Biology subjects

Siligardi, G.

Publications and source records attributed to Siligardi, G..

4 recordsLinked to original sources

bioRxiv2026

Structure-function studies of HRIKD-{triangleup}KI, a Minimal Kinase Domain of Human Heme-Regulated Inhibitor Kinase

EIF2 kinase heme-regulated inhibitor (HRI) is a novel target for haematological malignancies with modulators reported to trigger cell death via the HRI-eIF2-ATF4 pathway. We report a protocol for producing the minimal kinase domain of full-length human HRI, termed HRIKD-{Delta}KI, where the unstructured 140 amino acid (aa) kinase insert (KI) within HRI kinase domain (HRIKD) is replaced with a 2aa glycine/serine (GS) linker. X-ray crystal structures were determined of apo-HRIKD-{Delta}KI and of its complex with ATP at 2.1 & 2.5 [A] resolution respectively. Both structures display a canonical bi-lobal kinase fold. However, they remain in a non-productive state with a displaced C-helix, disassembled R-spine, and a disordered activation segment hindering the substrate site. Biophysical assays (fluorescence based thermal shift & Synchrotron Radiation Circular Dichroism) demonstrate HRIKD-{Delta}KI retains its functional ligand-binding conformation. All together, these findings define structural and ligand-binding features of HRI to support ongoing drug discovery efforts in blood cancer.

biochemistry↗

bioRxiv2026

Protein disorder controls allostery in DNA

Metabolism, gene expression, and signaling all require the adaptation of protein activity to the mixture of reactants and products in a cell. This trait to adapt, called allostery, is hardwired in the structure of proteins. Binding a ligand at one location in a protein can change distant locations, thus tuning protein activity. How allostery works has been subject of intense research since its discovery sixty years ago. The challenge is to understand the order of events that follow ligand-binding in the three-dimensional architecture of proteins. Here we simplify this task by studying allostery in DNA, a nearly one-dimensional system. DNA can transmit allosteric signals over many nanometers to generate cooperativity in the binding of transcription factors, an archetype of the long-range action of allostery. We found that binding of the transcription factor ComK amplifies intrinsic microsecond structural fluctuations in DNA many nanometers distant from the binding site. Yet, it is not protein binding per se, but the intrinsically disordered region (IDR) of the protein that amplifies these fluctuations. IDR removal does not only rigidify DNA, but it also abolishes allostery. The result is a structurally distorted protein-DNA complex that lost its function. These findings have important implications for our understanding of transcription activation and suggest a new functional role for IDRs in transcription factors.

biophysics↗

bioRxiv2025

Engineered heat-stable variants of Trypanosoma cruzi flagellar protein Tc24 enable serological detection of Chagas disease

Chagas disease, caused by the protozoan Trypanosoma cruzi, is one of the major neglected diseases globally, killing 12,000 people per year and silently infecting an estimated 7 million people worldwide. Current diagnostic methods are limited by cost, complexity and cold-chain requirements. The T. cruzi flagellar protein Tc24 is a promising antigen for serological tests but suffers of poor solubility and heat stability. Here, we computationally engineered and produced three variants of Tc24, which exhibit remarkable heat stability, up to 69{degrees}C, and express with higher solubility in E. coli compared to the wild-type protein, reducing production costs, eliminating the need for a cold chain and therefore facilitating cost-effective production and storage without refrigeration and even in lyophilised form. These variants remained remarkably stable for 70 days in solution at 25{degrees}C and successfully detected antibodies in human sera samples from Chagas disease patients from Northern and Southern regions of Latin America, demonstrating the preservation of their antigenicity. The best-performing engineered variant was incorporated into a prototype of lateral flow test, demonstrating potential for rapid, affordable and accessible Chagas disease diagnostics in resource-limited settings.

biochemistry↗

bioRxiv2021

A conformational switch controlling the toxicity of the prion protein

Prion infections cause conformational changes of PrPC and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ("H-latch") altering the flexibility of the 2-3 and {beta}2-2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation, prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is causally linked to prion neurotoxicity.

molecular biology↗