Co-expressed recombinant human Translin-Trax complex binds DNA

AbstractTrax, expressed alone aggregates into insoluble complexes, whereas upon co-expression with Translin becomes readily soluble and forms a stable heteromeric complex (∼430kDa) containing both proteins at nearly equimolar ratio. Based on the subunit molecular weights, estimated by MALDI-TOF-MS, the purified complex appears to comprise of either an octameric Translin plus a hexameric Trax (calculated MW 420kDa) or a heptamer each of Trax and Translin (calculated MW 425kDa) or a hexameric Translin plus an octameric Trax (calculated MW 431kDa). The complex binds single-stranded/double-stranded DNA. ssDNA gel-shifted complex shows both proteins at nearly equimolar ratio, suggesting that Translin “chaperones” Trax and forms heteromeric complex that is DNA binding competent

A membrane protein, EzrA, regulates assembly dynamics of FtsZ by interacting with the C-terminal tail of FtsZ

FtsZ polymerizes to form a dynamic ring structure called the Z-ring at the midcell of bacteria. EzrA, a membrane protein, has been shown to prevent the formation of aberrant Z-rings in the low GC Gram-positive bacteria by inhibiting FtsZ assembly. In this study, we show that Bacillus subtilis (B. subtilis) EzrA inhibited the assembly and bundling of B. subtilis FtsZ. It increased the critical concentration of FtsZ assembly and depolymerized the preformed FtsZ polymers in vitro. We obtained evidence suggesting that B. subtilis EzrA forms complex with B. subtilis FtsZ in vitro. EzrA was found to bind to FtsZ at a single site with a dissociation constant of 4.3±0.6µM. EzrA-FtsZ interaction has a significant electrostatic contribution as apparent from the effect of salt on their binding interactions. To elucidate the site of interaction between EzrA and FtsZ, we deleted 16 amino acid residues from the extreme C-terminal tail of B. subtilis FtsZ, which are conserved in FtsZ orthologues. EzrA did not inhibit the assembly of C-terminal truncated B. subtilis FtsZ. It also did not bind to the C-terminal truncated FtsZ detectably, suggesting that EzrA interacts with FtsZ through its conserved C-terminal tail residues. Further, a 17-residue synthetic peptide (365-382) of the C-terminal tail of FtsZ (CTP17) was used to probe the interaction of EzrA with the C-terminal tail of FtsZ. CTP17 bound to EzrA, inhibited the binding of EzrA to FtsZ, and surmounted the inhibitory effects of EzrA on the assembly of FtsZ in vitro. The data together showed that EzrA binds to the C-terminal tail of FtsZ. FtsA, a positive regulator of FtsZ assembly, is also known to interact with the C-terminal tail of FtsZ. The results indicated an interesting possibility that the assembly dynamics of FtsZ in the Z-ring is regulated by the competition between positive and negative regulators sharing the same binding site on FtsZ

SepF Increases the Assembly and Bundling of FtsZ Polymers and Stabilizes FtsZ Protofilaments by Binding along Its Length*

SepF (Septum Forming) protein has been recently identified through genetic studies, and it has been suggested to be involved in the division of Bacillus subtilis cells. We have purified functional B. subtilis SepF from the inclusion bodies overexpressed in Escherichia coli. Far-UV circular dichroism and fluorescence spectroscopic analysis involving the extrinsic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid suggested that the purified SepF had characteristics of folded proteins. SepF was found to promote the assembly and bundling of FtsZ protofilaments using three complimentary techniques, namely 90° light scattering, sedimentation, and transmission electron microscopy. SepF also decreased the critical concentration of FtsZ assembly, prevented the dilution-induced disassembly of FtsZ protofilaments, and suppressed the GTPase activity of FtsZ. Further, thick bundles of FtsZ protofilaments were observed using fluorescein isothiocyanate-labeled SepF (FITC-SepF). Interestingly, FITC-SepF was found to be uniformly distributed along the length of the FtsZ protofilaments, suggesting that SepF copolymerizes with FtsZ. SepF formed a stable complex with FtsZ, as evident from the gel filtration analysis. Using a C-terminal tail truncated FtsZ (FtsZΔ16) and a C-terminal synthetic peptide of B. subtilis FtsZ (366-382); we provided evidence indicating that SepF binds primarily to the C-terminal tail of FtsZ. The present work in concert with the available in vivo data support a model in which SepF plays an important role in regulating the assembly dynamics of the divisome complex; therefore, it may have an important role in bacterial cell division

Crystal structures of Drosophila mutant translin and characterization of translin variants reveal the structural plasticity of translin proteins

Translin protein is highly conserved in eukaryotes. Human translin binds both ssDNA and RNA. Its nucleic acid binding site results from a combination of basic regions in the octameric structure. We report here the first biochemical characterization of wild-type Drosophila melanogaster (drosophila) translin and a chimeric translin, and present 3.5 Å resolution crystal structures of drosophila P168S mutant translin from two crystal forms. The wild-type drosophila translin most likely exists as an octamer/decamer, and binds to the ssDNA Bcl-CL1 sequence. In contrast, ssDNA binding-incompetent drosophila P168S mutant translin exists as a tetramer. The structures of the mutant translin are identical in both crystal forms, and their C-terminal residues are disordered. The chimeric protein, possessing two nucleic acid binding motifs of drosophila translin, the C-terminal residues of human translin, and serine at position 168, attains the octameric state and binds to ssDNA. The present studies suggest that the oligomeric status of translin critically influences the DNA binding properties of translin proteins

Design of human ACE2 mimic miniprotein binders that interact with RBD of SARS-CoV-2 variants of concerns

The world of medicine demands from the research community solutions to the emerging problem of SARS-CoV-2 variants and other such potential global pandemics. With advantages of specificity over small molecule drugs and designability over antibodies, miniprotein therapeutics offers a unique solution to the threats of rapidly emerging SARS-CoV-2 variants. Unfortunately, most of the promising miniprotein binders are de novo designed and it is not viable to generate molecules for each new variant. Therefore in this study, we demonstrate a method for design of miniprotein mimics from the interaction interphase of human angiotensin converting enzyme 2 (ACE2). ACE2 is the natural interacting partner for the SARS-CoV-2 spike receptor binding domain (RBD) and acts as a recognition molecule for viral entry into the host cells. Starting with ACE2 N-terminal triple helix interaction interphase, we generated more than 70 miniprotein sequences. Employing Rosetta folding and docking scores we selected 10 promising miniprotein candidates amongst which 3 were found to be soluble in lab studies. Further, using molecular mechanics (MM) calculations on molecular dynamics (MD) trajectories we test interaction of miniproteins with RBD from various variants of concern (VOC). Presently, we report two key findings; miniproteins in this study are generated using less than 10 lab testing experiments, yet when tested through in-vitro experiments, they show submicro to nanomolar affinities towards SARS-CoV-2 RBD. Also in simulation studies, when compared with previously developed therapeutics, our miniproteins display remarkable ability to mimic ACE2 interphase; making them an ideal solution to the ever evolving problem of VOCs. Communicated by Ramaswamy H. Sarma</p

<i>In Vitro</i> Investigation Unveiling New Insights into the Antimalarial Mechanism of Chloroquine: Role in Perturbing Nucleation Events during Heme to β‑Hematin Transformation

Malaria parasites generate toxic heme during hemoglobin digestion, which is neutralized by crystallizing into inert hemozoin (β-hematin). Chloroquine blocks this detoxification process, resulting in heme-mediated toxicity in malaria parasites. However, the exact mechanism of chloroquine’s action remains unknown. This study investigates the impact of chloroquine on the transformation of heme into β-hematin. The results show that chloroquine does not completely halt the transformation process but rather slows it down. Additionally, chloroquine complexation with free heme does not affect substrate availability or inhibit β-hematin formation. Scanning electron microscopy (SEM) and X-ray powder diffraction (XRD) studies indicate that the size of β-hematin crystal particles and crystallites increases in the presence of chloroquine, suggesting that chloroquine does not impede crystal growth. These findings suggest that chloroquine delays hemozoin production by perturbing the nucleation events of crystals and/or the stability of crystal nuclei. Thus, contrary to prevailing beliefs, this study provides a new perspective on the working mechanism of chloroquine