Periplasmic Peptidoglycan Binding Proteins from Escherichia coli
Creators: name: Jalal, Ishrat Mohd.
Publisher: name: Science
Year: 2020
Subjects: subject: Peptidoglycan Layer
subject: peptidoglycan binding proteins
subject: PG
subject: Outer-membrane Protein A
subject: OmpA
subject: Peptidoglycan-associated lipoprotein
subject: Pal
subject: YfiB
subject: YfiRNB
subject: cyclic-di-GMP
subject: Biofilm
subject: solution NMR
subject: Biology--Microbiology
subject: Biology--Molecular
subject: Biology--Bioinformatics
subject: Biological Sciences

description: Gram –negative bacteria have a complex cell-envelope that is made up of the plasmamembrane, the peptidoglycan layer (PG) and the outer-membrane (OM). The role of the cell envelope is diverse where it can act as a selective chemical barrier, is responsible for defining cell shape, and allows the cell to sustain large mechanical loads. The properties of the envelope are highly dependent on the PG layer that is inter-connected to the OMvia structural proteins either through covalent or non-covalent interactions. The peptidoglycan binding proteins (PGBP) interact non-covalently with the mesodiaminopimelic acid (mDAP) residue of the peptidoglycan layer, that is unique to the Gram –ve bacteria. Several members of the PGBP proteins have previously beenidentified, such as OmpA, Pal and MotB. They all consist of a conserved OmpA_C like region in their C-terminal domain, but information on how these proteins interact within the periplasm is still emerging. As many traditional antibiotics have targeted thedestabilization of the peptidoglycan layer, the PGBPs may provide further opportunities towards the development of novel anti-infectious agents or immunomodulatory drugs. In this thesis we explore the role of the lipoprotein Pal from Escherichia coli, a bacterial target for antisepsis therapies, focusing on the OmpA_C like region. We analyzed Pal using solution NMR methods and showed that it was the only PGBP from E. coli that copurifiedwith the biosynthetic PG-precursor (PGp), similar to Haemophilus influenza Pal. In addition, NMR titration studies indicated that conserved residues of the OmpA_C like region in Pal took part in binding to various soluble peptidoglycan ligands. Ec.Pal wasalso shown to comprise of an N-terminal domain, containing a flexible linker region and an ?-1 helix, that is responsible for membrane association. This finding is important as it indicates its potential role towards dual orientation of this lipoprotein in the outermembrane. In addition, we have also studied a more recently discovered PGBP member, YfiB, another lipoprotein that is part of the YfiRNB cyclic-di-GMP regulating system. This unique cyclic dinucleotide plays a major role in bacterial biofilm formation.Through NMR analyses, we could show that wild-type YfiB from E. coli can interact with soluble peptidoglycan ligands through association with conserved residues within the OmpA_C like region, similar to Ec. Pal. Moreover, we also showed that the second messenger cyclic-di-GMP and its byproduct GMP, can bind to the peptidoglycan binding surface of YfiB, indicating its role in the possible allosteric regulation of the YfiRNB system. Furthermore, in a bioinformatics analysis the PGBPs were phylogenetically analyzed and a simplified relationship among these proteins was obtained using a set of highly homologous sequences. This improved our understanding of the relationshipsbetween YfiB and Pal, as well as with another E. coli PGBP YiaD. Lastly, an in-depth analysis of all the YfiB sequences retrieved from the Pfam database, together with an operon structural study, revealed some unique architectures of the yfiB gene locus, whichmay shed further light on its function as a member of the cyclic-di-GMP signaling system.