Christopher L. Johnson

Modulation of the ligand binding properties of the transcription repressor NmrA by GATA-containing DNA and site-directed mutagenesis

NmrA is a negative transcription‐regulating protein that binds to the C‐terminal region of the GATA transcription‐activating protein AreA. The proposed molecular mechanism of action for NmrA is to inhibit AreA binding to its target promoters. In contrast to this proposal, we report that a C‐terminal fragment of AreA can bind individually to GATA‐containing DNA and NmrA and that in the presence of a mixture of GATA‐containing DNA and NmrA, the AreA fragment binds preferentially to the GATA‐containing DNA in vitro. These observations are consistent with NmrA acting by an indirect route, such as by controlling entry into the nucleus. Deletion of the final nine amino acids of a C‐terminal fragment of AreA does not affect NmrA binding. Wild‐type NmrA binds NAD+(P+) with much greater affinity than NAD(P)H, despite the lack of the consensus GXXGXXG dinucleotide‐binding motif. However, introducing the GXXGXXG sequence into the NmrA double mutant N12G/A18G causes an ∼13‐fold increase in the KD for NAD+ and a 2.3‐fold increase for NADP+. An H37W mutant in NmrA designed to increase the interaction with the adenine ring of NAD+ has a decrease in KD of ∼4.5‐fold for NAD+ and a marginal 24% increase for NADP+. The crystal structure of the N12G/A18G mutant protein shows changes in main chain position as well as repositioning of H37, which disrupts contacts with the adenine ring of NAD+, changes which are predicted to reduce the binding affinity for this dinucleotide. The substitutions E193Q/D195N or Q202E/F204Y in the C‐terminal domain of NmrA reduced the affinity for a C‐terminal fragment of AreA, implying that this region of the protein interacts with AreA.

Low resolution structure and dynamics of a Colicin-Receptor complex determined by neutron scattering

Background: In order to kill E. coli, colicins need to cross the bacterial outer membrane. Results: Neutron scattering data show colicin N at the protein-lipid interface of its receptor OmpF. Conclusion: Colicins can unfold and penetrate membranes via the outside wall of their receptors. Significance: The protein-lipid interface may be the route that colicins take into the cell. Proteins that translocate across cell membranes need to overcome a significant hydrophobic barrier. This is usually accomplished via specialized protein complexes, which provide a polar transmembrane pore. Exceptions to this include bacterial toxins, which insert into and cross the lipid bilayer itself. We are studying the mechanism by which large antibacterial proteins enter Escherichia coli via specific outer membrane proteins. Here we describe the use of neutron scattering to investigate the interaction of colicin N with its outer membrane receptor protein OmpF. The positions of lipids, colicin N, and OmpF were separately resolved within complex structures by the use of selective deuteration. Neutron reflectivity showed, in real time, that OmpF mediates the insertion of colicin N into lipid monolayers. This data were complemented by Brewster Angle Microscopy images, which showed a lateral association of OmpF in the presence of colicin N. Small angle neutron scattering experiments then defined the three-dimensional structure of the colicin N-OmpF complex. This revealed that colicin N unfolds and binds to the OmpF-lipid interface. The implications of this unfolding step for colicin translocation across membranes are discussed.

Structural characterization of Salmonella typhimurium YeaZ, an M22 O-sialoglycoprotein endopeptidase homolog

The Salmonella typhimurium “yeaZ” gene (StyeaZ) encodes an essential protein of unknown function (StYeaZ), which has previously been annotated as a putative homolog of the Pasteurella haemolytica M22 O‐sialoglycoprotein endopeptidase Gcp. YeaZ has also recently been reported as the first example of an RPF from a gram‐negative bacterial species. To further characterize the properties of StYeaZ and the widely occurring MK‐M22 family, we describe the purification, biochemical analysis, crystallization, and structure determination of StYeaZ. The crystal structure of StYeaZ reveals a classic two‐lobed actin‐like fold with structural features consistent with nucleotide binding. However, microcalorimetry experiments indicated that StYeaZ neither binds polyphosphates nor a wide range of nucleotides. Additionally, biochemical assays show that YeaZ is not an active O‐sialoglycoprotein endopeptidase, consistent with the lack of the critical zinc binding motif. We present a detailed comparison of YeaZ with available structural homologs, the first reported structural analysis of an MK‐M22 family member. The analysis indicates that StYeaZ has an unusual orientation of the A and B lobes which may require substantial relative movement or interaction with a partner protein in order to bind ligands. Comparison of the fold of YeaZ with that of a known RPF domain from a gram‐positive species shows significant structural differences and therefore potentially distinctive RPF mechanisms for these two bacterial classes. Proteins 2006.

The antibacterial toxin colicin N binds to the inner core of lipopolysaccharide and close to its translocator protein

Colicins are a diverse family of large antibacterial protein toxins, secreted by and active against Escherichia coli and must cross their target cells outer membrane barrier to kill. To achieve this, most colicins require an abundant porin (e.g. OmpF) plus a low‐copy‐number, high‐affinity, outer membrane protein receptor (e.g. BtuB). Recently, genetic screens have suggested that colicin N (ColN), which has no high‐affinity receptor, targets highly abundant lipopolysaccharide (LPS) instead. Here we reveal the details of this interaction and demonstrate that the ColN receptor‐binding domain (ColN‐R) binds to a specific region of LPS close to the membrane surface. Data from in vitro studies using calorimetry and both liquid‐ and solid‐state NMR reveal the interactions behind the in vivo requirement for a defined oligosaccharide region of LPS. Delipidated LPS (LPSΔLIPID) shows weaker binding; and thus full affinity requires the lipid component. The site of LPS binding means that ColN will preferably bind at the interface and thus position itself close to the surface of its translocon component, OmpF. ColN is, currently, unique among colicins in requiring LPS and, combined with previous data, this implies that the ColN translocon is distinct from those of other known colicins.

Structural Analysis of the Recognition of the Negative Regulator NmrA and DNA by the Zinc Finger from the GATA-Type Transcription Factor AreA

Amongst the most common protein motifs in eukaryotes are zinc fingers (ZFs), which, although largely known as DNA binding modules, also can have additional important regulatory roles in forming protein:protein interactions. AreA is a transcriptional activator central to nitrogen metabolism in Aspergillus nidulans. AreA contains a GATA-type ZF that has a competing dual recognition function, binding either DNA or the negative regulator NmrA. We report the crystal structures of three AreA ZF-NmrA complexes including two with bound NAD(+) or NADP(+). The molecular recognition of AreA ZF-NmrA involves binding of the ZF to NmrA via hydrophobic and hydrogen bonding interactions through helices alpha1, alpha6 and alpha11. Comparison with an earlier NMR solution structure of AreA ZF-DNA complex by overlap of the AreA ZFs shows that parts of helices alpha6 and alpha11 of NmrA are positioned close to the GATA motif of the DNA, mimicking the major groove of DNA. The extensive overlap of DNA with NmrA explains their mutually exclusive binding to the AreA ZF. The presence of bound NAD(+)/NADP(+) in the NmrA-AreaA ZF complex, however, causes minimal structural changes. Thus, any regulatory effects on AreA function mediated by the binding of oxidised nicotinamide dinucleotides to NmrA in the NmrA-AreA ZF complex appear not to be modulated via protein conformational rearrangements.

Structure of the ribosomal interacting GTPase YjeQ from the enterobacterial species Salmonella typhimurium

The YjeQ class of P-loop GTPases assist in ribosome biogenesis and also bind to the 30S subunit of mature ribosomes. YjeQ ribosomal binding is GTP-dependent and thought to specifically direct protein synthesis, although the nature of the upstream signal causing this event in vivo is as yet unknown. The attenuating effect of YjeQ mutants on bacterial growth in Escherichia coli makes it a potential target for novel antimicrobial agents. In order to further explore the structure and function of YjeQ, the isolation, crystallization and structure determination of YjeQ from the enterobacterial species Salmonella typhimurium (StYjeQ) is reported. Whilst the overall StYjeQ fold is similar to those of the previously reported Thematoga maritima and Bacillus subtilis orthologues, particularly the GTPase domain, there are larger differences in the three OB folds. Although the zinc-finger secondary structure is conserved, significant sequence differences alter the nature of the external surface in each case and may reflect varying signalling pathways. Therefore, it may be easier to develop YjeQ-specific inhibitors that target the N- and C-terminal regions, disrupting the metabolic connectivity rather than the GTPase activity. The availability of coordinates for StYjeQ will provide a significantly improved basis for threading Gram-negative orthologue sequences and in silico compound-screening studies, with the potential for the development of species-selective drugs.

The unstructured domain of colicin N kills Escherichia coli

Bacteria often produce toxins which kill competing bacteria. Colicins, produced by and toxic to Escherichia coli bacteria are three‐domain proteins so efficient that one molecule can kill a cell. The C‐terminal domain carries the lethal activity and the central domain is required for surface receptor binding. The N‐terminal domain, required for translocation across the outer membrane, is always intrinsically unstructured. It has always been assumed therefore that the C‐terminal cytotoxic domain is required for the bactericidal activity. Here we report the unexpected finding that in isolation, the 90‐residue unstructured N‐terminal domain of colicin N is cytotoxic. Furthermore it causes ion leakage from cells but, unlike known antimicrobial peptides (AMPs) with this property, shows no membrane binding behaviour. Finally, its activity remains strictly dependent upon the same receptor proteins (OmpF and TolA) used by full‐length colicin N. This mechanism of rapid membrane disruption, via receptor mediated binding of a soluble peptide, may reveal a new target for the development of highly specific antibacterials.

The transcription repressor NmrA is subject to proteolysis by three Aspergillus nidulans proteases

The role of specific cleavage of transcription repressor proteins by proteases and how this may be related to the emerging theme of dinucleotides as cellular signaling molecules is poorly characterized. The transcription repressor NmrA of Aspergillus nidulans discriminates between oxidized and reduced dinucleotides, however, dinucleotide binding has no effect on its interaction with the zinc finger in the transcription activator AreA. Protease activity in A. nidulans was assayed using NmrA as the substrate, and was absent in mycelium grown under nitrogen sufficient conditions but abundant in mycelium starved of nitrogen. One of the proteases was purified and identified as the protein Q5BAR4 encoded by the gene AN2366.2. Fluorescence confocal microscopy showed that the nuclear levels of NmrA were reduced approximately 38% when mycelium was grown on nitrate compared to ammonium and absent when starved of nitrogen. Proteolysis of NmrA occurred in an ordered manner by preferential digestion within a C‐terminal surface exposed loop and subsequent digestion at other sites. NmrA digested at the C‐terminal site was unable to bind to the AreA zinc finger. These data reveal a potential new layer of control of nitrogen metabolite repression by the ordered proteolytic cleavage of NmrA. NmrA digested at the C‐terminal site retained the ability to bind NAD+ and showed a resistance to further digestion that was enhanced by the presence of NAD+. This is the first time that an effect of dinucleotide binding to NmrA has been demonstrated.

Interfacial Interactions of Pore-Forming Colicins

Colicins are water soluble toxins secreted by E. coli cells to kill other E. coli and related species. To do this they need to cross the outer membrane, periplasm and inner membrane. Pore forming colicins, as their name suggests form a voltage dependent pore in the inner membrane. This chapter deals with the interfaces, both lipid and protein, that the colicins experience as they make the short but complex journey that brings them to the point of pore formation. The succession of molecular interactions with lipid and protein receptors causes a series of conformational changes which allow these large > 40 kDa proteins to outwit the normally tight defensive shield of the target cell. This is done by combining general physico-chemical interfacial interactions, such as the use of amphipathic helical peptides, with precisely targeted protein-protein interactions involving both rigid and natively disordered protein domains.

The Two-State Prehensile Tail of the Antibacterial Toxin Colicin N

Intrinsically disordered regions within proteins are critical elements in many biomolecular interactions and signaling pathways. Antibacterial toxins of the colicin family, which could provide new antibiotic functions against resistant bacteria, contain disordered N-terminal translocation domains (T-domains) that are essential for receptor binding and the penetration of the Escherichia coli outer membrane. Here we investigate the conformational behavior of the T-domain of colicin N (ColN-T) to understand why such domains are widespread in toxins that target Gram-negative bacteria. Like some other intrinsically disordered proteins in the solution state of the protein, ColN-T shows dual recognition, initially interacting with other domains of the same colicin N molecule and later, during cell killing, binding to two different receptors, OmpF and TolA, in the target bacterium. ColN-T is invisible in the high-resolution x-ray model and yet accounts for 90 of the toxin’s 387 amino acid residues. To reveal its solution structure that underlies such a dynamic and complex system, we carried out mutagenic, biochemical, hydrodynamic and structural studies using analytical ultracentrifugation, NMR, and small-angle x-ray scattering on full-length ColN and its fragments. The structure was accurately modeled from small-angle x-ray scattering data by treating ColN as a flexible system, namely by the ensemble optimization method, which enables a distribution of conformations to be included in the final model. The results reveal, to our knowledge, for the first time the dynamic structure of a colicin T-domain. ColN-T is in dynamic equilibrium between a compact form, showing specific self-recognition and resistance to proteolysis, and an extended form, which most likely allows for effective receptor binding.