The structures of exocyst subunit Exo70p and the Exo84p C-terminal domains reveal a common motif

The exocyst is a large complex that is required for tethering vesicles at the final stages of the exocytic pathway in all eukaryotes. Here we present the structures of the Exo70p subunit of this complex and of the C-terminal domains of Exo84p, at 2.0-A and 2.85-A resolution, respectively. Exo70p forms a 160-A-long rod with a novel fold composed of contiguous alpha-helical bundles. The Exo84p C terminus also forms a long rod (80 A), which unexpectedly has the same fold as the Exo70p N terminus. Our structural results and our experimental observations concerning the interaction between Exo70p and other exocyst subunits or Rho3p GTPase are consistent with an architecture wherein exocyst subunits are composed of mostly helical modules strung together into long rods.     

The C-terminal domain of the MutL homolog from Neisseria gonorrhoeae forms an inverted homodimer

The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage.     

The sigma(70)-like motif: a eukaryotic RNA binding domain unique to a superfamily of proteins required for ribosome biogenesis

Little is understood about the role of nucleolar RNA binding proteins in ribosome biogenesis, although there is a clear need for them based on the strict folding requirements of the pre-rRNA. We have identified a superfamily of RNA binding proteins whose members are required for different stages of ribosome biogenesis. The Imp4 superfamily is composed of five individual families (Imp4, Rpf1, Rpf2, Brx1, and Ssf) that all possess the sigma(70)-like motif, a eukaryotic RNA binding domain with prokaryotic origins. The Imp4 superfamily members associate with RNAs that are consistent with their distinct roles in ribosome biogenesis and suggest the mechanisms by which they function.     

Functional interaction between RNA polymerase alpha subunit C-terminal domain and sigma70 in UP-element- and activator-dependent transcription

We show that the Escherichia coli RNA polymerase (RNAP) alpha subunit C-terminal domain (alphaCTD) functionally interacts with sigma(70) at a subset of UP-element- and activator-dependent promoters, we define the determinants of alphaCTD and sigma(70) required for the interaction, and we present a structural model for the interaction. The alphaCTD-sigma(70) interaction spans the upstream promoter and core promoter, thereby linking recognition of UP-elements and activators in the upstream promoter with recognition of the -35 element in the core promoter. We propose that the alphaCTD-sigma(70) interaction permits UP-elements and activators not only to "recruit" RNAP through direct interaction with alphaCTD, but also to "remodel" RNAP-core-promoter interaction through indirect, alphaCTD-bridged interactions with sigma(70).     

RpoN (sigma54) controls production of antifungal compounds and biocontrol activity in Pseudomonas fluorescens CHA0

Pseudomonas fluorescens CHA0 is an effective biocontrol agent of root diseases caused by fungal pathogens. The strain produces the antibiotics 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) that make essential contributions to pathogen suppression. This study focused on the role of the sigma factor RpoN (sigma54) in regulation of antibiotic production and biocontrol activity in P. fluorescens. An rpoN in-frame-deletion mutant of CHAO had a delayed growth, was impaired in the utilization of several carbon and nitrogen sources, and was more sensitive to salt stress. The rpoN mutant was defective for flagella and displayed drastically reduced swimming and swarming motilities. Interestingly, the rpoN mutant showed a severalfold enhanced production of DAPG and expression of the biosynthetic gene phlA compared with the wild type and the mutant complemented with monocopy rpoN+. By contrast, loss of RpoN function resulted in markedly lowered PLT production and plt gene expression, suggesting that RpoN controls the balance of the two antibiotics in strain CHA0. In natural soil microcosms, the rpoN mutant was less effective in protecting cucumber from a root rot caused by Pythium ultimum. Remarkably, the mutant was not significantly impaired in its root colonization capacity, even at early stages of root infection by Pythium spp. Taken together, our results establish RpoN for the first time as a major regulator of biocontrol activity in Pseudomonas fluorescens.     

PCR mutagenesis identifies a polymerase-binding sequence of sigma 54 that includes a sigma 70 homology region.

Sigma 54 is a minor bacterial sigma factor that is not a member of the sigma 70 family of proteins but binds the same core RNA polymerase. Previously, we identified a region of sigma 54 that is important for binding core polymerase. In this work, PCR mutagenesis was used to identify specific amino acids important for this binding. The results show that important residues are clustered most closely in a short sequence that was previously speculated to be potentially homologous to a sequence in sigma 70. The mutagenesis also identifies important residues in the flanking hydrophobic-acidic region of sigma 54, which is absent in sigma 70. Overall, the data indicate that sigma 54 binds core polymerase through a sequence homologous to that of sigma 70 but in addition uses unique motifs to modify this interaction.     

Ribosomal protein L7/L12 has a helix-turn-helix motif similar to that found in DNA-binding regulatory proteins.

Inspection of the structure of the C-terminal domain of ribosomal protein L7/L12 (1) reveals a helix-turn-helix motif similar to the one found in many DNA-binding regulatory proteins (2-5). The 19 alpha-carbon atoms of the L7/L12 alpha-helices superimpose on the DNA binding helices of CAP and cro with root-mean-square distances between corresponding alpha carbons of 1.45 and 1.55 A, respectively. These helices in L7/L12 are within a patch of highly conserved residues on the surface of L7/L12 whose role is as yet uncertain. We raise the possibility that they may constitute a binding site for nucleic acids, most probably RNA. Consistent with this hypothesis are calculations of the electrostatic charge potential surrounding the protein, which show a region of positive potential centered on the first of these helices.     

The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin

Ubiquitin binding proteins regulate the stability, function, and/or localization of ubiquitinated proteins. Here we report the crystal structures of the zinc-finger ubiquitin binding domain (ZnF UBP) from the deubiquitinating enzyme isopeptidase T (IsoT, or USP5) alone and in complex with ubiquitin. Unlike other ubiquitin binding domains, this domain contains a deep binding pocket where the C-terminal diglycine motif of ubiquitin is inserted, thus explaining the specificity of IsoT for an unmodified C terminus on the proximal subunit of polyubiquitin. Mutations in the domain demonstrate that it is required for optimal catalytic activation of IsoT. This domain is present in several other protein families, and the ZnF UBP domain from an E3 ligase also requires the C terminus of ubiquitin for binding. These data suggest that binding the ubiquitin C terminus may be necessary for the function of other proteins.     

The three-dimensional structure of the C-terminal DNA-binding domain of human Ku70.

The proteins Ku70 (69.8 kDa) and Ku80 (82.7 kDa) form a heterodimeric complex that is an essential component of the nonhomologous end joining DNA double-strand break repair pathway in mammalian cells. Interaction of Ku with DNA is central for the functions of Ku. Ku70, which is mainly responsible for the DNA binding activity of the Ku heterodimer, contains two DNA-binding domains. We have solved the solution structure of the Ku80-independent DNA-binding domain of Ku70 encompassing residues 536-609 using nuclear magnetic resonance spectroscopy. Residues 536-560 are highly flexible and have a random structure but form specific interactions with DNA. Residues 561-609 of Ku70 form a well defined structure with 3 alpha-helices and also interact with DNA. The three-dimensional structure indicates that all conserved hydrophobic residues are in the hydrophobic core and therefore may be important for structural integrity. Most of the conserved positively charged residues are likely to be critical for DNA recognition. The C-terminal DNA-binding domain of Ku70 contains a helix-extended strand-helix motif, which occurs in other nucleic acid-binding proteins and may represent a common nucleic acid binding motif.