Structure-function Studies Of Magnetospirillum Gryphiswaldense Ferric Uptake Regulator Fur And Equine Arteritis Virus Helicase Nsp10

Iron is critical to many fundamental biological processes, including DNA synthesis, respiration, and the tricarboxylic acid cycle, etc. Unfortunately, although iron is essential for life, high concentration of iron is toxic for the cell by formation of highly reactive radicals via the Haber-Weiss reaction. Most bacteria maintain a remarkably precise control over cytoplasmic iron level through ferric uptake regulator (Fur). Recent studies identified and characterized a fur gene in Magnetospirillum gryphiswaldense strain MSR-1 and demonstrated that it can directly regulate expression of several key genes involved in iron transport and oxygen metabolism, in addition, it also functions in magnetosome formation. MSR-1 fur was also able to complement a fur mutant of E. coli in iron-responsive manner in vivo. Previous study also solved the structure of MSR-1 apo-Fur, which consists of a DNA binding domain (DBD), a dimer domain (DD) and a hinge region connecting these two domains. MSR-1 apo-Fur is a dimer independent of metal ions in crystal.Herein, we provide the evidences that MSR-1 apo-Fur also exists as a dimer independent of metal ions in solution by means of size exclusion chromatography and analytical ultracentrifugation. MSR-1 Fur can specially bind to a 25 bp sequence of the promoter region of feoABl (feoABl operator) and E. coli Fur box under iron-rich conditions. We further determined the structure of MSR-1 holo-Fur. The structure reveals that MSR-1 Fur has two different iron binding sites, S1 and S2, both of which hexa-coordinate metal ions in an octahedral geometry. S1 which has higher metal ion binding affinity lies between DBD and DD, while S2 is buried within DD. The binding of metal ion induces the conformational arrangement of Fur and then positions the recognition helix of DBD in the orientation which enables MSR-1 Fur to recognize target DNA. Although SI is sufficient for MSR-1 Fur to bind DNA in vitro, both S1 and S2 are essential for function of MSR-1 Fur in vivo.To investigate the mechanism of DNA recognition by MSR-1 Fur, we further determined the structure of MSR-1 Fur in complex with feoABl operator or E. coli Fur box, respectively. These structures combined with biochemical experiment and in vivo assay depict that Arg57 specially recognizes base G and Lys 15 inserts into a narrow minor groove region with enhanced negative electrostatic potential. Computational simulation indicated that this region is intrinsically narrow, demonstrating that MSR-1 Fur specially reads DNA shape. Structural comparison revealed that the key residues involved in DNA interaction are conserved across bacterial Fur proteins. This analysis indicates that the mechanism of DNA recognition by Fur is conserved in bacteria. In summary, the first part of this thesis combines providing important insights into mechanism of metal ion activation and operator recognition of the ferric uptake regulator Fur.Helicases are ATP-dependent motor proteins capable of moving along their nucleic acid substrates and unwinding duplexed regions. These enzymes are known to be critical players in a wide variety of biological processes and are encoded by all organisms, as well as positive-stranded RNA (+RNA) viruses. On the basis of sequence comparison, helicases have been classified into six superfamilies (SF1 to SF6). Most of the structural and functional information of SF1 helicase is from cellular proteins. Helicase of nidovirus is one of the two most conserved nidovirus non-structural proteins. Previous studies identified that equine arteritis virus (EAV, which is a kind of nidovirus) helicase nsp10 which belongs to SF1, is essential to genome RNA synthesis (replication) and subgenomic (sg) mRNA synthesis (transcription). A unique feature of nidoviral helicases is the presence of an N-terminal (predicted) complex zinc-binding domain (ZBD) which is not found in any other RNA viruses.Herein, we over-expressed the truncated recombinant EAV nsp10 (nsp10A) in E. coli, and proved that nsp10A still retains ATPase and helicase activity as that of full-length protein. Moreover, we determined the high-resolution structure of nsp10A, alone and in complex with nucleic acid poly(dT). A previously uncharacterized domain 1B connects helicase core domains 1A and 2A to ZBD, which consists of a novel RING-like module and a treble-clef zinc finger, together coordinating three Zn2+. Structural characterization, mutagenesis and biochemistry revealed that nucleic acid binding is sequence-independent and that helicase activity depends on the extensive relay of interactions through the ZBD and helicase domains. Previous reported nonsense mutant directly or indirectly disrupts the interaction between ZBD and helicase core domain. We also determined the structure of porcine reproductive and respiratory syndrome virus (PRRSV) helicase, the structural conservation in arterivirus was revealed by structural comparison of EAV nsp10 and PRRSV nsp10. Structural comparison also revealed that the arterivirus helicase structurally resembles the cellular helicase human Upfl which is involved in quality control of mRNAs through nonsense-mediated mRNA decay, suggesting that virus-encoded helicases may be involved in the post-transcriptional quality control of extremely large ORF1ab (9.5 kb-21 kb) of nidovirus.In conclusion, in the second part of this thesis we provide the first structural characterization of a key nidovirus enzyme and an antiviral drug target. On the basis of sequence conservation, many of these structural observations will be highly helpful for the study of helicases in other nidoviruses, including coronaviruses. The structural similarity between nsp10 and Upfl establishes a new connection between the research on viral and cellular helicases, which could be mutually insightful for understanding the evolution and function of this group of vitally important enzymes.