Author: Megan Frisk

Author: Stella M. Hurtley

Author: Phil Szuromi

Author: Julia Fahrenkamp-Uppenbrink

Author: Kristen L. Mueller

Author: Jelena Stajic

Author: Pamela J. Hines

Author: Pamela J. Hines

Author: Peter Stern

Author: Valda Vinson

Author: Sacha Vignieri

Author: Phil Szuromi

Author: Caroline Ash

Author: Beverly A. Purnell

Author: Keith T. Smith

Author: Brad Wible

Author: Paula A. Kiberstis

The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and adaptive immune defenses by hijacking the adaptor protein 1 (AP-1) and AP-2 clathrin adaptors. We found that HIV-1 Nef and the guanosine triphosphatase Arf1 induced trimerization and activation of AP-1. Here we report the cryo–electron microscopy structures of the Nef- and Arf1-bound AP-1 trimer in the active and inactive states. A central nucleus of three Arf1 molecules organizes the trimers. We combined the open trimer with a known dimer structure and thus predicted a hexagonal assembly with inner and outer faces that bind the membranes and clathrin, respectively. Hexagons were directly visualized and the model validated by reconstituting clathrin cage assembly. Arf1 and Nef thus play interconnected roles in allosteric activation, cargo recruitment, and coat assembly, revealing an unexpectedly intricate organization of the inner AP-1 layer of the clathrin coat. Authors: Qing-Tao Shen, Xuefeng Ren, Rui Zhang, Il-Hyung Lee, James H. Hurley

Responding to stimuli, nucleotide-binding domain and leucine-rich repeat–containing proteins (NLRs) oligomerize into multiprotein complexes, termed inflammasomes, mediating innate immunity. Recognition of bacterial pathogens by NLR apoptosis inhibitory proteins (NAIPs) induces NLR family CARD domain–containing protein 4 (NLRC4) activation and formation of NAIP-NLRC4 inflammasomes. The wheel-like structure of a PrgJ-NAIP2-NLRC4 complex determined by cryogenic electron microscopy at 6.6 angstrom reveals that NLRC4 activation involves substantial structural reorganization that creates one oligomerization surface (catalytic surface). Once activated, NLRC4 uses this surface to catalyze the activation of an inactive NLRC4, self-propagating its active conformation to form the wheel-like architecture. NAIP proteins possess a catalytic surface matching the other oligomerization surface (receptor surface) of NLRC4 but not those of their own, ensuring that one NAIP is sufficient to initiate NLRC4 oligomerization. Authors: Zehan Hu, Qiang Zhou, Chenlu Zhang, Shilong Fan, Wei Cheng, Yue Zhao, Feng Shao, Hong-Wei Wang, Sen-Fang Sui, Jijie Chai

The NLR family apoptosis inhibitory proteins (NAIPs) bind conserved bacterial ligands, such as the bacterial rod protein PrgJ, and recruit NLR family CARD-containing protein 4 (NLRC4) as the inflammasome adapter to activate innate immunity. We found that the PrgJ-NAIP2-NLRC4 inflammasome is assembled into multisubunit disk-like structures through a unidirectional adenosine triphosphatase polymerization, primed with a single PrgJ-activated NAIP2 per disk. Cryo–electron microscopy (cryo-EM) reconstruction at subnanometer resolution revealed a ~90° hinge rotation accompanying NLRC4 activation. Unlike in the related heptameric Apaf-1 apoptosome, in which each subunit needs to be conformationally activated by its ligand before assembly, a single PrgJ-activated NAIP2 initiates NLRC4 polymerization in a domino-like reaction to promote the disk assembly. These insights reveal the mechanism of signal amplification in NAIP-NLRC4 inflammasomes. Authors: Liman Zhang, Shuobing Chen, Jianbin Ruan, Jiayi Wu, Alexander B. Tong, Qian Yin, Yang Li, Liron David, Alvin Lu, Wei Li Wang, Carolyn Marks, Qi Ouyang, Xinzheng Zhang, Youdong Mao, Hao Wu