Search Results (4)

A robust cell envelope is the first line of protection for an infecting pathogen when encountering the immune defense of its host. In Gram-positive organisms, LytR-CpsA-Psr (LCP) family proteins play a major role in the synthesis and assembly of the cell envelope. While these proteins could be considered for potential new drug targets, not enough is known about how they function to support the integrity of the cell wall. Streptococcus agalactiae (group B streptococcus or GBS) is known to encode at least three LCP family proteins, including CpsA, LytR (BrpA) and Psr. Using strains of GBS that have mutations in two of the three LCP proteins, we were able to determine a role for these proteins in GBS cell wall integrity. The results presented here demonstrate that the absence of Psr results in a decreased growth rate, decreased viability over time, inconsistent cocci morphology and diminished cell wall integrity, as well as an increased penicillin susceptibility, decreased capsule levels and attenuation in virulence in a zebrafish model of infectious disease. A strain that is missing two of the LCP family proteins, CpsA and Psr, exhibits an increase in these defective phenotypes, indicating that CpsA and Psr are partially redundant in function. Full article

The human transcription factor FOXO3 (a member of the ‘forkhead’ family of transcription factors) controls a variety of cellular functions that make it a highly relevant target for intervention in anti-cancer and anti-aging therapies. FOXO3 is a mostly intrinsically disordered protein (IDP). Absence of knowledge of its structural properties outside the DNA-binding domain constitutes a considerable obstacle to a better understanding of structure/function relationships. Here, I present extensive molecular dynamics (MD) simulation data based on implicit solvation models of the entire FOXO3/DNA complex, and accelerated MD simulations under explicit solvent conditions of a central region of particular structural interest (FOXO3^120–530). A new graphical tool for studying and visualizing the structural diversity of IDPs, the Local Compaction Plot (LCP), is introduced. The simulations confirm the highly disordered nature of FOXO3 and distinguish various degrees of folding propensity. Unexpectedly, two ‘linker’ regions immediately adjacent to the DNA-binding domain are present in a highly extended conformation. This extended conformation is not due to their amino acid composition, but rather is caused by electrostatic repulsion of the domains connected by the linkers. FOXO3 is thus an IDP present in an unusually extended conformation to facilitate interaction with molecular interaction partners. Full article

Membrane proteins are an important class of macromolecules found in all living organisms and many of them serve as important drug targets. In order to understand their biological and biochemical functions and to exploit them for structure-based drug design, high-resolution and accurate structures of membrane proteins are needed, but are still rarely available, e.g., predominantly from X-ray crystallography, and more recently from single particle cryo-EM — an increasingly powerful tool for membrane protein structure determination. However, while protein-lipid interactions play crucial roles for the structural and functional integrity of membrane proteins, for historical reasons and due to technological limitations, until recently, the primary method for membrane protein crystallization has relied on detergents. Bicelle and lipid cubic phase (LCP) methods have also been used for membrane protein crystallization, but the first step requires detergent extraction of the protein from its native cell membrane. The resulting, crystal structures have been occasionally questioned, but such concerns were generally dismissed as accidents or ignored. However, even a hint of controversy indicates that methodological drawbacks in such structural research may exist. In the absence of caution, structures determined using these methods are often assumed to be correct, which has led to surprising hypotheses for their mechanisms of action. In this communication, several examples of structural studies on membrane proteins or complexes will be discussed: Resistance-Nodulation-Division (RND) family transporters, microbial rhodopsins, Tryptophan-rich Sensory Proteins (TSPO), and Energy-Coupling Factor (ECF) type ABC transporters. These analyses should focus the attention of membrane protein structural biologists on the potential problems in structure determination relying on detergent-based methods. Furthermore, careful examination of membrane proteins in their native cell environments by biochemical and biophysical techniques is warranted, and completely detergent-free systems for membrane protein research are crucially needed. Full article

Lactobacillus plantarum is one of the most predominant species in the human gut microbiota of healthy individuals. We have previously characterized some probiotic features of L. plantarum LM3, as the high resistance to different stress, the binding ability toward some extracellular matrix proteins and plasminogen and the immunomodulatory role of the surface expressed adhesin EnoA1. We have also identified the flmA, flmB and flmC genes, coding for putative proteins named FlmA, FlmB and FlmC, whose null mutations partially impaired biofilm development; the L. plantarum LM3–6 strain, carrying a deletion in flmC, showed a high rate of autolysis, supporting the hypothesis that FlmC might be involved in cell wall integrity. Here, we report the in-silico characterization of ΔTM-FlmC, a portion of the FlmC protein. The protein has been also expressed, purified and characterized by means of CD spectroscopy, ICP-mass and UHPLC-HRMS. The obtained experimental data validated the predicted model unveiling also the presence of a bound lipid molecule and of a Mg(II) ion. Overall, we provide strong evidences that ΔTM-FlmC belongs to the LytR-CpsA-Psr (LCP) family of domains and is involved in cell envelope biogenesis. Full article