Paolo Carloni

Accurate and efficient description of protein vibrational dynamics: Comparing molecular dynamics and Gaussian models

Current all‐atom potential based molecular dynamics (MD) allows the identification of a proteins functional motions on a wide‐range of timescales, up to few tens of nanoseconds. However, functional, large‐scale motions of proteins may occur on a timescale currently not accessible by all‐atom potential based MD. To avoid the massive computational effort required by this approach, several simplified schemes have been introduced. One of the most satisfactory is the Gaussian network approach based on the energy expansion in terms of the deviation of the protein backbone from its native configuration. Here, we consider an extension of this model that captures in a more realistic way the distribution of native interactions due to the introduction of effective side‐chain centroids. Since their location is entirely determined by the protein backbone, the model is amenable to the same exact and computationally efficient treatment as previous simpler models. The ability of the model to describe the correlated motion of protein residues in thermodynamic equilibrium is established through a series of successful comparisons with an extensive (14 ns) MD simulation based on the AMBER potential of HIV‐1 protease in complex with a peptide substrate. Thus, the model presented here emerges as a powerful tool to provide preliminary, fast yet accurate characterizations of protein near‐native motion. Proteins 2004.

Drug resistance in HIV-1 protease: Flexibility-assisted mechanism of compensatory mutations.

The emergence of drug‐resistant variants is a serious side effect associated with acquired immune deficiency syndrome therapies based on inhibition of human immunodeficiency virus type 1 protease (HIV‐1 PR). In these variants, compensatory mutations, usually located far from the active site, are able to affect the enzymatic activity via molecular mechanisms that have been related to differences in the conformational flexibility, although the detailed mechanistic aspects have not been clarified so far. Here, we perform multinanosecond molecular dynamics simulations on L63P HIV‐1 PR, corresponding to the wild type, and one of its most frequently occurring compensatory mutations, M46I, complexed with the substrate and an enzymatic intermediate. The quality of the calculations is established by comparison with the available nuclear magnetic resonance data. Our calculations indicate that the dynamical fluctuations of the mutated enzyme differ from those in the wild type. These differences in the dynamic properties of the adducts with the substrate and with the gem‐diol intermediate might be directly related to variations in the enzymatic activity and therefore offer an explanation of the observed changes in catalytic rate between wild type and mutated enzyme. We anticipate that this “flexibility‐assisted” mechanism might be effective in the vast majority of compensatory mutations, which do not change the electrostatic properties of the enzyme.

Water and potassium dynamics inside the KcsA K+ channel

Molecular dynamics simulations and electrostatic modeling are used to investigate structural and dynamical properties of the potassium ions and of water molecules inside the KcsA channel immersed in a membrane‐mimetic environment. Two potassium ions, initially located in the selectivity filter binding sites, maintain their position during 2 ns of dynamics. A third potassium ion is very mobile in the water‐filled cavity. The protein appears engineered so as to polarize water molecules inside the channel cavity. The resulting water induced dipole and the positively charged potassium ion within the cavity are the key ingredients for stabilizing the two K+ ions in the binding sites. These two ions experience single file movements upon removal of the potassium in the cavity, confirming the role of the latter in ion transport through the channel.

Role of protein frame and solvent for the redox properties of azurin from Pseudomonas aeruginosa

We have coupled hybrid quantum mechanics (density functional theory; Car–Parrinello)/molecular mechanics molecular dynamics simulations to a grand-canonical scheme, to calculate the in situ redox potential of the Cu2+ + e− → Cu+ half reaction in azurin from Pseudomonas aeruginosa. An accurate description at atomistic level of the environment surrounding the metal-binding site and finite-temperature fluctuations of the protein structure are both essential for a correct quantitative description of the electronic properties of this system. We report a redox potential shift with respect to copper in water of 0.2 eV (experimental 0.16 eV) and a reorganization free energy λ = 0.76 eV (experimental 0.6–0.8 eV). The electrostatic field of the protein plays a crucial role in fine tuning the redox potential and determining the structure of the solvent. The inner-sphere contribution to the reorganization energy is negligible. The overall small value is mainly due to solvent rearrangement at the protein surface.

Role of Conformational Fluctuations in the Enzymatic Reaction of HIV-1 Protease

The emergence of compensatory drug-resistant mutations in HIV-1 protease challenges the common view of the reaction mechanism of this enzyme. Here, we address this issue by performing classical and ab initio molecular dynamics simulations (MD) on a complex between the enzyme and a peptide substrate. The classical MD calculation reveals large-scale protein motions involving the flaps and the cantilever. These motions modulate the conformational properties of the substrate at the cleavage site. The ab initio calculations show in turn that substrate motion modulates the activation free energy barrier of the enzymatic reaction dramatically. Thus, the catalytic power of the enzyme does not arise from the presence of a pre-organized active site but from the protein mechanical fluctuations. The implications of this finding for the emergence of drug-resistance are discussed.

Substrate Binding Mechanism of HIV-1 Protease from Explicit-Solvent Atomistic Simulations

The binding mechanism of a peptide substrate (Thr-Ile-Met-Met-Gln-Arg, cleavage site p2-NC of the viral polyprotein) to wild-type HIV-1 protease has been investigated by 1.6 micros biased all-atom molecular dynamics simulations in explicit water. The configuration space has been explored biasing seven reaction coordinates by the bias-exchange metadynamics technique. The structure of the Michaelis complex is obtained starting from the substrate outside the enzyme within a backbone rmsd of 0.9 A. The calculated free energy of binding is -6 kcal/mol, and the kinetic constants for association and dissociation are 1.3 x 10(6) M(-1) s(-1) and 57 s(-1), respectively, consistent with experiments. In the main binding pathway, the flaps of the protease do not open sizably. The substrate slides inside the enzyme cavity from the tight lateral channel. This may contrast with the natural polyprotein substrate which is expected to bind by opening the flaps. Thus, mutations might influence differently the binding kinetics of peptidomimetic ligands and of the natural substrate.

Targeting biomolecular flexibility with metadynamics.

Metadynamics calculations allow investigating structure, plasticity, and energetics in a variety of biological processes spanning from molecular docking to protein folding. Recent theoretical developments have led to applications to increasingly complex systems and processes stepping up the biological relevance of the problem solved. Here, after summarizing recent technical advances and applications, we give a perspective of the method as a tool for enzymology and for the prediction of NMR and other spectroscopic data.

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.

Adaptive protein evolution grants organismal fitness by improving catalysis and flexibility

Protein evolution is crucial for organismal adaptation and fitness. This process takes place by shaping a given 3-dimensional fold for its particular biochemical function within the metabolic requirements and constraints of the environment. The complex interplay between sequence, structure, functionality, and stability that gives rise to a particular phenotype has limited the identification of traits acquired through evolution. This is further complicated by the fact that mutations are pleiotropic, and interactions between mutations are not always understood. Antibiotic resistance mediated by β-lactamases represents an evolutionary paradigm in which organismal fitness depends on the catalytic efficiency of a single enzyme. Based on this, we have dissected the structural and mechanistic features acquired by an optimized metallo-β-lactamase (MβL) obtained by directed evolution. We show that antibiotic resistance mediated by this enzyme is driven by 2 mutations with sign epistasis. One mutation stabilizes a catalytically relevant intermediate by fine tuning the position of 1 metal ion; whereas the other acts by augmenting the protein flexibility. We found that enzyme evolution (and the associated antibiotic resistance) occurred at the expense of the protein stability, revealing that MβLs have not exhausted their stability threshold. Our results demonstrate that flexibility is an essential trait that can be acquired during evolution on stable protein scaffolds. Directed evolution aided by a thorough characterization of the selected proteins can be successfully used to predict future evolutionary events and design inhibitors with an evolutionary perspective.

Conformational flexibility of the catalytic Asp dyad in HIV-1 protease: An ab initio study on the free enzyme.

The enzyme protease from the human immunodeficiency virus type 1 (HIV‐1 PR) is one of the main targets for therapeutic intervention in AIDS. Computer modeling is useful for probing the binding of novel ligands, yet empirical force field‐based methods have encountered problems in adequately describing interactions of the catalytic aspartyl pair. In this work we use ab initio dynamic methods to study the molecular interactions and the conformational flexibility of the Asp dyad in the free enzyme. Calculations are performed on model complexes that include, besides the Asp dyad, the conserved Thr26 and Gly27 residues and water molecules present in the active site channel. Our calculations provide proton location and binding mode of the active‐site water molecule, which turn out to be different from those of the eukariotic isoenzyme. Furthermore, the calculations reproduce well the structural features of the aspartyl dyad in the protein. Finally, they allow the identification of both dipole/charge interactions and a low‐barrier hydrogen bond as important stabilizing factors for the peculiar conformation of the active site. These findings are consistent with site‐directed mutagenesis experiments on the 27, 27` positions ( Bagossi et al. , Protein Eng 1996;9:997–1003). The electric field of the protein frame (included in some of the calculations) does not affect significantly the chemical bonding at the cleavage site. Proteins 2000;39:26–36.