Gianni De Fabritiis

Complete reconstruction of an enzyme-inhibitor binding process by molecular dynamics simulations

The understanding of protein–ligand binding is of critical importance for biomedical research, yet the process itself has been very difficult to study because of its intrinsically dynamic character. Here, we have been able to quantitatively reconstruct the complete binding process of the enzyme-inhibitor complex trypsin-benzamidine by performing 495 molecular dynamics simulations of free ligand binding of 100 ns each, 187 of which produced binding events with an rmsd less than 2 Å compared to the crystal structure. The binding paths obtained are able to capture the kinetic pathway of the inhibitor diffusing from solvent (S0) to the bound (S4) state passing through two metastable intermediate states S2 and S3. Rather than directly entering the binding pocket the inhibitor appears to roll on the surface of the protein in its transition between S3 and the final binding pocket, whereas the transition between S2 and the bound pose requires rediffusion to S3. An estimation of the standard free energy of binding gives ΔG° = -5.2 ± 0.4 kcal/mol (cf. the experimental value -6.2 kcal/mol), and a two-states kinetic model kon = (1.5 ± 0.2) × 108 M-1 s-1 and koff = (9.5 ± 3.3) × 104 s-1 for unbound to bound transitions. The ability to reconstruct by simple diffusion the binding pathway of an enzyme-inhibitor binding process demonstrates the predictive power of unconventional high-throughput molecular simulations. Moreover, the methodology is directly applicable to other molecular systems and thus of general interest in biomedical and pharmaceutical research.

Foundations of dissipative particle dynamics

We derive a mesoscopic modeling and simulation technique that is very close to the technique known as dissipative particle dynamics. The model is derived from molecular dynamics by means of a systematic coarse-graining procedure. This procedure links the forces between the dissipative particles to a hydrodynamic description of the underlying molecular dynamics (MD) particles. In particular, the dissipative particle forces are given directly in terms of the viscosity emergent from MD, while the interparticle energy transfer is similarly given by the heat conductivity derived from MD. In linking the microscopic and mesoscopic descriptions we thus rely on the macroscopic or phenomenological description emergent from MD. Thus the rules governing this form of dissipative particle dynamics reflect the underlying molecular dynamics; in particular, all the underlying conservation laws carry over from the microscopic to the mesoscopic description. We obtain the forces experienced by the dissipative particles together with an approximate form of the associated equilibrium distribution. Whereas previously the dissipative particles were spheres of fixed size and mass, now they are defined as cells on a Voronoi lattice with variable masses and sizes. This Voronoi lattice arises naturally from the coarse-graining procedure, which may be applied iteratively and thus represents a form of renormalization-group mapping. It enables us to select any desired local scale for the mesoscopic description of a given problem. Indeed, the method may be used to deal with situations in which several different length scales are simultaneously present. We compare and contrast this particulate model with existing continuum fluid dynamics techniques, which rely on a purely macroscopic and phenomenological approach. Simulations carried out with the present scheme show good agreement with theoretical predictions for the equilibrium behavior.

Emergence of Multiple EGFR Extracellular Mutations during Cetuximab Treatment in Colorectal Cancer

Purpose: Patients with colorectal cancer who respond to the anti-EGFR antibody cetuximab often develop resistance within several months of initiating therapy. To design new lines of treatment, the molecular landscape of resistant tumors must be ascertained. We investigated the role of mutations in the EGFR signaling axis on the acquisition of resistance to cetuximab in patients and cellular models. Experimental Design: Tissue samples were obtained from 37 patients with colorectal cancer who became refractory to cetuximab. Colorectal cancer cells sensitive to cetuximab were treated until resistant derivatives emerged. Mutational profiling of biopsies and cell lines was performed. Structural modeling and functional analyses were performed to causally associate the alleles to resistance. Results: The genetic profile of tumor specimens obtained after cetuximab treatment revealed the emergence of a complex pattern of mutations in EGFR, KRAS, NRAS, BRAF, and PIK3CA genes, including two novel EGFR ectodomain mutations (R451C and K467T). Mutational profiling of cetuximab-resistant cells recapitulated the molecular landscape observed in clinical samples and revealed three additional EGFR alleles: S464L, G465R, and I491M. Structurally, these mutations are located in the cetuximab-binding region, except for the R451C mutant. Functionally, EGFR ectodomain mutations prevent binding to cetuximab but a subset is permissive for interaction with panitumumab. Conclusions: Colorectal tumors evade EGFR blockade by constitutive activation of downstream signaling effectors and through mutations affecting receptor–antibody binding. Both mechanisms of resistance may occur concomitantly. Our data have implications for designing additional lines of therapy for patients with colorectal cancer who relapse upon treatment with anti-EGFR antibodies. Clin Cancer Res; 21(9); 2157–66. ©2015 AACR.

Kinetic characterization of the critical step in HIV-1 protease maturation

HIV maturation requires multiple cleavage of long polyprotein chains into functional proteins that include the viral protease itself. Initial cleavage by the protease dimer occurs from within these precursors, and yet only a single protease monomer is embedded in each polyprotein chain. Self-activation has been proposed to start from a partially dimerized protease formed from monomers of different chains binding its own N termini by self-association to the active site, but a complete structural understanding of this critical step in HIV maturation is missing. Here, we captured the critical self-association of immature HIV-1 protease to its extended amino-terminal recognition motif using large-scale molecular dynamics simulations, thus confirming the postulated intramolecular mechanism in atomic detail. We show that self-association to a catalytically viable state requires structural cooperativity of the flexible β-hairpin “flap” regions of the enzyme and that the major transition pathway is first via self-association in the semiopen/open enzyme states, followed by enzyme conformational transition into a catalytically viable closed state. Furthermore, partial N-terminal threading can play a role in self-association, whereas wide opening of the flaps in concert with self-association is not observed. We estimate the association rate constant (kon) to be on the order of ∼1 × 104 s−1, suggesting that N-terminal self-association is not the rate-limiting step in the process. The shown mechanism also provides an interesting example of molecular conformational transitions along the association pathway.

Induced Effects of Sodium Ions on Dopaminergic G-Protein Coupled Receptors

G-protein coupled receptors, the largest family of proteins in the human genome, are involved in many complex signal transduction pathways, typically activated by orthosteric ligand binding and subject to allosteric modulation. Dopaminergic receptors, belonging to the class A family of G-protein coupled receptors, are known to be modulated by sodium ions from an allosteric binding site, although the details of sodium effects on the receptor have not yet been described. In an effort to understand these effects, we performed microsecond scale all-atom molecular dynamics simulations on the dopaminergic D2 receptor, finding that sodium ions enter the receptor from the extracellular side and bind at a deep allosteric site (Asp2.50). Remarkably, the presence of a sodium ion at this allosteric site induces a conformational change of the rotamer toggle switch Trp6.48 which locks in a conformation identical to the one found in the partially inactive state of the crystallized human β2 adrenergic receptor. This study provides detailed quantitative information about binding of sodium ions in the D2 receptor and reports a possibly important sodium-induced conformational change for modulation of D2 receptor function.

Explicit solvent dynamics and energetics of HIV‐1 protease flap opening and closing

An accurate description of the conformational dynamics of the β‐hairpin flaps of HIV‐1 protease is of central importance in elucidating the functional recognition of the enzyme by ligands. Using all‐atom molecular dynamics simulations in explicit solvent, with a total of 461 trajectories of ∼50 ns each, we report the closed, semiopen, open, and wide‐open flap conformation of the free wild‐type protease. The free energy of flap opening and closing from the semiopen state is 0.9 ± 0.2 and 2.4 ± 0.4 kcal/mol, respectively. The mean relaxation time of opening is ∼8 ns, in good agreement with NMR data. The explicit solvent simulations quantitatively confirm the hypothesis that the semiopen state is the dominant population in the free protease whilst fast flap tip fluctuations lead frequently to an open state. More pronounced flap rearrangements lead to a rare wide‐open state with the catalytic site completely exposed to the solvent. The structures of the different flap conformations provided herein are of general interest for improved drug design of HIV‐1 protease, in particular, the wide‐open conformation could be favored by the large Gag and GagPol polyprotein chains. Strategies that take into account multiple flap‐gating mechanisms may lead to more effective inhibitors. Proteins 2010.

High-throughput molecular dynamics: the powerful new tool for drug discovery.

Molecular dynamics simulations are capable of resolving molecular recognition processes with chemical accuracy, but their practical application is popularly considered limited to the timescale accessible to a single simulation, which is far below biological timescales. In this perspective article, we propose that the true limiting factor for molecular dynamics is rather the high hardware and electrical power costs, which constrain not only the length of runs but also the number that can be performed concurrently. As a result of innovation in accelerator processors and high-throughput protocols, the cost of molecular dynamics sampling has been dramatically reduced and we argue that molecular dynamics simulation is now placed to become a key technology for in silico drug discovery in terms of binding pathways, poses, kinetics and affinities.

Spontaneous inward opening of the dopamine transporter is triggered by PIP2-regulated dynamics of the N-terminus.

We present the dynamic mechanism of concerted motions in a full-length molecular model of the human dopamine transporter (hDAT), a member of the neurotransmitter/sodium symporter (NSS) family, involved in state-to-state transitions underlying function. The findings result from an analysis of unbiased atomistic molecular dynamics simulation trajectories (totaling >14 μs) of the hDAT molecule immersed in lipid membrane environments with or without phosphatidylinositol 4,5-biphosphate (PIP2) lipids. The N-terminal region of hDAT (N-term) is shown to have an essential mechanistic role in correlated rearrangements of specific structural motifs relevant to state-to-state transitions in the hDAT. The mechanism involves PIP2-mediated electrostatic interactions between the N-term and the intracellular loops of the transporter molecule. Quantitative analyses of collective motions in the trajectories reveal that these interactions correlate with the inward-opening dynamics of hDAT and are allosterically coupled to the known functional sites of the transporter. The observed large-scale motions are enabled by specific reconfiguration of the network of ionic interactions at the intracellular end of the protein. The isomerization to the inward-facing state in hDAT is accompanied by concomitant movements in the extracellular vestibule and results in the release of an Na+ ion from the Na2 site and destabilization of the substrate dopamine in the primary substrate binding S1 site. The dynamic mechanism emerging from the findings highlights the involvement of the PIP2-regulated interactions between the N-term and the intracellular loop 4 in the functionally relevant conformational transitions that are also similar to those found to underlie state-to-state transitions in the leucine transporter (LeuT), a prototypical bacterial homologue of the NSS.

Kinetic modulation of a disordered protein domain by phosphorylation.

Phosphorylation is a major post-translational mechanism of regulation that frequently targets disordered protein domains, but it remains unclear how phosphorylation modulates disordered states of proteins. Here we determine the kinetics and energetics of a disordered protein domain the kinase-inducible domain (KID) of the transcription factor CREB and that of its phosphorylated form pKID, using high-throughput molecular dynamic simulations. We identify the presence of a metastable, partially ordered state with a 60-fold slowdown in conformational kinetics that arises due to phosphorylation, kinetically stabilizing residues known to participate in an early binding intermediate. We show that this effect is only partially reconstituted by mutation to glutamate, indicating that the phosphate is uniquely required for the long-lived state to arise. This mechanism of kinetic modulation could be important for regulation beyond conformational equilibrium shifts.

Mesoscopic dynamics of Voronoi fluid particles

We compare and contrast two recently reported mesoscopic fluid particle models based on a two-dimensional Voronoi tessellation. Both models describe a Newtonian fluid at mesoscopic scales where fluctuations are important. From the requirement of thermodynamic consistency, the equilibrium distribution function is given through the Einstein distribution function. We compute from the Einstein distribution the equilibrium distribution function for a single fluid particle. We observe excellent agreement between the simulation results for the proposed models and the theoretical distribution function.