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Engineering transcriptional regulation in Escherichia coli using an archaeal TetR-family transcription factor Vrije Universiteit Brussel

David Sybers, Amber Bernauw, Hassan Ramadan Mohamed Ahmed Maklad, Diala El Masri, Daniel Charlier, Marjan De Mey, Indra Bervoets, Eveline Peeters

Synthetic biology requires well-characterized biological parts that can be combined into functional modules. One type of biological parts are transcriptional regulators and their cognate operator elements, which enable to either generate an input-specific response or are used as actuator modules. A range of regulators has already been characterized and used for orthogonal gene expression engineering, however, previous efforts have mostly ...

Bistable expression of a toxin-antitoxin system located in a cryptic prophage of Escherichia coliO157:H7 Vrije Universiteit Brussel

Dukas Jurenas, Nathan Fraikin, Frédéric Goormaghtigh, Pieter De Bruyn, Alexandra Vandervelde, Thomas Jove, Daniel Charlier, Remy Loris, Laurence Van Melderen
Type II toxin-antitoxin systems (TA) are classically composed of two genes encoding a toxic protein and a cognate antitoxin protein. Both genes are organized in an operon whose transcription is autoregulated at the level of transcription by the antitoxin-toxin complex thanks to the antitoxin DNA-binding domain. Here we investigated the transcriptional regulation of a particular toxin-antitoxin (TA) system located in the immunity region of a ...

Nanobody-aided crystallization of the transcription regulator PaaR2 from Escherichia coli O157:H7 Vrije Universiteit Brussel

Pieter De Bruyn, Marusa Prolic Kalinsek, Alexandra Vandervelde, Milan Malfait, Yann Sterckx, Frank Sobott, San Hadzi, Els Pardon, Jan Steyaert, Remy Loris, et al.
paaR2-paaA2-parE2 is a three-component toxin-antitoxin module found in prophage CP-993P of Escherichia coli O157:H7. Transcription regulation of this module occurs via the 123 amino acid regulator PaaR2 which forms a large oligomeric structure. Despite appearing to be well-folded, PaaR2 as well as its N-terminal DNA binding domain withstand crystallization. Native mass spectrometry was used to screen for nanobodies that form a unique complex and ...

The sequence-ensemble relationships in fuzzy protein complexes Vrije Universiteit Brussel

San Hadzi, Remy Loris, Jurij Lah
Intrinsically disordered proteins (IDPs) interact with globular proteins through variety of mechanisms, resulting in structurally heterogeneous ensembles known as fuzzy complexes. While there exists a reasonable comprehension on how IDP sequence determines the unbound IDP ensemble, little is known about what shapes the structural ensembles of IDPs bound to their targets. Using a statistical-thermodynamic model we show that the target-bound ...

Alternative dimerization is required for activity and inhibition of the HEPN ribonuclease RnlA Vrije Universiteit Brussel

Gabriela Garcia Rodriguez, Daniel Charlier, Dorien Wilmaerts, Jan Michiels, Remy Loris

The rnlAB toxin-antitoxin operon from Escherichia coli functions as an anti-phage defense system. RnlA was identified as a member of the HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain) superfamily of ribonucleases. The activity of the toxin RnlA requires tight regulation by the antitoxin RnlB, the mechanism of which remains unknown. Here we show that RnlA exists in an equilibrium between two different homodimer states: an ...

Entropic pressure controls olgomerization of Vibrio cholerae ParD2 antitoxin Vrije Universiteit Brussel

Gabriela Garcia Rodriguez, Yana Girardin, Oleksandr Volkov, Ranjan Kumar Singh, Gopinath Muruganandam, Jeroen Van Dyck, Frank Sobott, Wim Versées, Daniel Charlier, Remy Loris, et al.
ParD2 is the antitoxin component of the parDE2 toxin-antitoxin module from Vibrio cholerae and consists of an ordered DNA binding domain followed by an intrinsically disordered ParE-neutralizing domain. In absence of the C-terminal IDP domain, VcParD2 crystallizes as a doughnut-shaped hexadecamer formed by the association of eight dimers. This assembly is stabilized via hydrogen bonds and salt bridges rather than hydrophobic contacts. In ...

Crystal and solution structure of NDRG1, a membrane-binding protein linked to myelination and tumour suppression Vrije Universiteit Brussel

Venla Mustonen, Gopinath Muruganandam, Remy Loris, Petri Kursula, Salla Ruskamo
N‐myc downstream‐regulated gene 1 (NDRG1) is a tumour suppressor involved in vesicular trafficking and stress response. NDRG1 participates in peripheral nerve myelination, and mutations in the NDRG1 gene lead to Charcot‐Marie‐Tooth neuropathy. The 43‐kDa NDRG1 is considered as an inactive member of the α/β hydrolase superfamily. In addition to a central α/β hydrolase fold domain, NDRG1 consists of a short N terminus and a C‐terminal region with ...

Prokaryote toxin-antitoxin modules: complex regulation of an unclear function Vrije Universiteit Brussel

Pieter De Bruyn, Yana Girardin, Remy Loris
Toxin-antitoxin (TA) modules are small operons in bacteria and archaea that encode a metabolic inhibitor (toxin) and a matching regulatory protein (antitoxin). While their biochemical activities are often well defined, their biological functions remain unclear. In type II TA modules, both toxin and antitoxin are proteins, and the antitoxin inhibits the biochemical activity of the toxin via complex formation with the toxin. The different TA ...

Arc self‐association and formation of virus‐like capsids are mediated by an N‐terminal helical coil motif Vrije Universiteit Brussel

Maria Eriksen, Oleksii Nikolaienko, Erik Hallin, Sverre Grødem, Helene Bustad, Marte Flydal, Ian Merski, Tomohisa Hosokawa, Daniela Lascu, Shreeram Akerkar, et al.

Activity-regulated cytoskeleton-associated protein (Arc) is a protein interaction hub with diverse roles in intracellular neuronal signaling, and important functions in neuronal synaptic plasticity, memory, and postnatal cortical development. Arc has homology to retroviral Gag protein and is capable of self-assembly into virus-like capsids implicated in the intercellular transfer of RNA. However, the molecular basis of Arc self-association ...

A nanobody toolbox targeting dimeric coiled-coil modules for functionalization of designed protein origami structures Vrije Universiteit Brussel

Andrea Majerle, San Hadzi, Jana Aupič, Tadej Satler, Fabio Lapenta, Žiga Strmšek, Jurij Lah, Remy Loris, Roman Jerala
Coiled-coils (CC) are widely used in protein design since their sequence-structure relationship is quite well-understood. In the CC protein origami (CCPO) approach a polypeptide chain is composed from a defined sequence of orthogonal CC building segments which fold into different polyhedral shapes such as tetrahedron, triangular prism, and four-sided pyramid. While noninteracting residues of the CCs can be engineered, additional ...