Emiliano Ippoliti

Bioinorganic Chemistry of Parkinson's Disease: Structural Determinants for the Copper-Mediated Amyloid Formation of Alpha-Synuclein

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinsons disease (PD). A central, unresolved question in the pathophysiology of PD relates to the role of AS-metal interactions in amyloid fibril formation and neurodegeneration. Our previous works established a hierarchy in alpha-synuclein-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. Two independent, non-interacting copper-binding sites were identified at the N-terminal region of AS, with significant difference in their affinities for the metal ion. In this work we have solved unknown details related to the structural binding specificity and aggregation enhancement mediated by Cu(II). The high-resolution structural characterization of the highest affinity N-terminus AS-Cu(II) complex is reported here. Through the measurement of AS aggregation kinetics we proved conclusively that the copper-enhanced AS amyloid formation is a direct consequence of the formation of the AS-Cu(II) complex at the highest affinity binding site. The kinetic behavior was not influenced by the His residue at position 50, arguing against an active role for this residue in the structural and biological events involved in the mechanism of copper-mediated AS aggregation. These new findings are central to elucidate the mechanism through which the metal ion participates in the fibrillization of AS and represent relevant progress in the understanding of the bioinorganic chemistry of PD.

Water at hydrophobic interfaces delays proton surface-to-bulk transfer and provides a pathway for lateral proton diffusion

Fast lateral proton migration along membranes is of vital importance for cellular energy homeostasis and various proton-coupled transport processes. It can only occur if attractive forces keep the proton at the interface. How to reconcile this high affinity to the membrane surface with high proton mobility is unclear. Here, we tested whether a minimalistic model interface between an apolar hydrophobic phase (n-decane) and an aqueous phase mimics the biological pathway for lateral proton migration. The observed diffusion span, on the order of tens of micrometers, and the high proton mobility were both similar to the values previously reported for lipid bilayers. Extensive ab initio simulations on the same water/n-decane interface reproduced the experimentally derived free energy barrier for the excess proton. The free energy profile GH+ adopts the shape of a well at the interface, having a width of two water molecules and a depth of 6 ± 2RT. The hydroniums in direct contact with n-decane have a reduced mobility. However, the hydroniums in the second layer of water molecules are mobile. Their in silico diffusion coefficient matches that derived from our in vitro experiments, (5.7 ± 0.7) × 10-5 cm2 s-1. Conceivably, these are the protons that allow for fast diffusion along biological membranes.

Conformational fluctuations of UreG, an intrinsically disordered enzyme.

UreG proteins are small GTP binding (G) proteins that catalyze the hydrolysis of GTP necessary for the maturation of urease, a virulence factor in bacterial pathogenesis. UreG proteins are the first documented cases of intrinsically disordered enzymes. The comprehension of the dynamics of folding-unfolding events occurring in this protein could shed light on the enzymatic mechanism of UreG. Here, we used the recently developed replica exchange with solute tempering (REST2) computational methodology to explore the conformational space of UreG from Helicobacter pylori (HpUreG) and to identify its structural fluctuations. The same simulation and analysis protocol has been applied to HypB from Methanocaldococcus jannaschii (MjHypB), which is closely related to UreG in both sequence and function, even though it is not intrinsically disordered. A comparison of the two systems reveals that both HpUreG and MjHypB feature a substantial rigidity of the protein regions involved in catalysis, justifying its residual catalytic activity. On the other hand, HpUreG tends to unfold more than MjHypB in portions involved in protein-protein interactions with metallochaperones necessary for the formation of multiprotein complexes known to be involved in urease activation.

Structure and Dynamics of Oligonucleotides in the Gas Phase

By combining ion-mobility mass spectrometry experiments with sub-millisecond classical and ab initio molecular dynamics we fully characterized, for the first time, the dynamic ensemble of a model nucleic acid in the gas phase under electrospray ionization conditions. The studied oligonucleotide unfolds upon vaporization, loses memory of the solution structure, and explores true gas-phase conformational space. Contrary to our original expectations, the oligonucleotide shows very rich dynamics in three different timescales (multi-picosecond, nanosecond, and sub-millisecond). The shorter timescale dynamics has a quantum mechanical nature and leads to changes in the covalent structure, whereas the other two are of classical origin. Overall, this study suggests that a re-evaluation on our view of the physics of nucleic acids upon vaporization is needed.

Structural Determinants of Cisplatin and Transplatin Binding to the Met-Rich Motif of Ctr1: A Computational Spectroscopy Approach

The cellular uptake of cisplatin and of other platinum-based drugs is mediated by the high-affinity copper transporter Ctr1. The eight-residue long peptide called Mets7 (MTGMKGMS) mimics one of extracellular methionine (Met)-rich motifs of Ctr1. It is an excellent model for investigating the interaction of platinum drugs with Ctr1 under in vitro and in vivo conditions. Some of us have shown that (i) Cisplatin loses all of its ligands upon reaction with Mets7 and the metal ion binds to the three Met residues and completes its coordination shell with a fourth ligand that can be a chloride or a water/hydroxyl oxygen. (ii) Transplatin loses only the chlorido ligands, which are replaced by Met residues. Here, we provide information on the structural determinants of cisplatin/Mets7 and transplatin/Mets7 adducts by computational methods. The predictions are validated against EXAFS, NMR, and CD spectra. While EXAFS gives information restricted to the metal coordination shell, NMR provides information extended to residue atoms around the coordination shell, and finally, CD provides information about the overall conformation of the peptide. This allows us to elucidate the different reaction modes of cisplatin and transplatin toward the peptide, as well as to propose the platinated peptides [PtX](+)-(M*TGM*KGM*S) (X = Cl(-), OH(-)) and trans[Pt(NH3)2](2+)-(M*TGM*KGMS) as the most relevant species occurring in water solution.

Counterion Redistribution upon Binding of a Tat-Protein Mimic to HIV-1 TAR RNA.

Binding of proteins and small molecules to RNA involves many electrostatic interactions, which may alter the distribution of ions around the RNA molecule. Here, we use molecular dynamics simulations to investigate how binding of a cyclic peptide mimic of the HIV-1 Tat protein affects the ionic distribution around the HIV-1 TAR RNA element. The calculations reproduce the structural properties observed in NMR studies of TAR and its complex. They also provide insight into the rearrangement of counterions during the molecular recognition events leading to the formation of the protein/RNA complex.

Structural biology of cisplatin complexes with cellular targets: the adduct with human copper chaperone atox1 in aqueous solution.

Cisplatin is one of the most used anticancer drugs. Its cellular influx and delivery to target DNA may involve the copper chaperone Atox1 protein. Although the mode of binding is established by NMR spectroscopy measurements in solution-the Pt atom binds to Cys12 and Cys15 while retaining the two ammine groups-the structural determinants of the adduct are not known. Here a structural model by hybrid Car-Parrinello density functional theory-based QM/MM simulations is provided. The platinated site minimally modifies the fold of the protein. The calculated NMR and CD spectral properties are fully consistent with the experimental data. Our in silico/in vitro approach provides, together with previous studies, an unprecedented view into the structural biology of cisplatin-protein adducts.

Proton Dynamics in Protein Mass Spectrometry

Native electrospray ionization/ion mobility-mass spectrometry (ESI/IM-MS) allows an accurate determination of low-resolution structural features of proteins. Yet, the presence of proton dynamics, observed already by us for DNA in the gas phase, and its impact on protein structural determinants, have not been investigated so far. Here, we address this issue by a multistep simulation strategy on a pharmacologically relevant peptide, the N-terminal residues of amyloid-β peptide (Aβ(1-16)). Our calculations reproduce the experimental maximum charge state from ESI-MS and are also in fair agreement with collision cross section (CCS) data measured here by ESI/IM-MS. Although the main structural features are preserved, subtle conformational changes do take place in the first ∼0.1 ms of dynamics. In addition, intramolecular proton dynamics processes occur on the picosecond-time scale in the gas phase as emerging from quantum mechanics/molecular mechanics (QM/MM) simulations at the B3LYP level of theory. We conclude that proton transfer phenomena do occur frequently during fly time in ESI-MS experiments (typically on the millisecond time scale). However, the structural changes associated with the process do not significantly affect the structural determinants.

Role of the Membrane Dipole Potential for Proton Transport in Gramicidin A Embedded in a DMPC Bilayer

The membrane potential at the water/phospholipid interfaces may play a key role for proton conduction of gramicidin A (gA). Here we address this issue by Density Functional Theory-based molecular dynamics and metadynamics simulations. The calculations, performed on gA embedded in a solvated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) model membrane environment (about 2,000 atoms), indicate that (i) the membrane dipole potential rises at the channel mouth by ∼0.4 V. A similar value has been measured for gA embedded in a DMPC monolayer; (ii) the calculated free energy barrier is located at the channel entrance, consistent with experiments comparing gA proton conduction in different bilayers. The electronic structures of the proton ligands (water molecules and peptide units) are similar to those in the bulk solvent. Based on these results, we suggest an important role of the membrane dipole potential for the free energy barrier of proton permeation of gA. This may provide a rationale for the large increase in the rate of proton conduction under application of a transmembrane voltage, as observed experimentally. Our calculations might suggest also a role for proton desolvation for the permeation process. This role has already emerged from EVB calculations on gA embedded in a model membrane.

Designing the Sniper: Improving Targeted Human Cytolytic Fusion Proteins for Anti-Cancer Therapy via Molecular Simulation

Targeted human cytolytic fusion proteins (hCFPs) are humanized immunotoxins for selective treatment of different diseases including cancer. They are composed of a ligand specifically binding to target cells genetically linked to a human apoptosis-inducing enzyme. hCFPs target cancer cells via an antibody or derivative (scFv) specifically binding to e.g., tumor associated antigens (TAAs). After internalization and translocation of the enzyme from endocytosed endosomes, the human enzymes introduced into the cytosol are efficiently inducing apoptosis. Under in vivo conditions such enzymes are subject to tight regulation by native inhibitors in order to prevent inappropriate induction of cell death in healthy cells. Tumor cells are known to up-regulate these inhibitors as a survival mechanism resulting in escape of malignant cells from elimination by immune effector cells. Cytosolic inhibitors of Granzyme B and Angiogenin (Serpin P9 and RNH1, respectively), reduce the efficacy of hCFPs with these enzymes as effector domains, requiring detrimentally high doses in order to saturate inhibitor binding and rescue cytolytic activity. Variants of Granzyme B and Angiogenin might feature reduced affinity for their respective inhibitors, while retaining or even enhancing their catalytic activity. A powerful tool to design hCFPs mutants with improved potency is given by in silico methods. These include molecular dynamics (MD) simulations and enhanced sampling methods (ESM). MD and ESM allow predicting the enzyme-protein inhibitor binding stability and the associated conformational changes, provided that structural information is available. Such “high-resolution” detailed description enables the elucidation of interaction domains and the identification of sites where particular point mutations may modify those interactions. This review discusses recent advances in the use of MD and ESM for hCFP development from the viewpoints of scientists involved in both fields.