SymProFold: Structural prediction of symmetrical biological assemblies.

Symmetry in nature often emerges from self-assembly processes and serves a wide range of functions. Cell surface layers (S-layers) form symmetrical lattices on many bacterial and archaeal cells, playing essential roles such as facilitating cell adhesion, evading the immune system, and protecting against environmental stress. However, the experimental structural characterization of these S-layers is challenging due to their self-assembly properties and high sequence variability. In this study, we introduce the SymProFold pipeline, which utilizes the high accuracy of AlphaFold-Multimer predictions to derive symmetrical assemblies from protein sequences, specifically focusing on two-dimensional S-layer arrays and spherical viral capsids. The pipeline tests all known symmetry operations observed in these systems (p1, p2, p3, p4, and p6) and identifies the most likely symmetry for the assembly. The predicted models were validated using available experimental data at the cellular level, and additional crystal structures were obtained to confirm the symmetry and interfaces of several SymProFold assemblies. Overall, the SymProFold pipeline enables the determination of symmetric protein assemblies linked to critical functions, thereby opening possibilities for exploring functionalities and designing targeted applications in diverse fields such as nanotechnology, biotechnology, medicine, and materials and environmental sciences.

The molecular architecture of Lactobacillus S-layer: Assembly and attachment to teichoic acids.

S-layers are crystalline arrays found on bacterial and archaeal cells. Lactobacillus is a diverse family of bacteria known especially for potential gut health benefits. This study focuses on the S-layer proteins from Lactobacillus acidophilus and Lactobacillus amylovorus common in the mammalian gut. Atomic resolution structures of Lactobacillus S-layer proteins SlpA and SlpX exhibit domain swapping, and the obtained assembly model of the main S-layer protein SlpA aligns well with prior electron microscopy and mutagenesis data. The S-layer's pore size suggests a protective role, with charged areas aiding adhesion. A highly similar domain organization and interaction network are observed across the Lactobacillus genus. Interaction studies revealed conserved binding areas specific for attachment to teichoic acids. The structure of the SlpA S-layer and the suggested incorporation of SlpX as well as its interaction with teichoic acids lay the foundation for deciphering its role in immune responses and for developing effective treatments for a variety of infectious and bacteria-mediated inflammation processes, opening opportunities for targeted engineering of the S-layer or lactobacilli bacteria in general.

Unmasking crucial residues in adipose triglyceride lipase for coactivation with comparative gene identification-58.

Lipolysis is an essential metabolic process that releases unesterified fatty acids from neutral lipid stores to maintain energy homeostasis in living organisms. Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis and can be coactivated upon interaction with the protein comparative gene identification-58 (CGI-58). The underlying molecular mechanism of ATGL stimulation by CGI-58 is incompletely understood. Based on analysis of evolutionary conservation, we used site directed mutagenesis to study a C-terminally truncated variant and full-length mouse ATGL providing insights in the protein coactivation on a per-residue level. We identified the region from residues N209-N215 in ATGL as essential for coactivation by CGI-58. ATGL variants with amino acids exchanges in this region were still able to hydrolyze triacylglycerol at the basal level and to interact with CGI-58, yet could not be activated by CGI-58. Our studies also demonstrate that full-length mouse ATGL showed higher tolerance to specific single amino acid exchanges in the N209-N215 region upon CGI-58 coactivation compared to C-terminally truncated ATGL variants. The region is either directly involved in protein-protein interaction or essential for conformational changes required in the coactivation process. Three-dimensional models of the ATGL/CGI-58 complex with the artificial intelligence software AlphaFold demonstrated that a large surface area is involved in the protein-protein interaction. Mapping important amino acids for coactivation of both proteins, ATGL and CGI-58, onto the 3D model of the complex locates these essential amino acids at the predicted ATGL/CGI-58 interface thus strongly corroborating the significance of these residues in CGI-58-mediated coactivation of ATGL.

Vibrio cholerae's ToxRS bile sensing system.

The seventh pandemic of the diarrheal cholera disease, which began in 1960, is caused by the Gram-negative bacterium Vibrio cholerae. Its environmental persistence provoking recurring sudden outbreaks is enabled by V. cholerae's rapid adaption to changing environments involving sensory proteins like ToxR and ToxS. Located at the inner membrane, ToxR and ToxS react to environmental stimuli like bile acid, thereby inducing survival strategies for example bile resistance and virulence regulation. The presented crystal structure of the sensory domains of ToxR and ToxS in combination with multiple bile acid interaction studies, reveals that a bile binding pocket of ToxS is only properly folded upon binding to ToxR. Our data proposes an interdependent functionality between ToxR transcriptional activity and ToxS sensory function. These findings support the previously suggested link between ToxRS and VtrAC-like co-component systems. Besides VtrAC, ToxRS is now the only experimentally determined structure within this recently defined superfamily, further emphasizing its significance. In-depth analysis of the ToxRS complex reveals its remarkable conservation across various Vibrio species, underlining the significance of conserved residues in the ToxS barrel and the more diverse ToxR sensory domain. Unravelling the intricate mechanisms governing ToxRS's environmental sensing capabilities, provides a promising tool for disruption of this vital interaction, ultimately inhibiting Vibrio's survival and virulence. Our findings hold far-reaching implications for all Vibrio strains that rely on the ToxRS system as a shared sensory cornerstone for adapting to their surroundings.

Cholera is a contagious diarrheal disease that leads to about 20,000 to 140,000 yearly deaths. It is caused by a bacterium called Vibrio cholerae, which can survive in harsh conditions and many environments. It often contaminates water, where it lives in an energy-conserving mode. But when humans consume Vibrio cholerae-contaminated water or food, the bacterium can sense its new environment and switch into a high-energy consuming state, causing fever, diarrhea, and vomiting. Vibrio cholerae recognizes bile acid in the human stomach, which signals that the bacterium has reached ideal conditions for causing disease. So far, it has been unclear, how exactly the bacterium detects bile acid. Understanding how these bacteria sense bile acid, could help scientists develop new ways to prevent cholera outbreaks or treat infections. Gubensäk et al. analysed two proteins from the Vibrio cholerae bacterium, called ToxR and ToxS, which are located below the bacteria's protective membrane. More detailed analyses showed that the two proteins bind together, forming a bile-binding pocket. When correctly assembled, this bile-sensing machine detects bile concentrations in the body, allowing the bacterium to adapt to the local conditions. Using crystal structures, a series of interaction studies, and modeling software, Gubensäk et al. detailed step-by-step how the two proteins sense bile acid and help the bacteria adapt and thrive in the human body. The results confirm the results of previous studies that implicated ToxR and ToxS in bile sensing and provide new details about the process. Scientists may use this information to develop new ways to interfere with the bacteria's bile-sensing and gut adaptation processes. They may also use the information to screen for existing drugs that block bile sensing and then test as cholera treatments or prevention strategies in clinical trials. New cholera treatment or prevention approaches that don't rely on antibiotics may help public health officials respond to growing numbers of cholera outbreaks and to prevent the spread of antibiotic-resistant bacteria.

The Crystal Structure of Mouse Ces2c, a Potential Ortholog of Human CES2, Shows Structural Similarities in Substrate Regulation and Product Release to Human CES1.

Members of the carboxylesterase 2 (Ces2/CES2) family have been studied intensively with respect to their hydrolytic function on (pro)drugs, whereas their physiological role in lipid and energy metabolism has been realized only within the last few years. Humans have one CES2 gene which is highly expressed in liver, intestine, and kidney. Interestingly, eight homologous Ces2 (Ces2a to Ces2h) genes exist in mice and the individual roles of the corresponding proteins are incompletely understood. Mouse Ces2c (mCes2c) is suggested as potential ortholog of human CES2. Therefore, we aimed at its structural and biophysical characterization. Here, we present the first crystal structure of mCes2c to 2.12 Å resolution. The overall structure of mCes2c resembles that of the human CES1 (hCES1). The core domain adopts an α/ß hydrolase-fold with S230, E347, and H459 forming a catalytic triad. Access to the active site is restricted by the cap, the flexible lid, and the regulatory domain. The conserved gate (M417) and switch (F418) residues might have a function in product release similar as suggested for hCES1. Biophysical characterization confirms that mCes2c is a monomer in solution. Thus, this study broadens our understanding of the mammalian carboxylesterase family and assists in delineating the similarities and differences of the different family members.

Small Things Matter: The 11.6-kDa TraB Protein is Crucial for Antibiotic Resistance Transfer Among Enterococci.

Conjugative transfer is the most important means for spreading antibiotic resistance genes. It is used by Gram-positive and Gram-negative bacteria, and archaea as well. Conjugative transfer is mediated by molecular membrane-spanning nanomachines, so called Type 4 Secretion Systems (T4SS). The T4SS of the broad-host-range inc18-plasmid pIP501 is organized in a single operon encoding 15 putative transfer proteins. pIP501 was originally isolated from a clinical Streptococcus agalactiae strain but is mainly found in Enterococci. In this study, we demonstrate that the small transmembrane protein TraB is essential for pIP501 transfer. Complementation of a markerless pIP501∆traB knockout by traB lacking its secretion signal sequence did not fully restore conjugative transfer. Pull-downs with Strep-tagged TraB demonstrated interactions of TraB with the putative mating pair formation proteins, TraF, TraH, TraK, TraM, and with the lytic transglycosylase TraG. As TraB is the only putative mating pair formation complex protein containing a secretion signal sequence, we speculate on its role as T4SS recruitment factor. Moreover, structural features of TraB and TraB orthologs are presented, making an essential role of TraB-like proteins in antibiotic resistance transfer among Firmicutes likely.

Structure of the Reductase Domain of a Fungal Carboxylic Acid Reductase and Its Substrate Scope in Thioester and Aldehyde Reduction.

The synthesis of aldehydes from carboxylic acids has long been a challenge in chemistry. In contrast to the harsh chemically driven reduction, enzymes such as carboxylic acid reductases (CARs) are considered appealing biocatalysts for aldehyde production. Although structures of single- and didomains of microbial CARs have been reported, to date no full-length protein structure has been elucidated. In this study, we aimed to obtain structural and functional information regarding the reductase (R) domain of a CAR from the fungus Neurospora crassa (Nc). The NcCAR R-domain revealed activity for N-acetylcysteamine thioester (S-(2-acetamidoethyl) benzothioate), which mimics the phosphopantetheinylacyl-intermediate and can be anticipated as the minimal substrate for thioester reduction by CARs. The determined crystal structure of the NcCAR R-domain reveals a tunnel that putatively harbors the phosphopantetheinylacyl-intermediate, which is in good agreement with docking experiments performed with the minimal substrate. In vitro studies were performed with this highly purified R-domain and NADPH, demonstrating carbonyl reduction activity. The R-domain was able to accept not only a simple aromatic ketone but also benzaldehyde and octanal, which are typically considered to be the final product of carboxylic acid reduction by CAR. Also, the full-length NcCAR reduced aldehydes to primary alcohols. In conclusion, aldehyde overreduction can no longer be attributed exclusively to the host background.

Accurate prediction of protein structures and interactions using a three-track neural network.

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.