Bruno Rizzuti

Using simulations to provide the framework for experimental protein folding studies.

Molecular dynamics simulations are a powerful theoretical tool to model the protein folding process in atomistic details under realistic conditions. Combined with a number of experimental techniques, simulations provide a detailed picture of how a protein folds or unfolds in the presence of explicit solvent and other molecular species, such as cosolvents, osmolytes, cofactors, active binding partners or inert crowding agents. The denaturing effects of temperature, pressure and external mechanical forces can also be probed. Qualitative and quantitative agreement with experiment contributes to a comprehensive molecular picture of protein states along the folding/unfolding pathway. The variety of systems examined reveals key features of the protein folding process.

Early stage aggregation of human serum albumin in the presence of metal ions

The heat induced aggregation of human serum albumin (HSA) with and without an equimolar amount of Cu(II) and Zn(II) was investigated by using optical absorption, fluorescence, AFM and EPR spectroscopy. Turbidity experiments as a function of temperature indicate that the protein aggregation occurs after the melting of the protein. The kinetic of HSA aggregation, investigated between 60 and 70°C by monitoring the optical density changes at 400nm on a 180min time window, shows an exponential growth with a rate that increases with the temperature. Fluorescence of the thioflavin T evidences a significant increase of the intensity at 480nm at increasing incubation time. These results combined with AFM experiments show that the protein aggregates are elongated oligomers with fibrillar-like features. The absence of a lag-phase suggests that the early stage aggregation of HSA follows a downhill pathway that does not require the formation of an organized nucleus. The presence of Cu(II) and Zn(II) ions does not affect the thermally induced aggregation process and the morphology of HSA aggregates. The result is compatible with the binding of the metal ions to the protein in the native state and with the high conformational stability of HSA.

Thermally induced denaturation and aggregation of BLG-A: effect of the Cu 2+ and Zn 2+ metal ions

There is growing evidence that metal ions can accelerate the aggregation process of several proteins. This process, associated with several neuro-degenerative diseases, has been reported also for non-pathological proteins. In the present work, the effects of copper and zinc ions on the denaturation and aggregation processes of β-lactoglobulin A (BLG-A) are investigated by differential scanning calorimetry (DSC), fluorescence, electron paramagnetic resonance (EPR) and optical density. The DSC profiles reveal that the thermal behaviour of BLG-A is a complex process, strongly dependent on the protein concentration. For concentrations ≤0.13 mM, the thermogram shows an endothermic peak at 84.3°C, corresponding to denaturation; for concentrations >0.13 mM an exothermic peak also appears, above 90°C, related to the aggregation of the denaturated BLG-A molecules. The thioflavin T fluorescence indicates that the thermally induced aggregates show fibrillar features. The presence of either equimolar Cu2+ or Zn2+ ions in the protein solution has different effects. In particular, copper binds to the protein in the native state, as evidenced by EPR experiments, and destabilizes BLG-A by decreasing the denaturation temperature by about 10°C, whereas zinc ions probably perturb the partially denaturated state of the protein. The kinetics of BLG-A aggregation shows that both metal ions abolish the lag phase before the aggregation starts. Moreover, the rate of the process is 4.6-fold higher in the presence of copper, whereas the effect of zinc is negligible. The increase of the aggregation rate, induced by copper, may be due to a site-specific binding of the metal ion on the protein.

Stability of trans-Resveratrol Associated with Transport Proteins

Spectrophotometry and fluorescence combined with docking and molecular dynamics simulations are used to study the effect of the carrier proteins β-lactoglobulin and human serum albumin on the degradative trans-to-cis conversion of resveratrol. The spectroscopic measurements quantify the concentration of resveratrol isoforms after 2 h of irradiation with light at 340 nm, showing that their ratio depends linearly on temperature between 20 and 50 °C and obeys an Arrhenius law with activation energies of photoisomerization of 7.8 and 11.2 kcal/mol for β-lactoglobulin and albumin, respectively, compared to 5.1 kcal/mol in solution. Thus, both proteins protect trans-resveratrol from degradation, with albumin being more effective than β-lactoglobulin. The computational techniques clarify details of the binding of trans-resveratrol to the proteins and show that the stabilizing effect correlates with an increase of the dihedral order parameter of the ligand. These findings suggest that transport proteins are viable carriers to stabilize and deliver resveratrol in vivo in the biologically effective trans form.

Evidence of reduced flexibility in disulfide bridge-depleted azurin: a molecular dynamics simulation study.

Two molecular dynamics simulations have been performed for 2 ns, at room temperature, on fully hydrated wild type and Cys3Ala/Cys26Ala double-mutant azurin, to investigate the role of the unique disulfide bridge on the structure and dynamics of the protein. The results show that the removal of the [bond]SS[bond] bond does not affect the structural features of the protein, whereas alterations of the dynamical properties are observed. The root mean square fluctuations of the atomic positions are, on average, considerably reduced in the azurin mutant with respect to the wild type form. The number of intramolecular hydrogen bonds between protein backbone atoms that are lost during the simulation, with respect to the starting configuration, are reduced in the absence of the disulfide bond. The analysis of the dynamical cross-correlation map, characterising the protein co-ordinated internal motions, demonstrates in the mutated azurin a significant decrease in anti-correlated displacements between protein residues, with the only exception occurring in the region of the mutation sites. The overall findings show a relevant reduction in flexibility as a consequence of the disulfide bridge depletion in azurin, suggesting that the [bond]SS[bond] bond is a structural element which significantly contributes to the dynamic properties of the native protein.

Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma.

Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the ‘fuzzy’ interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs.

Catalytic activity of copper ions in the amyloid fibrillation of β-lactoglobulin

The self-assembly of proteins and polypeptides in amyloid fibrillar aggregates is rapidly emerging as a promising route towards the fabrication of nano-objects with controlled morphologies and properties. Transition metal ions are known to play an important but elusive role in the amyloid fibrillation associated with neurodegenerative diseases such as Alzheimers and Parkinsons diseases. We have considered the effect of copper ions Cu2+ on the nanoscale morphology and fibrillation kinetics of β-lactoglobulin (βLG), a model protein not related to amyloid diseases, which denatures and self-assembles in nanofibrils upon heating at low pH and ionic strength. We found that an increasing level of Cu2+ decreases the enthalpy of denaturation and significantly increases the rate of fibril nucleation, also producing a small increase of the fibril elongation rate. Cu2+ acts as a catalytic agent during protein denaturation and fibrillation, without binding to βLG before or after heating, and produces only minor changes in fibril morphology. Beside possible implications for amyloid pathologies in vivo, our results suggest that transition metal ions can be used to control the self-assembly of protein-based nano-objects in vitro.

Multiple binding modes of ibuprofen in human serum albumin identified by absolute binding free energy calculations

Human serum albumin possesses multiple binding sites and transports a wide range of ligands that include the anti-inflammatory drug ibuprofen. A complete map of the binding sites of ibuprofen in albumin is difficult to obtain in traditional experiments, because of the structural adaptability of this protein in accommodating small ligands. In this work, we provide a set of predictions covering the geometry, affinity of binding and protonation state for the pharmaceutically most active form (S-isomer) of ibuprofen to albumin, by using absolute binding free energy calculations in combination with classical molecular dynamics (MD) simulations and molecular docking. The most favorable binding modes correctly reproduce several experimentally identified binding locations, which include the two Sudlows drug sites (DS2 and DS1) and the fatty acid binding sites 6 and 2 (FA6 and FA2). Previously unknown details of the binding conformations were revealed for some of them, and formerly undetected binding modes were found in other protein sites. The calculated binding affinities exhibit trends which seem to agree with the available experimental data, and drastically degrade when the ligand is modeled in a protonated (neutral) state, indicating that ibuprofen associates with albumin preferentially in its charged form. These findings provide a detailed description of the binding of ibuprofen, help to explain a wide range of results reported in the literature in the last decades, and demonstrate the possibility of using simulation methods to predict ligand binding to albumin.

Fatty acid binding into the highest affinity site of human serum albumin observed in molecular dynamics simulation

Multiple molecular dynamics simulations were performed to investigate the association of stearic acid into the highest affinity binding site of human serum albumin. All binding events ended with a rapid (<10 ps) lock-in of the fatty acid due to formation of a hydrogen bond with Tyr401. The kinetics and energetics of the penetration process both depended linearly on the positional shift of the fatty acid, with an average insertion time and free energy reduction of, respectively, 32 ± 20 ps and 0.70 ± 0.15 kcal/mol per methylene group absorbed. Binding events of longer duration (tbind>1 ns) were characterized by a slow exploration of the pocket entry and, frequently, of a nearby protein crevice corresponding to a metastable state along the route to the binding site. Taken all together, these findings reconstruct the following pathway for the binding process of stearic acid: (i) contact with the protein surface, possibly facilitated by the presence of an intermediate location, (ii) probing of the site entry, (iii) insertion into the protein, and (iv) lock-in at the final position. This general description may also apply to other long-chain fatty acids binding into any of the high-affinity sites of albumin, or to specific sites of other lipid-binding proteins.

Molecular simulations of β-lactoglobulin complexed with fatty acids reveal the structural basis of ligand affinity to internal and possible external binding sites

The interaction of saturated fatty acids of different length (C8:0 to C18:0) with β‐lactoglobulin (βLG) was investigated by molecular dynamics simulation and docking approaches. The results show that the presence of such ligands in the hydrophobic central cavity of βLG, known as the protein calyx, determines an enhancement of atomic fluctuations compared with the unliganded form, especially for loops at the entrance of the binding site. Concerted motions are evidenced for protein regions that could favor the binding of ligands. The mechanism of anchoring of fatty acids of different length is similar for the carboxylate head‐group, through electrostatic interactions with the side chains of Lys60/Lys69. The key protein residues to secure the hydrocarbon chain are Phe105/Met107, which adapt their conformation upon ligand binding. In particular, Phe105 provides an additional hydrophobic clamp only for the tail of the two fatty acids with the longest chains, palmitic, and stearic acid, which are known to bind βLG with a high affinity. The search of additional external binding sites for fatty acids, distinct from the calyx, was also carried out for palmitic acid. Two external sites with a lower affinity were identified as secondary sites, one consisting in a hydrophobic cavity allowing two distinct binding modes for the fatty acid, and the other corresponding to a surface crevice close to the protein α‐helix. The overall results provide a comprehensive picture of the dynamical behavior of βLG in complex with fatty acids, and elucidate the structural basis of the binding of these physiological ligands. Proteins 2014; 82:2609–2619.