Biology subjects

Alcorlo, M.

Publications and source records attributed to Alcorlo, M..

4 recordsLinked to original sources

bioRxiv2024

Structural insights into peptidoglycan hydrolysis by the FtsEX system in Escherichia coli during cell division

Bacterial cell division relies on precise peptidoglycan (PG) remodelling, a process orchestrated by the FtsEX complex. Comprised of FtsE and FtsX, this complex collaborates with EnvC, a periplasmic lytic enzyme activator, to regulate septal PG hydrolysis by amidases like AmiB. While recent structural investigations, particularly of Pseudomonas aeruginosa FtsEX (PaeFtsEX), have shed light on complex interactions and proposed activation mechanisms, the structural intricacies governing PG degradation by the FtsEX complex and EnvC in Escherichia coli cytokinesis remain unexplored. In this study, we present a comprehensive biochemical and structural analysis of E. coli FtsEX complexes, unveiling a key role for ATP in complex stabilization that extends across bacterial species. Upon EnvC binding, ATPase activity markedly increases. High-resolution structures of EcoFtsEX, both in the presence and absence of EnvC, reveal a symmetrical conformation of EcoFtsEX capable of accommodating the inherent asymmetry of EnvC, mediated by flexible loops within the periplasmic domain. Our negative-staining imaging showcases an elongated EcoFtsEX/EnvC/AmiB complex reminiscent of the PaeFtsEX system. These findings collectively provide intricate insights into the regulation of PG cleavage by FtsEX in E. coli - a pivotal model system used in pilot genetic studies, suggesting a conserved mechanism for precise hydrolase activation in bacteria.

microbiology↗

bioRxiv2020

Structural characterization of the essential cell division protein FtsE and its interaction with FtsX in Streptococcus pneumoniae

FtsEX is a membrane complex widely conserved across diverse bacterial genera and involved in critical processes such as recruitment of division proteins and in spatial and temporal regulation of muralytic activity during cell division or sporulation. FtsEX is a member of the ABC transporter superfamily, where FtsX is an integral membrane protein and FtsE is an ATPase, required for mechanotransmission of the signal from the cytosol through the membrane, to regulate the activity of cell-wall hydrolases in the periplasm. Both proteins are essential in the major human respiratory pathogenic bacterium, Streptococcus pneumoniae and interact with the modular peptidoglycan hydrolase PcsB at the septum. Here, we report the high-resolution structures of pneumococcal FtsE in complex with different nucleotides. Structural analysis reveals that FtsE contains all the conserved structural motifs associated with ATPase activity, and allowed interpretation of the in vivo dimeric arrangement in both ADP and ATP states. Interestingly, three specific FtsE regions were identified with high structural plasticity that shape the cavity in which the cytosolic region of FtsX would be inserted. The residues corresponding to the FtsX coupling helix, responsible for FtsE contact, were identified and validated by in vivo mutagenesis studies showing that this interaction is essential for cell growth and proper morphology. IMPORTANCEBacterial cell division is a central process that requires exquisite orchestration of both the cell wall biosynthetic and lytic machineries. The essential membrane complex FtsEX, widely conserved across bacteria, play a central role by recruiting proteins to the divisome apparatus and by regulating periplasmic muralytic activity from the cytosol. FtsEX is a member of the Type VII family of the ABC-superfamily but instead transporter, couple ATP hydrolysis by FtsE to mechanically transduce a conformational signal to activate PG hydrolases. So far, no structural information is available for FtsE. Here we provide the structural characterization of FtsE confirming its ATPase nature and revealing regions with high structural plasticity key for FtsX binding. The complementary region in FtsX has been also identified and validated in vivo. Our results provide evidences on how difference between ATP and ADP states in FtsE would dramatically alter FtsEX interaction with PG hydrolase PcsB in pneumococcal division.

biochemistry↗

bioRxiv2019

Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall.

In oval shaped Streptococcus pneumoniae, septal and longitudinal peptidoglycan synthesis is performed by independent functional complexes; the divisome and the elongasome. Penicillin binding proteins (PBPs) were long considered as the key peptidoglycan synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class B PBPs work together with non-PBP glycosyltransferases (FtsW and RodA) to make up the core peptidoglycan synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question. Here we utilize the peptidoglycan hydrolase CbpD to show that class A PBPs have an autonomous role during cell wall synthesis in S. pneumoniae. Purified CbpD was shown to target the septum of S. pneumoniae cells. Using assays to specifically inhibit PBP2x, we demonstrate that CbpD specifically target nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes or repairs nascent peptidoglycan synthesized by FtsW/PBP2x. Our results support a model in which pneumococcal peptidoglycan is made by three functional entities, the divisome, the elongasome and a peptidoglycan-repairing or -remodelling complex consisting of bifunctional PBPs. To our knowledge this is the first time a specific function has been identified for class A PBPs in bacterial cell wall synthesis.

microbiology↗

bioRxiv2018

Structure of the large extracellular loop of FtsX and its interaction with the essential peptidoglycan hydrolase PcsB in Streptococcus pneumoniae

Streptococcus pneumoniae is a leading killer of infants and immunocompromised adults and has become increasingly resistant to major antibiotics. Therefore, the development of new antibiotic strategies is desperately needed. Targeting bacterial cell division is one such strategy, specifically targeting essential proteins for the synthesis and breakdown of peptidoglycan. One complex important to this process is FtsEX. FtsEX comprises an integral membrane protein (FtsX) and cytoplasmic ATPase (FtsE) that resembles an ATP-binding cassette (ABC) transporter. Here, we present NMR solution structural and crystallographic models of the large extracellular domain of FtsX, denoted ECL1. The structure of ECL1 reveals an upper extended {beta}-hairpin and a lower -helical lobe, each extending from a mixed -{beta} core. The helical lobe mediates a physical interaction with the peptidoglycan hydrolase PcsB, via the coiled-coil domain of PcsB (PcsB-CC). Characterization of S. pneumoniae D39 derived strains harboring mutations in the -helical lobe shows that this subdomain is essential for cell viability and required for proper cell division of S. pneumoniae.\n\nIMPORTANCEFtsX is a ubiquitous bacterial integral membrane protein involved in cell division that regulates the activity of peptidoglycan (PG) hydrolases. FtsX is representative of a large group of ABC3 superfamily proteins that function as \"mechanotransmitters,\" proteins that relay signals from inside to the outside of the cell. Here we present a structural characterization of the large extracellular loop (ECL1) of FtsX from the human opportunistic pathogen Streptococcus pneumoniae. We show a direct interaction between the peptidoglycan hydrolase PcsB and FtsX, and demonstrate that this interaction is essential for cell viability. As such, FtsX represents an attractive, conserved target for the development of new classes of antibiotics.

microbiology↗