Shedding Light on the Interaction of Human Anti-Apoptotic Bcl-2 Protein with Ligands through Biophysical and in Silico Studies.

Bcl-2 protein is involved in cell apoptosis and is considered an interesting target for anti-cancer therapy. The present study aims to understand the stability and conformational changes of Bcl-2 upon interaction with the inhibitor venetoclax, and to explore other drug-target regions. We combined biophysical and in silico approaches to understand the mechanism of ligand binding to Bcl-2. Thermal shift assay (TSA) and urea electrophoresis showed a significant increase in protein stability upon venetoclax incubation, which is corroborated by molecular docking and molecular dynamics simulations. An 18 °C shift in Bcl-2 melting temperature was observed in the TSA, corresponding to a binding affinity multiple times higher than that of any other reported Bcl-2 inhibitor. This protein-ligand interaction does not implicate alternations in protein conformation, as suggested by SAXS. Additionally, bioinformatics approaches were used to identify deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) of Bcl-2 and their impact on venetoclax binding, suggesting that venetoclax interaction is generally favored against these deleterious nsSNPs. Apart from the BH3 binding groove of Bcl-2, the flexible loop domain (FLD) also plays an important role in regulating the apoptotic process. High-throughput virtual screening (HTVS) identified 5 putative FLD inhibitors from the Zinc database, showing nanomolar affinity toward the FLD of Bcl-2.

Structural insights of an LCP protein-LytR-from Streptococcus dysgalactiae subs. dysgalactiae through biophysical and in silico methods.

The rise of antibiotic-resistant bacterial strains has become a critical health concern. According to the World Health Organization, the market introduction of new antibiotics is alarmingly sparse, underscoring the need for novel therapeutic targets. The LytR-CpsA-Psr (LCP) family of proteins, which facilitate the insertion of cell wall glycopolymers (CWGPs) like teichoic acids into peptidoglycan, has emerged as a promising target for antibiotic development. LCP proteins are crucial in bacterial adhesion and biofilm formation, making them attractive for disrupting these processes. This study investigated the structural and functional characteristics of the LCP domain of LytR from Streptococcus dysgalactiae subsp. dysgalactiae. The protein structure was solved by X-ray Crystallography at 2.80 Å resolution. Small-angle X-ray scattering (SAXS) data were collected to examine potential conformational differences between the free and ligand-bound forms of the LytR LCP domain. Additionally, docking and molecular dynamics (MD) simulations were used to predict the interactions and conversion of ATP to ADP and AMP. Experimental validation of these predictions was performed using malachite green activity assays. The determined structure of the LCP domain revealed a fold highly similar to those of homologous proteins while SAXS data indicated potential conformational differences between the ligand-free and ligand-bound forms, suggesting a more compact conformation during catalysis, upon ligand binding. Docking and MD simulations predicted that the LytR LCP domain could interact with ADP and ATP and catalyze their conversion to AMP. These predictions were experimentally validated by malachite green activity assays, confirming the protein's functional versatility. The study provides significant insights into the structural features and functional capabilities of the LCP domain of LytR from S. dysgalactiae subsp. dysgalactiae. These findings pave the way for designing targeted therapies against antibiotic-resistant bacteria and offer strategies to disrupt bacterial biofilm formation.

Screening of Buffers and Additives for Protein Stabilization by Thermal Shift Assay: A Practical Approach.

Thermal shift assay (TSA), also commonly designed by differential scanning fluorimetry (DSF) or ThermoFluor, is a technique relatively easy to implement and perform, useful in a myriad of applications. In addition to versatility, it is also rather inexpensive, making it suitable for high-throughput approaches. TSA uses a fluorescent dye to monitor the thermal denaturation of the protein under study and determine its melting temperature (Tm). One of its main applications is to identify the best buffers and additives that enhance protein stability.Understanding the TSA operating mode and the main methodological steps is a central key to designing effective experiments and retrieving meaningful conclusions. This chapter intends to present a straightforward TSA protocol, with different troubleshooting tips, to screen effective protein stabilizers such as buffers and additives, as well as data treatment and analysis. TSA results provide conditions in which the protein of interest is stable and therefore suitable to carry out further biophysical and structural characterization.

Using Small-angle X-ray Scattering to Characterize Biological Systems: A General Overview and Practical Tips.

Small-angle X-ray Scattering (SAXS) is a versatile and powerful technique with applications in a wide range of fields. The continuous improvements in hardware, data analysis software, and standards for validation significantly contributed to increase its popularity and, nowadays, SAXS is a well-established method. SAXS allows to study flexible and dynamic systems (e.g., proteins and other biomolecules) in solution, providing information about their size and shape. Contrary to other structural characterization methods, SAXS has no limitations on the size of the particle under study and can be used in integrated approaches to reveal important insights otherwise difficult to obtain regarding folding-unfolding, conformational changes, movement of flexible regions, and the formation of complexes.This chapter, in addition to a concise overview on the methodology, intends to systematically enumerate the main steps involved in sample preparation and data collection, processing and analysis including useful practical notes to identify and overcome common bottlenecks. This way, a less experienced user can use the content of the chapter as a starting point to properly design and perform a successful SAXS experiment.

Singularities of Pyogenic Streptococcal Biofilms - From Formation to Health Implication.

Biofilms are generally defined as communities of cells involved in a self-produced extracellular matrix adhered to a surface. In biofilms, the bacteria are less sensitive to host defense mechanisms and antimicrobial agents, due to multiple strategies, that involve modulation of gene expression, controlled metabolic rate, intercellular communication, composition, and 3D architecture of the extracellular matrix. These factors play a key role in streptococci pathogenesis, contributing to therapy failure and promoting persistent infections. The species of the pyogenic group together with Streptococcus pneumoniae are the major pathogens belonging the genus Streptococcus, and its biofilm growth has been investigated, but insights in the genetic origin of biofilm formation are limited. This review summarizes pyogenic streptococci biofilms with details on constitution, formation, and virulence factors associated with formation.