Structural basis for flagellin-induced NAIP5 activation.

The NAIP (NLR family apoptosis inhibitory protein)/NLRC4 (NLR family CARD containing protein 4) inflammasome senses Gram-negative bacterial ligand. In the ligand-bound state, the winged helix domain of NAIP forms a steric clash with NLRC4 to open it up. However, how ligand binding activates NAIP is less clear. Here, we investigated the dynamics of the ligand-binding region of inactive NAIP5 and solved the cryo-EM structure of NAIP5 in complex with its specific ligand, FliC from flagellin, at 2.9-Å resolution. The structure revealed a "trap and lock" mechanism in FliC recognition, whereby FliC-D0C is first trapped by the hydrophobic pocket of NAIP5, then locked in the binding site by ID (insertion domain) and C-terminal tail of NAIP5. The FliC-D0N domain further inserts into ID to stabilize the complex. According to this mechanism, FliC triggers the conformational change of NAIP5 by bringing multiple flexible domains together.

Structural basis for flagellin induced NAIP5 activation.

The NAIP/NLRC4 inflammasome is activated when NAIP binds to a gram-negative bacterial ligand. Initially, NAIP exists in an inactive state with a wide-open conformation. Upon ligand binding, the winged helix domain (WHD) of NAIP is activated and forms steric clash with NLRC4 to open it up. However, how ligand binding induces the conformational change of NAIP is less clear. To understand this process, we investigated the dynamics of the ligand binding region of inactive NAIP5 and solved the cryo-EM structure of NAIP5 in complex with its specific ligand, FliC from flagellin, at 2.93 Å resolution. The structure revealed a "trap and lock" mechanism in FliC recognition, whereby FliC-D0C is first trapped by the hydrophobic pocket of NAIP5, then locked in the binding site by the insertion domain (ID) and C-terminal tail (CTT) of NAIP5. The FliC-D0N domain further inserts into the loop of ID to stabilize the complex. According to this mechanism, FliC activates NAIP5 by bringing multiple flexible domains together, particularly the ID, HD2, and LRR domains, to form the active conformation and support the WHD loop in triggering NLRC4 activation.

Mechanism of NAIP-NLRC4 inflammasome activation revealed by cryo-EM structure of unliganded NAIP5.

The nucleotide-binding domain (NBD), leucine rich repeat (LRR) domain containing protein family (NLR family) apoptosis inhibitory proteins (NAIPs) are cytosolic receptors that play critical roles in the host defense against bacterial infection. NAIPs interact with conserved bacterial ligands and activate the NLR family caspase recruitment domain containing protein 4 (NLRC4) to initiate the NAIP-NLRC4 inflammasome pathway. Here we found the process of NAIP activation is completely different from NLRC4. Our cryo-EM structure of unliganded mouse NAIP5 adopts an unprecedented wide-open conformation, with the nucleating surface fully exposed and accessible to recruit inactive NLRC4. Upon ligand binding, the winged helix domain (WHD) of NAIP5 undergoes roughly 20° rotation to form a steric clash with the inactive NLRC4, which triggers the conformational change of NLRC4 from inactive to active state. We also show the rotation of WHD places the 17-18 loop at a position that directly bind the active NLRC4 and stabilize the NAIP5-NLRC4 complex. Overall, these data provide structural mechanisms of inactive NAIP5, the process of NAIP5 activation and NAIP-dependent NLRC4 activation.

Structural aspects of the MHC expression control system.

The major histocompatibility complex (MHC) spans innate and adaptive immunity by presenting antigenic peptides to CD4+ and CD8+ T cells. Multiple transcription factors form an enhanceosome complex on the MHC promoter and recruit transcriptional machinery to activate gene transcription. Immune signals such as interferon-γ (IFN-γ) control MHC level by up-regulating components of the enhanceosome complex. As MHC plays crucial roles in immune regulation, alterations in the MHC enhanceosome structure will alter the pace of rapid immune responses at the transcription level and lead to various diseases related to the immune system. In this review, we discuss the current understanding of the MHC enhanceosome, with a focus on the structures of MHC enhanceosome components and the molecular basis of MHC enhanceosome assembly.

Crystal structure of yeast xylose reductase in complex with a novel NADP-DTT adduct provides insights into substrate recognition and catalysis.

Aldose reductases (ARs) belonging to the aldo-keto reductase (AKR) superfamily catalyze the conversion of carbonyl substrates into their respective alcohols. Here we report the crystal structures of the yeast Debaryomyces nepalensis xylose reductase (DnXR, AKR2B10) in the apo form and as a ternary complex with a novel NADP-DTT adduct. Xylose reductase, a key enzyme in the conversion of xylose to xylitol, has several industrial applications. The enzyme displayed the highest catalytic efficiency for l-threose (138 ± 7 mm-1 ·s-1 ) followed by d-erythrose (30 ± 3 mm-1 ·s-1 ). The crystal structure of the complex reveals a covalent linkage between the C4N atom of the nicotinamide ring of the cosubstrate and the S1 sulfur atom of DTT and provides the first structural evidence for a protein mediated NADP-low-molecular-mass thiol adduct. We hypothesize that the formation of the adduct is facilitated by an in-crystallo Michael addition of the DTT thiolate to the specific conformation of bound NADPH in the active site of DnXR. The interactions between DTT, a four-carbon sugar alcohol analog, and the enzyme are representative of a near-cognate product ternary complex and provide significant insights into the structural basis of aldose binding and specificity and the catalytic mechanism of ARs. DATABASE: Structural data are available in the PDB under the accession numbers 5ZCI and 5ZCM.