Biology subjects

Kojic, M.

Publications and source records attributed to Kojic, M..

4 recordsLinked to original sources

bioRxiv2025

Charge segregation in the C-terminal linker of FtsZ enables Z-ring scaling with prokaryotic cell width

Bacterial cytokinesis is orchestrated by the Z-ring, a cytoskeletal structure formed by treadmilling filaments of the tubulin-like GTPase FtsZ. During assembly, filament curvature must match the cell diameter, yet how curvature is encoded in FtsZ remains unclear. Using in vitro reconstitution with purified proteins and supported lipid bilayers, we identify FtsZs intrinsically disordered C-terminal linker (CTL) as a curvature tuner that determines the Z-ring diameter. Increasing CTL charge segregation ({kappa}) progressively reduces Z-ring diameter without altering treadmilling dynamics, monomer turnover, or GTPase activity, indicating that {kappa} modulates filament curvature rather than polymerization kinetics. Linker-dilution experiments, in which wild-type FtsZ is mixed with a CTL-deletion mutant ({Delta}CTL), further show that inter-CTL electrostatic interactions straighten filaments, while weakening these contacts increases curvature and compacts rings. To test whether this mechanism is conserved, we analyzed 4,603 prokaryotic FtsZ sequences and found that CTL charge segregation quantified by {kappa} - but not CTL length, fraction of charged residues, or net charge per residue - correlates with cell width: at low {kappa}, cells show a broad range of widths, whereas high {kappa} values exclude wide cells. Co-expression of {Delta}CTL variants in vivo perturbs Z-ring positioning and impairs growth, consistent with a curvature mismatch and defects during assembly. Together, these results suggest a simple evolutionary mechanism in which CTL charge patterning encodes intrinsic filament curvature, enabling Z-ring geometry to scale with cell diameter and linking protein sequences to bacterial morphology.

biochemistry↗

bioRxiv2023

PRC domain-containing proteins modulate FtsZ-based archaeal cell division

Dividing cells into two daughter cells is a complicated process that in bacteria and eukaryotes requires many proteins to work together. For archaea that divide via an FtsZ-based mechanism, only three proteins of the cell division machinery could so far be identified. These are two tubulin homologs, FtsZ1, FtsZ2 and the membrane anchor of FtsZ2, SepF. Here, we investigate additional archaeal cell division proteins that were identified by immunoprecipitation of SepF. These proteins comprise a single PRC-barrel domain and strictly co-occur with FtsZ. Two out of three PRC-barrel domain containing proteins found in Haloferax volcanii, CdpB1 and CdpB2 localize to the site of cell division in a SepF-dependent manner. Moreover, depletions and deletions cause severe cell division defects, generating drastically enlarged cells. Fluorescence microscopy of tagged FtsZ1, FtsZ2 and SepF in CdpB1/2 deletion strains revealed that the divisome is unusually disordered and not organized into a distinct ring-like structure at the cell centre. Biochemical analysis of CdpB homologs from different archaeal species showed that SepF interacts directly with CdpB1, which in turn binds to CdpB2, forming a tripartite complex. A crystal structure of CdpB1 and B2 recapitulated these interactions and suggested how these proteins might form filaments, possibly aligning SepF and therefore the FtsZ2 ring during cell division. In summary, we demonstrate that PRC domain proteins play essential roles in FtsZ based cell division in archaea.

microbiology↗

bioRxiv2021

Construction of redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides

Many protein expression and purification systems are commercially available to provide a sufficient amount of pure, soluble and active native protein, such as the pMAL system based on E. coli maltose binding protein tag (MBP). Adding specific amino acid tags to the N- or C-terminus of the protein increases solubility and facilitates affinity purification of proteins. However, many of expressed tagged proteins consequently lose functionality, particularly small peptides such as antimicrobial peptides (AMPs). Objective of this study was to redesign the pMAL expression vector in order to increase the efficacy of MBP tag separation from native peptides. Redesign of the pMAL expression vector included introduction of the His6 tag and the enterokinase cleavage site downstream from the original MBP tag and Xa cleavage site enabling purification of native and active peptide (P) following two-step affinity chromatography. In the first step the entire MBP-His6-P fusion protein is purified through binding to Ni-NTA agarose. In the second step, the purification was performed by adding mixture of amylose and Ni-NTA agarose resins following cleavage of the fusion protein with active His6 tagged enterokinase. This removes MBP-His6 and His6-enterokinase leaving pure native protein in solution. The redesigned pMAL vectors were optimized for cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides expression. Two-step purification protocol was successfully applied in purification of active native AMPs, lactococcin A and human {beta}-defensin. Taken together, we established the optimal conditions and pipeline for overexpression and purification of large amount of native peptides, that can be implemented in any laboratory.

bioengineering↗

bioRxiv2018

Lactococcin B is inactivated by intrinsic proteinase PrtP digestion in Lactococcus lactis subsp. lactis BGMN1-501

In our previous study, we showed that PrtP is able to impair bacteriocin LcnB activity despite being produced by the same organism, and even if they were encoded by the same plasmid. However, exact cleavage site within LcnB bacteriocin, as well as the activity of the resulting peptides remained unknown. Here we further explored the interplay between these two proteins and defined, using mass spectrometry, that the hydrolysis occurs between the sixth and seventh amino acid on the N terminus of LcnB. Although it was suspected that the cleaved form of LcnB could retain some level of activity, both chemically synthesized and recombinant variant of truncated LcnB exhibited no antimicrobial activity. Wild type form of LcnB was recombinantly overexpressed using the same expression system, its antimicrobial activity was tested before and after the treatment with PrtP proteinase, and the degradation products were analyzed with reverse-phase high-pressure liquid chromatography. The results confirmed the inactivity of the truncated LcnB and additionally corroborated the PrtP cleavage site in LcnB bacteriocin.\n\nImportanceLactococcal enzyme PrtP, considered as a growth promoting factor, is involved in casein breakdown and enabling bacteria efficient growth in amino acids-poor, but protein-rich media. However, its interaction with bacteriocins was not known until recently. Bacteriocin LcnB can also be considered as growth promoting factor, since its known physiological role mirrors in preventing competing bacteria of reaching high growth densities. In this manuscript, we define the exact peptide bond inside the bacteriocin LcnB which is recognized by PrtP. This N-terminal removal of six amino acids completely inactivates the bacteriocin. The biological function of such action remains elusive. It is unexpected that in the same strain one enzyme inactivates a protein important for survival, unless it is some type of regulation.

microbiology↗