Structure of the NLRP1 caspase recruitment domain suggests potential mechanisms for its association with procaspase‐1

Abstract

The NLRP1 inflammasome responds to microbial challenges such as Bacillus anthracis infection and is implicated in autoimmune disease such as vitiligo. Human NLRP1 contains both an N-terminal pyrin domain (PYD) and a C-terminal caspase recruitment domain (CARD), with the latter being essential for its association with the downstream effector procaspase-1. Here we report a 2.0 Å crystal structure of the human NLRP1 CARD as a fusion with the maltose-binding protein. The structure reveals the six-helix bundle fold of the NLRP1 CARD, typical of the death domain superfamily. The charge surface of the NLRP1 CARD structure and a procaspase-1 CARD model suggests potential mechanisms for their association through electrostatic attraction.

Design of an expression system to enhance MBP-mediated crystallization

“…The short linker of VD residues followed by the first helical residues of the target proteins may help enhance crystallization. Even though this strategy has resulted in success for some of our targets such as the NLRP1 CARD47, and CARD8 CARD48, and the AIM2 PYD domain47 (Fig. 2b,c), it has failed for other targets such as the zebrafish IGBP1-CARD and human NLRP1-PYD (sequences are listed in Table S2).…”

“…Expression and purification of the MBP-tagged proteins have been described previously4748. Briefly, transformed BL21 (DE3) Codon Plus RIPL cells (Stratagene, Santa Clara, CA) were grown at 37 °C and protein expression was induced at 18 °C for at least two hours with 0.3 mM IPTG.…”

“…Inspired by the successful examples of using MBP as a crystallization chaperone, we designed an expression vector (V28E) coding an E. coli MBP protein1346 followed by a very short linker with amino acid sequence of valine and aspartate encoded by the Sal I restriction enzyme site (GTCGAC). This vector has limited success in expression and crystallization of several fusion proteins for PYD and CARD domains474849. Here we sought to systematically explore the usage of linkers of various lengths and test their effects on crystallization of death domain superfamily members.…”

Structural aspects of molecular recognition in the immune system. Part II: Pattern recognition receptors (IUPAC Technical Report)

“…These structures reveal a high degree of conservation with a fold comprising a 6-helix bundle. The structures of several CARDs determined by crystallography or NMR spectroscopy have also been reported [214,[219][220][221][222]. These domains also adopt a compact 6-helix bundle structure in crystals and in solution.…”

International Union of Basic and Clinical Pharmacology. XCVI. Pattern Recognition Receptors in Health and Disease

“…At the atomic level, structural data exist for the nucleotide-binding domain (NBD)-LRR of NLRC4 (Hu et al, 2013), the LRRs of NLRX1 and NLRP1, and the effector domains of NOD1 (CARD), NLRC5 (atypical CARD), NLRP1 (CARD and pyrin domain [PYD]) (Hiller et al, 2003), and NLRP3, 4, 7, 10, 12, and 14 (all PYD) (Coussens et al, 2007;Manon et al, 2007;Srimathi et al, 2008;Pinheiro et al, 2010Pinheiro et al, , 2011Bae and Park, 2011;Eibl et al, 2012Eibl et al, , 2014Su et al, 2013;Jin et al, 2013;Gutte et al, 2014). Although we still await the structure of an NLR in complex with either its ligand or a downstream signaling adaptor, these structures have provided important insight into the molecular functionality of NLR signaling regulation and transduction.…”