Biology subjects

Giachin, G.

Publications and source records attributed to Giachin, G..

5 recordsLinked to original sources

bioRxiv2024

Metal ion activation and DNA recognition by the Deinococcus radiodurans manganese sensor DR2539

The accumulation of manganese ions is crucial for scavenging reactive oxygen species (ROS) and protecting the proteome of Deinococcus radiodurans (Dr). However, metal homeostasis still needs to be tightly regulated to avoid toxicity. DR2539, a dimeric transcription regulator, plays a key role in Dr manganese homeostasis. Despite comprising three well-conserved domains: a DNA binding domain, a dimerization domain, and an ancillary domain, both the metal ion activation mechanism and the DNA recognition mechanism remain elusive. In this study, we present biophysical analyses and the structure of the dimerization and DNA binding domains of DR2539 in its holo form and in complex with the 21 bp pseudo-palindromic repeat of the dr1709 promotor region. These findings shed light into the activation and recognition mechanisms. The dimer presents eight manganese binding sites that induce structural conformations essential for DNA binding. The analysis of the protein-DNA interfaces elucidates the significance of Tyr59 and helix H3 sequence in the interaction with the DNA. Finally, the structure in solution as determined by small angle X-ray scattering experiments and supported by AlphaFold modelling provides a model illustrating the conformational changes induced upon metal binding.

biophysics↗

bioRxiv2023

The acidic intrinsically disordered region of the inflammatory mediator HMGB1 mediates fuzzy interactions with chemokine CXCL12

Chemokines engage in heterodimeric interactions to activate or dampen their cognate receptors in inflammatory conditions. The chemokine CXCL12 forms with the alarmin HMGB1 a patho-physiologically relevant heterocomplex (HMGB1[bullet]CXCL12), whose formation synergically promotes the inflammatory response elicited by the G-protein coupled receptor CXCR4. However, the molecular details of complex formation were still elusive. Through an integrative structural approach (NMR, AUC, ITC, MST, SAXS) we show that HMGB1[bullet]CXCL12 represents the first fuzzy chemokines heterocomplex reported so far. HMGB1 and CXCL12 form a dynamic equimolar assembly, rather than involving one HMGB1 and two CXCL12 molecules as previously assumed, with structured and unstructured HMGB1 regions recognizing the dimerization surface of CXCL12. We uncover an unexpected role of the acidic intrinsically disordered region (IDR) in heterocomplex formation and provide the first evidence that the acidic IDR facilitates the ternary HMGB1*CXCL12*CXCR4 interaction on the cell surface. Thus, the interaction of HMGB1 with CXCL12 diverges radically from the classical rigid heterophilic chemokine-chemokine dimerization. Simultaneous interference with the multiple interactions within HMGB1[bullet]CXCL12 complex formation might offer novel pharmacological strategies to inhibit its detrimental activity in inflammatory conditions.

biochemistry↗

bioRxiv2021

A novel conformational state for SARS-CoV-2 main protease

The SARS-CoV-2 main protease (Mpro) has a pivotal role in mediating viral genome replication and transcription of coronavirus, making it a promising target for drugs against Covid-19 pandemic. Here we present a crystal structure of Mpro disclosing new structural features of key regions of the enzyme. We show that the oxyanion loop, involved in substrate recognition and enzymatic activity, can adopt a new conformation, which is stable and significantly different from the known ones. In this new state the S1 subsite of the substrate binding region is completely reshaped and a new cavity near the S2 subsite is created. This new structural information expands the knowledge of the conformational space available to Mpro, paving the way for the design of novel classes of inhibitors specifically designed to target this unprecedented binding site conformation, thus enlarging the chemical space for urgent antiviral drugs against Covid-19 pandemic.

biochemistry↗

bioRxiv2019

Structural basis for osmotic regulation of the DNA binding properties of H-NS proteins

H-NS proteins act as osmotic sensors translating changes in osmolarity into altered DNA binding properties, thus, regulating enterobacterial genome organization and genes transcription. The molecular mechanism underlying the switching process and its conservation among H-NS family members remains elusive.\n\nHere, we focus on the H-NS family protein MvaT from P. aeruginosa and demonstrate experimentally that its protomer exists in two different conformations, corresponding to two different functional states. In the half-opened state (dominant at low salt) the protein forms filaments along DNA, in the fully opened state (dominant at high salt) the protein bridges DNA. This switching is a direct effect of ionic strengths on electrostatic interactions between the appositively charged DNA binding and N-terminal domains of MvaT. The asymmetric charge distribution and intramolecular interactions are conserved among the H-NS family of proteins. Therefore, our study establishes a general paradigm for the molecular mechanistic basis of the osmosensitivity of H-NS proteins.

biochemistry↗

bioRxiv2018

Deciphering copper coordination in the animal prion protein amyloidogenic domain

Prions are pathological isoforms of the cellular prion protein (PrPC) responsible for transmissible spongiform encephalopathies (TSE). PrPC interacts with copper through unique octarepeat and non-octarepeat (non-OR) binding sites. Previous works on human PrPC suggest that copper binding to the non-OR region may have a role during prion conversion. The molecular details of copper coordination within the non-OR region are not well characterized. By means of small angle X-ray scattering (SAXS) and extended X-ray absorption fine structure (EXAFS) spectroscopy, we have investigated the Cu(II) structural effects on the protein folding and its coordination geometries when bound to the non-OR region of recombinant PrPC (recPrP) from animal species considered high or less resistant to TSE. As TSE-resistant model, we used ovine PrPC carrying the protective polymorphism at residues A136, R154 and R171 (OvPrP ARR); while as highly TSE-susceptible PrPC models we employed OvPrP with polymorphism V136, R154 and Q171 (OvPrP VRQ) and Bank vole recPrP (BvPrP). Our results reveal that Cu(II) affects the structural plasticity of the non-OR region leading to a more compacted conformation of recPrP. We also identified two Cu(II) coordinations in the non-OR region of these animal species. In type-1 coordination present in OvPrP ARR, Cu(II) is coordinated by four residues (S95, Q98, M109 and H111). Conversely, the type-2 coordination is present in OvPrP VRQ and BvPrP, where Cu(II) is coordinated by three residues (Q98, M109 and H111) and by one water molecule, making the non-OR region more flexible and open to the solvent. These changes in copper coordination in prion resistant and susceptible species provide new insights into the molecular mechanisms governing the resistance or susceptibility of certain species to TSE.

biochemistry↗