X-ray crystallographic characterization and phasing of an NtrC homologue

Abstract

The ZraR (HydG) protein is a 441-amino-acid protein with three functional domains and is homologous to the general nitrogenregulatory protein NtrC that regulates nitrogen assimilation in many bacteria. The AAA and DNA-binding domains (residues 141±441) of the ZraR protein from Salmonella typhimurium were crystallized using the sitting-drop vapour-diffusion method. X-ray diffraction data from the native crystal have been collected to 3.0 A Ê resolution. Initial phasing was successfully performed by the SIRAS method using derivativatized crystals soaked in 1 mM ethylmercuric phosphate. Preliminary structural analysis shows the presence of a hexamer in the asymmetric unit. Model building is in progress.

ATP-dependent Transcriptional Activation by Bacterial PspF AAA+Protein

“…6,13 The recent structural data showed that PspF, NtrC1 and ZraA organise into higher oligomeric rings, as hexamers in the cases of PspF and ZraA and as heptamer for NtrC1. 10,11 The dimensions of the oligomeric ring structures agree with that of hexamers from other AAA þ proteins, including NSF, involved in various membrane fusion events 29 and the DNA helicase in Holliday junction resolution, RuvB. 30 Functional and structural results therefore now firmly place EBP in the AAA þ family.…”

“…The recently published crystal structures of N-terminally truncated versions of NtrC1 from Aquifex aeolicus and ZraR from Salmonella typhimurium, both homologous to NtrC from Escherichia coli within the AAA þ domain of NtrC, show heptameric and hexameric ring organisations, respectively. 10,11 Residues in NtrC and DctD that were predicted to correspond to residues of the Walker A and Walker B motifs were shown to be involved in nucleotide binding and hydrolysis, respectively, and are in agreement with P-loop NTPases. 12 -14 The phage shock protein F (PspF) of E. coli regulates the s 54 -dependent expression of the psp operon, which is involved in the adaptation to various cellular stresses.…”

“…NtrC requires phosphorylation of the receiver domain for higher oligomer formation and the receiver domain is likely to be involved in the packing of the higher oligomeric form, 6 whereas the AAA þ domains of PspF, ZraA and NtrC1 C suffice to form hexamers and heptamers, respectively. 10,11,18 This difference may contribute to minor differences in the packing of protomers that could have important effects on the atomic geometry of the catalytic site. It would be interesting to know what role the corresponding residue of R168 of PspF plays in other EBPs.…”

“…To determine the PspF 1 -275 :ADP-AlF x ratio, PspF 1 -275 -ADP -AlF x was formed in the presence of [8][9][10][11][12][13][14] …”

“…[8][9][10][11][12][13][14] C]ADP -AlF x per dimer of PspF1 -275 in the dodecameric oligomer. To this end, PspF 1 -275 was incubated with NaF,[8][9][10][11][12][13][14] C]ADP and AlCl 3 under conditions where PspF 1 -275 forms homogeneous oligomers, as evidenced by the elution of a single symmetrical peak during gel-filtration chromatography (see above).…”