signalling Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, United Kingdom.
authors.forename: Louise
authors.initials: L
authors.lastname: Holland
authors.affiliation: Structural Biology Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, United Kingdom.
authors.forename: Evangelos
authors.initials: E
authors.lastname: Christodoulou
authors.affiliation: Structural Biology Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, United Kingdom.
authors.forename: Simone
authors.initials: S
authors.lastname: Kunzelmann
authors.affiliation: Molecular Structure of Cell Signalling Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, United Kingdom.
authors.forename: Diego
authors.initials: D
authors.lastname: Esposito
authors.affiliation: Molecular Structure of Cell Signalling Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, United Kingdom.
authors.forename: Katrin
authors.identifier: http://orcid.org/0000-0002-7698-4435
authors.initials: K
authors.lastname: Rittinger
mesh_terms: D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000737:chemistry / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D000070576:NLR Proteins; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D053838:Scattering, Small Angle; D016415:Sequence Alignment; D014961:X-Ray Diffraction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D000070576:NLR Proteins; C427903:NLRP1 protein, human; D009711:Nucleotides; D011994:Recombinant Proteins; D000119:Acetylmuramyl-Alanyl-Isoglutamine; D000255:Adenosine Triphosphate