Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes, such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur–DNA binding remain incompletely understood. Here, we report high-resolution structures of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the Fur–feoAB1 operator complex and the Fur–Pseudomonas aeruginosa Fur box complex. Apo-Fur is a transition metal ion-independent dimer whose binding induces profound conformational changes and confers DNA-binding ability. Structural characterization, mutagenesis, biochemistry and in vivo data reveal that Fur recognizes DNA by using a combination of base readout through direct contacts in the major groove and shape readout through recognition of the minor-groove electrostatic potential by lysine. The resulting conformational plasticity enables Fur binding to diverse substrates. Our results provide insights into metal ion activation and substrate recognition by Fur that suggest pathways to engineer magnetotactic bacteria and antipathogenic drugs.

β‐barrel outer membrane proteins (β‐OMPs) play critical roles in nutrition acquisition, protein import/export, and other fundamental biological processes. The assembly of β‐OMPs in Gram‐negative bacteria is mediated by the β‐barrel assembly machinery (BAM) complex, yet its precise mechanism remains elusive. Here, we report two structures of the BAM complex in detergents and in nanodisks, and two crystal structures of the BAM complex with bound substrates. Structural analysis indicates that the membrane compositions surrounding the BAM complex could modulate its overall conformations, indicating low energy barriers between different conformational states and a highly dynamic nature of the BAM complex. Importantly, structures of the BAM complex with bound substrates and the related functional analysis show that the first β‐strand of the BamA β‐barrel (β1BamA) in the BAM complex is associated with the last but not the first β‐strand of a β‐OMP substrate via antiparallel β‐strand interactions. These observations are consistent with the β‐signal hypothesis during β‐OMP biogenesis, and suggest that the β1BamA strand in the BAM complex may interact with the last β‐strand of an incoming β‐OMP substrate upon their release from the chaperone‐bound state.

Endoribonuclease (NendoU) is unique and conserved as a major genetic marker in nidoviruses that infect vertebrate hosts. Arterivirus nonstructural protein 11 (nsp11) was shown to have NendoU activity and play essential roles in the viral life cycle. Here, we report three crystal structures of porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus (EAV) nsp11 mutants. The structures of arterivirus nsp11 contain two conserved compact domains: the N-terminal domain (NTD) and C-terminal domain (CTD). The structures of PRRSV and EAV endoribonucleases are similar and conserved in the arterivirus, but they are greatly different from that of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoV), representing important human pathogens in the Nidovirales order. The catalytic center of NendoU activity is located in the CTD, where a positively charged groove is next to the key catalytic residues conserved in nidoviruses. Although the NTD is nearly identical, the catalytic region of the arterivirus nsp11 family proteins is remarkably flexible, and the oligomerization may be concentration dependent. In summary, our structures provide new insight into this key multifunctional NendoU family of proteins and lay a foundation for better understanding of the molecular mechanism and antiviral drug development.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus are two major members of the arterivirus family. PRRSV, a leading swine pathogen, causes reproductive failure in breeding stock and respiratory tract illness in young pigs. Due to the lack of a suitable vaccine or effective drug treatment and the quick spread of these viruses, infected animals either die quickly or must be culled. PRRSV costs the swine industry around $644 million annually in the United States and almost €1.5 billion in Europe every year. To find a way to combat these viruses, we focused on the essential viral nonstructural protein 11 (nsp11). nsp11 is associated with multiple functions, such as RNA processing and suppression of the infected host innate immunity system. The three structures solved in this study provide new insight into the molecular mechanisms of this crucial protein family and will benefit the development of new treatments against these deadly viruses.

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