Novel, tightly structurally related N-myristoyltransferase inhibitors display equally potent yet distinct inhibitory mechanisms.

N-myristoyltransferases (NMTs) catalyze essential acylations of N-terminal alpha or epsilon amino groups of glycines or lysines. Here, we reveal that peptides tightly fitting the optimal glycine recognition pattern of human NMTs are potent prodrugs relying on a single-turnover mechanism. Sequence scanning of the inhibitory potency of the series closely reflects NMT glycine substrate specificity rules, with the lead inhibitor blocking myristoylation by NMTs of various species. We further redesigned the series based on the recently recognized lysine-myristoylation mechanism by taking advantage of (1) the optimal peptide chassis and (2) lysine side chain mimicry with unnatural enantiomers. Unlike the lead series, the inhibitory properties of the new compounds rely on the protonated state of the side chain amine, which stabilizes a salt bridge with the catalytic base at the active site. Our study provides the basis for designing first-in-class NMT inhibitors tailored for infectious diseases and alternative active site targeting.

Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation.

Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-α-acetylation (NTA) and ε-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs and KATs, respectively), although the possibility that the same GCN5-related N-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localized GNATs, which possess a dual specificity. All characterized GNAT family members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinct KA and relaxed NTA specificities. Furthermore, inactivation of GNAT2 leads to significant NTA or KA decreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation process in vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryotic GNATs may also possess these previously underappreciated broader enzymatic activities.

Structural insights into Helicobacter pylori oncoprotein CagA interaction with ß1 integrin.

Infection with the gastric pathogen Helicobacter pylori is a risk factor for the development of gastric cancer. Pathogenic strains of H. pylori carry a type IV secretion system (T4SS) responsible for the injection of the oncoprotein CagA into host cells. H. pylori and its cag-T4SS exploit α5ß1 integrin as a receptor for CagA translocation. Injected CagA localizes to the inner leaflet of the host cell membrane, where it hijacks host cell signaling and induces cytoskeleton reorganization. Here we describe the crystal structure of the N-terminal ~100-kDa subdomain of CagA at 3.6 Å that unveils a unique combination of folds. The core domain of the protein consists of an extended single-layer ß-sheet stabilized by two independent helical subdomains. The core is followed by a long helix that forms a four-helix helical bundle with the C-terminal domain. Mapping of conserved regions in a set of CagA sequences identified four conserved surface-exposed patches (CSP1-4), which represent putative hot-spots for protein-protein interactions. The proximal part of the single-layer ß-sheet, covering CSP4, is involved in specific binding of CagA to the ß1 integrin, as determined by yeast two-hybrid and in vivo competition assays in H. pylori cell-culture infection studies. These data provide a structural basis for the first step of CagA internalization into host cells and suggest that CagA uses a previously undescribed mechanism to bind ß1 integrin to mediate its own translocation.

Structures of the tumor necrosis factor alpha inducing protein Tipalpha: a novel virulence factor from Helicobacter pylori.

Helicobacter pylori secretes a unique virulence factor, Tipalpha, that enters gastric cells and both stimulates the production of the TNF-alpha and activates the NF-kappaB pathway. The structures of a truncated version of Tipalpha (TipalphaN34) in two crystal forms are presented here. Tipalpha adopts a novel beta(1)alpha(1)alpha(2)beta(2)beta(3)alpha(3)alpha(4) topology that can be described as a combination of three domains. A first region consists in a short flexible extension, a second displays a dodecin-like fold and a third is a helical bundle domain similar to the sterile alpha motif (SAM). Analysis of the oligomerisation states of TipalphaN34 in the crystals and in solution suggests that the disulfide bridges could hold together Tipalpha monomers during their secretion in the gastric environment.

In HspA from Helicobacter pylori vicinal disulfide bridges are a key determinant of domain B structure.

Helicobacter pylori produces a heat shock protein A (HspA) that is unique to this bacteria. While the first 91 residues (domain A) of the protein are similar to GroES, the last 26 (domain B) are unique to HspA. Domain B contains eight histidines and four cysteines and was suggested to bind nickel. We have produced HspA and two mutants: Cys94Ala and Cys94Ala/Cys111Ala and identified the disulfide bridge pattern of the protein. We found that the cysteines are engaged in three disulfide bonds: Cys51/Cys53, Cys94/Cys111 and Cys95/Cys112 that result in a unique closed loop structure for the domain B.

Structural basis of the nickel response in Helicobacter pylori: crystal structures of HpNikR in Apo and nickel-bound states.

The survival of Helicobacter pylori in the human stomach critically relies on the availability and use of nickel, an absolute cofactor of the important virulence determinant urease. Nickel-responsive gene regulation is mediated by HpNikR, a protein belonging to the ribbon-helix-helix family of transcriptional regulators. Unlike its homologues, HpNikR acts as both a repressor and an activator within an acid adaptation cascade. We report the crystal structure of the full-length HpNikR in a nickel-free conformation and two nickel-bound structures obtained in different conditions: Ni1-HpNikR and Ni2-HpNikR. Apo-HpNikR shows the same global fold as its bacterial homologues although with an unusual closed trans-conformation and asymmetrical quaternary arrangement. The structure of Ni1-HpNikR in the presence of nickel has two different sides, one showing nickel binding similar to that of known NikRs and the other reflecting an intermediate state. The structure of Ni2-HpNikR obtained using a shorter exposure to nickel provides another snapshot of the nickel incorporation. Altogether, the three structures have allowed us to determine the route for nickel within HpNikR and reveal the cooperativity between the tetramerization domain and the DNA-binding domain. Experiments using point mutations of HpnikR residues involved in nickel internalisation confirm that these residues are critical for HpNikR functions in vivo.