This review is an attempt to retrace the chronicle that starts from the discovery of the role of nickel as the essential metal ion in urease for the enzymatic catalysis of urea, a key step in the biogeochemical cycle of nitrogen on Earth, to the most recent progress in understanding the chemistry of this historical enzyme. Data and facts are presented through the magnifying lenses of the authors, using their best judgment to filter and elaborate on the many facets of the research carried out on this metalloenzyme over the years. The tale is divided in chapters that discuss and describe the results obtained in the subsequent leaps in the knowledge that led from the discovery of a biological role for Ni to the most recent advancements in the comprehension of the relationship between the structure and function of urease. This review is intended not only to focus on the bioinorganic chemistry of this beautiful metal-based catalysis, but also, and maybe primarily, to evoke inspiration and motivation to further explore the realm of bio-based coordination chemistry.

Human α-Synuclein (αSyn) is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of αSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between αSyn's unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy–based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant αSyn. The conformational heterogeneity of monomeric αSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and “β-like” structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the “β-like” structures significantly increased in different conditions promoting the aggregation of αSyn: the presence of Cu²⁺, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric αSyn conformers, positively correlated with conditions known to promote the formation of aggregates. A new tool is thus made available to test directly the influence of mutations and pharmacological strategies on the conformational equilibrium of monomeric αSyn. Natively unstructured proteins defy the classical “one sequence–one structure” paradigm of protein science. In pathological conditions, monomers of these proteins can aggregate in the cell, a process that underlies neurodegenerative diseases such as Alzheimer and Parkinson. A key step in the aggregation process—the formation of misfolded intermediates—remains obscure. To shed light on this process, we characterized the folding and conformational diversity of αSyn, a natively unstructured protein involved in Parkinson disease, by mechanically stretching single molecules of this protein and recording their mechanical properties. These experiments permitted us to observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the αSyn sample. We found that one class of conformations, “β-like” structures, is directly related to αSyn aggregation. In fact, their relative abundance increases drastically in three different conditions known to promote the formation of αSyn fibrils. We expect that a critical concentration of αSyn with a “β-like” structure must be reached to trigger fibril formation. This critical concentration is therefore controlled by a chemical equilibrium. Novel pharmacological strategies can now be tailored to act upstream, before the aggregation process ensues, by targeting this equilibrium. To this end, single-molecule force spectroscopy can be an effective tool to tailor and test new pharmacological agents.

Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro and have identified 62 additional compounds with IC₅₀ values below 1 μM and profiled their selectivity toward chymotrypsin and 3CL-Pro from the Middle East respiratory syndrome virus. A subset of eight inhibitors showed anticytopathic effect in a Vero-E6 cell line, and the compounds thioguanosine and MG-132 were analyzed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Å, showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity.

The interaction with the host plasminogen/plasmin system represents a novel component in the molecular cross-talk between bifidobacteria and human host. Here, we demonstrated that the plasminogen-binding bifidobacterial speciesB. longum,B. bifidum,B. breveandB. lactisshare the key glycolytic enzyme enolase as a surface receptor for human plasminogen. Enolase was visualized on the cell surface of the model strainB. lactisBI07. The His-tagged recombinant protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. By site-directed mutagenesis we demonstrated that the interaction between theB. lactisBI07 enolase and human plasminogen involves an internal plasminogen-binding site homologous to that of pneumococcal enolase. According to our data, the positively charged residues Lys-251 and Lys-255, as well as the negatively charged Glu-252, of theB. lactisBI07 enolase are crucial for plasminogen binding. Acting as a human plasminogen receptor, the bifidobacterial surface enolase is suggested to play an important role in the interaction process with the host.