Bacillus subtilis HelD, an RNA Polymerase Interacting Helicase, Forms Amyloid-Like Fibrils

HelD, an RNA polymerase binding protein from Bacillus subtilis, stimulates transcription and helps in timely adaptation of cells under diverse environmental conditions. At present, no structural information is available for HelD. In the current study, we performed size exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS) which suggests that HelD is predominantly monomeric and globular in solution. Using combination of size exclusion chromatography and analytical ultracentrifugation, we also show that HelD has a tendency to form higher order oligomers in solution. CD experiments suggest that HelD has both α-helical (∼35%) and β sheet (∼26%) secondary structural elements. Thermal melting experiments suggest that even at 90°C, there is only about 30% loss in secondary structural contents with Tm of 44°C. However, with the increase in temperature, there was a gain in the β-sheet content and significant irreversible loss of α-helical content. Using a combination of X-ray fiber diffraction analysis, and dye based assays including Thioflavin-T based fluorescence and Congo red binding assays, we discovered that HelD forms amyloid-like fibrils at physiologically relevant conditions in vitro. Using confocal imaging, we further show that HelD forms amyloid inclusions in Escherichia coli. Bioinformatics-based sequence analysis performed using three independent web-based servers suggests that HelD has more than 20 hot-spots spread across the sequence that may aid the formation of amyloid-like fibrils. This discovery adds one more member to the growing list of amyloid or amyloid-like fibril forming cytosolic proteins in bacteria. Future studies aimed at resolving the function of amyloid-like fibrils or amyloid inclusions may help better understand their role, if any, in the bacterial physiology

Mycobacterium tuberculosis CarD, an essential global transcriptional regulator forms amyloid-like fibrils

CarD is an essential global transcription regulator from Mycobacterium tuberculosis (Mtb) that binds RNA polymerase and activates transcription by stabilizing the transcription initiation complex. Available crystal structures have captured two distinct, monomeric and domain-swapped homodimeric, oligomeric states of CarD. However, the actual oligomeric state of CarD in solution and its biological relevance has remained unclear. Here, we confirm the presence of the homodimeric state of CarD in solution by using synchrotron-based small-angle X-ray scattering. Furthermore, by using biochemical and biophysical experiments, in addition to mass-spectrometry, transmission electron microscopy, and confocal imaging, we show that CarD is the first soluble cytosolic protein in Mtb which displays the tendency to form amyloid-like fibrils both in vitro as well as in vivo. We demonstrate that the deletion of the fourteen N-terminal residues involved in domain-swapping hampers amyloid formation, thus, suggesting that domain-swapping is crucial in amyloidogenesis. The discovery of the amyloidogenic property of an essential cytosolic global transcription regulator, CarD, in a pathogenic bacteria will further open up new frontiers in research.Peer reviewe

Structural Basis of <i>Clostridium perfringens</i> Enterotoxin Activation and Oligomerization by Trypsin

Clostridium perfringens enterotoxin (CpE) is a β-pore forming toxin that disrupts gastrointestinal homeostasis in mammals by binding membrane protein receptors called claudins. Although structures of CpE fragments bound to claudins have been determined, the mechanisms that trigger CpE activation and oligomerization that lead to the formation of cytotoxic β-pores remain undetermined. Proteolysis of CpE in the gut by trypsin has been shown to play a role in this and subsequent cytotoxicity processes. Here, we report solution structures of full-length and trypsinized CpE using small-angle X-ray scattering (SAXS) and crystal structures of trypsinized CpE and its C-terminal claudin-binding domain (cCpE) using X-ray crystallography. Mass spectrometry and SAXS uncover that removal of the CpE N-terminus by trypsin alters the CpE structure to expose areas that are normally unexposed. Crystal structures of trypsinized CpE and cCpE reveal unique dimer interfaces that could serve as oligomerization sites. Moreover, comparisons of these structures to existing ones predict the functional implications of oligomerization in the contexts of cell receptor binding and β-pore formation. This study sheds light on trypsin’s role in altering CpE structure to activate its function via inducing oligomerization on its path toward cytotoxic β-pore formation. Its findings can incite new approaches to inhibit CpE-based cytotoxicity with oligomer-disrupting therapeutics

Potential Inhibitors of SARS-CoV-2 Main Protease (M^pro) Identified from the Library of FDA-Approved Drugs Using Molecular Docking Studies

The Corona Virus Infectious Disease-2019 (COVID-19) outbreak originated at Wuhan, China, in December 2019. It has already spread rapidly and caused more than 6.5 million deaths worldwide. Its causal agent is a beta-coronavirus named SARS-CoV-2. Many efforts have already been made to develop new vaccines and drugs against these viruses, but over time, it has changed its molecular nature and evolved into more lethal variants, such as Delta and Omicron. These will lead us to target its more-conserved proteins. The sequences’ BLAST and crystal structure of the main protease Mpro suggest a high sequence and structural conservation. Mpro is responsible for the proteolytic maturation of the polyprotein essential for the viral replication and transcription, which makes it an important drug target. Discovery of new drug molecules may take years before getting to the clinics. So, considering urgency, we performed molecular docking studies using FDA-approved drugs to identify molecules that could potentially bind to the substrate-binding site and inhibit SARS-CoV-2’s main protease (Mpro). We used the Glide module in the Schrödinger software suite to perform molecular docking studies, followed by MM-GBSA-based energy calculations to score the hit molecules. Molecular docking and manual analysis suggest that several drugs may bind and potentially inhibit Mpro. We also performed molecular simulations studies for selected compounds to evaluate protein–drug interactions. Considering bioavailability, lesser toxicity, and route of administration, some of the top-ranked drugs, including lumefantrine (antimalarial), dipyridamole (coronary vasodilator), dihydroergotamine (used for treating migraine), hexoprenaline (anti asthmatic), riboflavin (vitamin B2), and pantethine (vitamin B5) may be taken forward for further in vitro and in vivo experiments to investigate their therapeutic potential