Biology subjects

Gjorgjevikj, D.

Publications and source records attributed to Gjorgjevikj, D..

3 recordsLinked to original sources

bioRxiv2023

Sm-like protein Rof inhibits transcription termination factor Rho by binding site obstruction and conformational insulation

Transcription termination factor {rho} is a hexameric, RNA-dependent NTPase that can adopt active closed-ring and inactive open-ring conformations. The Sm-like protein Rof, a homolog of the RNA chaperone Hfq, inhibits {rho}-dependent termination in vivo but recapitulation of this activity in vitro has proven difficult and the precise mode of Rof action is presently unknown. Our electron microscopic structures of {rho}-Rof and {rho}-RNA complexes show that Rof undergoes pronounced conformational changes to bind {rho} at the protomer interfaces, undercutting {rho} conformational dynamics associated with ring closure and occluding extended primary RNA-binding sites that are also part of interfaces between {rho} and RNA polymerase. Consistently, Rof impedes {rho} ring closure, {rho}-RNA interactions, and {rho} association with transcription elongation complexes. Structure-guided mutagenesis coupled with functional assays confirmed that the observed {rho}-Rof interface is required for Rof-mediated inhibition of cell growth and {rho}-termination in vitro. Bioinformatic analyses revealed that Rof is restricted to Pseudomonadota and that the {rho}-Rof interface is conserved. Genomic contexts of rof differ between Enterobacteriaceae and Vibrionaceae, suggesting distinct modes of Rof regulation. We hypothesize that Rof and other cellular anti-terminators silence {rho} under diverse, but yet to be identified, stress conditions when unrestrained transcription termination by {rho} would be lethal.

biochemistry↗

bioRxiv2023

Widespread gene regulator Psu inhibits transcription termination factor rho by forced hyper-oligomerization

Many bacteriophages modulate the host transcription machinery for efficient expression of their own genomes. Phage P4 polarity suppression protein, Psu, is a building block of the viral capsid and inhibits the hexameric transcription termination factor, {rho}, by presently unknown mechanisms. We elucidated cryogenic electron microscopy structures of {rho}-Psu complexes, showing that Psu dimers laterally clamp two inactive, open {rho} rings and promote their expansion to higher-oligomeric states. Systematic ATPase, nucleotide binding and nucleic acid binding studies revealed that Psu hinders {rho} ring closure and traps nucleotides in their binding pockets on {rho}. Structure-guided mutagenesis in combination with growth, pull-down and termination assays further delineated the functional {rho}-Psu interfaces. Bioinformatic analyses suggested that, in addition to guarding its own genome against {rho}, Psu enables expression of diverse phage-defense systems commonly found in P4-like mobile genetic elements across bacteria. Thus, Psu is a widespread gene regulator that inhibits {rho} via forced hyper-oligomerization.

molecular biology↗

bioRxiv2021

Target recognition in tandem WW domains: complex structures for parallel and antiparallel ligand orientation in h-FBP21 tandem WW

Protein-protein interactions often rely on specialized recognition domains, such as WW domains, which bind to specific proline-rich sequences. The specificity of these protein-protein interactions can be increased by tandem repeats, i.e. two WW domains connected by a linker. With a flexible linker, the WW domains can move freely with respect to each other. Additionally, the tandem WW domains can bind in two different orientations to their target sequences. This makes the elucidation of complex structures of tandem WW domains extremely challenging. Here, we identify and characterize two complex structures of the tandem WW domain of human formin-binding protein 21 and a peptide sequence from its natural binding partner, the core-splicing protein SmB/B. The two structures differ in the ligand orientation, and consequently also in the relative orientation of the two WW domains. We analyze and probe the interactions in the complexes by molecular simulations and NMR experiments. The workflow to identify the complex structures uses molecular simulations, density-based clustering and peptide docking. It is designed to systematically generate possible complex structures for repeats of recognition domains. These structures will help us to understand the synergistic and multivalency effects that generate the astonishing versatility and specificity of protein-protein interactions.

molecular biology↗