POTRA domains of the TamA insertase interact with the outer membrane and modulate membrane properties.

The outer membrane (OM) of gram-negative bacteria serves as a vital organelle that is densely populated with OM proteins (OMPs) and plays pivotal roles in cellular functions and virulence. The assembly and insertion of these OMPs into the OM represent a fundamental process requiring specialized molecular chaperones. One example is the translocation and assembly module (TAM), which functions as a transenvelope chaperone promoting the folding of specific autotransporters, adhesins, and secretion systems. The catalytic unit of TAM, TamA, comprises a catalytic ß-barrel domain anchored within the OM and three periplasmic polypeptide-transport-associated (POTRA) domains that recruit the TamB subunit. The latter acts as a periplasmic ladder that facilitates the transport of unfolded OMPs across the periplasm. In addition to their role in recruiting the auxiliary protein TamB, our data demonstrate that the POTRA domains mediate interactions with the inner surface of the OM, ultimately modulating the membrane properties. Through the integration of X-ray crystallography, molecular dynamic simulations, and biomolecular interaction methodologies, we located the membrane-binding site on the first and second POTRA domains. Our data highlight a binding preference for phosphatidylglycerol, a minor lipid constituent present in the OM, which has been previously reported to facilitate OMP assembly. In the context of the densely OMP-populated membrane, this association may serve as a mechanism to secure lipid accessibility for nascent OMPs through steric interactions with existing OMPs, in addition to creating favorable conditions for OMP biogenesis.

Characterization of Hsp90 Co-Chaperone p23 Cleavage by Caspase-7 Uncovers a Peptidase-Substrate Interaction Involving Intrinsically Disordered Regions.

Caspases are cysteinyl peptidases involved in inflammation and apoptosis during which hundreds of proteins are cleaved by executioner caspase-3 and -7. Despite the fact that caspase-3 has a higher catalytic activity, caspase-7 is more proficient at cleaving poly(ADP ribose) polymerase 1 (PARP1) because it uses an exosite within its N-terminal domain (NTD). Here, we demonstrate that molecular determinants also located in the NTD enhance the recognition and proteolysis of the Hsp90 co-chaperone p23. Structure-activity relationship analyses using mutagenesis of the caspase-7 NTD and kinetics show that residues 36-45 of caspase-7, which overlap with residues necessary for efficacious PARP1 cleavage, participate in p23 recognition. We also demonstrate using chimeric and truncated proteins that the caspase-7 NTD binds close to the cleavage site in the C-terminal tail of p23. Moreover, because p23 is cleaved at a site bearing a P4 Pro residue (PEVD142↓G), which is far from the optimal sequence, we tested all residues at that position and found notable differences in the preference of caspase-7 and magnitude of differences between residues compared to the results of studies that have used small peptidic substrate libraries. Finally, bioinformatics shows that the regions we identified in caspase-7 and p23 are intrinsically disordered regions that contain molecular recognition features that permit a transient interaction between these two proteins. In summary, we characterized the binding mode for a caspase that is tailored to the specific recognition and cleavage of a substrate, highlighting the importance of studying the peptidase-substrate pair to understand the modalities of substrate recognition by caspases.

Epigenetic control of polyamines by the prion [PSI+].

Prion proteins are found in mammals and yeast, and can transmit diseases and encode heritable phenotypic traits. In Saccharomyces cerevisiae, eRF3, Rnq1, Ure2 and Swil are functional proteins with a soluble conformation that can switch to a non-functional, amyloid conformation denoted as [PSI+], [PIN+], [URE3] and [SWI+], respectively. The prion [PSI+] corresponds to an aggregated conformation of the translational release factor eRF3, which suppresses nonsense codons. [PSI+] modifies cellular fitness and induces several phenotypes according to the genetic background. An elegant series of studies has demonstrated that several [PSI+]-induced phenotypes occur as a consequence of decreased translational termination efficiency. However, the genes whose expression levels are controlled by [PSI+] remain largely unknown. Here, we show that [PSI+] enhances expression of antizyme, a negative regulator of cellular polyamines, by modulating the +1 frameshifting required for its expression. Our study also demonstrates that [PSI+] greatly affects cellular polyamines in yeast. We show that modification of the cellular content of polyamines by the prion accounts for half of the [PSI+]-induced phenotypes. Antizyme is the first protein to be described for which expression of its functional form is stimulated by [PSI+].

Unraveling the crystal structure of the HpaA adhesin: insights into cell adhesion function and epitope localization of a Helicobacter pylori vaccine candidate.

Helicobacter pylori is a bacterium that exhibits strict host restriction to humans and non-human primates, and the bacterium is widely acknowledged as a significant etiological factor in the development of chronic gastritis, peptic ulcers, and gastric cancers. The pathogenic potential of this organism lies in its adeptness at colonizing the gastric mucosa, which is facilitated by a diverse repertoire of virulence factors, including adhesins that promote the attachment of the bacteria to the gastric epithelium. Among these adhesins, HpaA stands out due to its conserved nature and pivotal role in establishing H. pylori colonization. Moreover, this lipoprotein holds promise as an antigen for the development of effective H. pylori vaccines, thus attracting considerable attention for in-depth investigations into its molecular function and identification of binding determinants. Here, we present the elucidation of the crystallographic structure of HpaA at 2.9 Å resolution. The folding adopts an elongated protein shape, which is distinctive to the Helicobacteraceae family, and features an apical domain extension that plays a critical role in the cell-adhesion activity on gastric epithelial cells. Our study also demonstrates the ability of HpaA to induce TNF-α expression in macrophages, highlighting a novel role as an immunoregulatory effector promoting the pro-inflammatory response in vitro. These findings not only contribute to a deeper comprehension of the multifaceted role of HpaA in H. pylori pathogenesis but also establish a fundamental basis for the design and development of structure-based derivatives, aimed at enhancing the efficacy of H. pylori vaccines. IMPORTANCE: Helicobacter pylori is a bacterium that can cause chronic gastritis, peptic ulcers, and gastric cancers. The bacterium adheres to the lining of the stomach using proteins called adhesins. One of these proteins, HpaA, is particularly important for H. pylori colonization and is considered a promising vaccine candidate against H. pylori infections. In this work, we determined the atomic structure of HpaA, identifying a characteristic protein fold to the Helicobacter family and delineating specific amino acids that are crucial to support the attachment to the gastric cells. Additionally, we discovered that HpaA can trigger the production of TNF-α, a proinflammatory molecule, in macrophages. These findings provide valuable insights into how H. pylori causes disease and suggest that HpaA has a dual role in both attachment and immune activation. This knowledge could contribute to the development of improved vaccine strategies for preventing H. pylori infections.