Davide Provasi

PLUMED: a portable plugin for free-energy calculations with molecular dynamics

Here we present a program aimed at free-energy calculations in molecular systems. It consists of a series of routines that can be interfaced with the most popular classical molecular dynamics (MD) codes through a simple patching procedure. This leaves the possibility for the user to exploit many different MD engines depending on the system simulated and on the computational resources available. Free-energy calculations can be performed as a function of many collective variables, with a particular focus on biological problems, and using state-of-the-art methods such as metadynamics, umbrella sampling and Jarzynski-equation based steered MD. The present software, written in ANSI-C language, can be easily interfaced with both Fortran and C/C++ codes.

Closed headpiece of integrin αIIbβ3 and its complex with an αIIbβ3-specific antagonist that does not induce opening.

The platelet integrin α(IIb)β(3) is essential for hemostasis and thrombosis through its binding of adhesive plasma proteins. We have determined crystal structures of the α(IIb)β(3) headpiece in the absence of ligand and after soaking in RUC-1, a novel small molecule antagonist. In the absence of ligand, the α(IIb)β(3) headpiece is in a closed conformation, distinct from the open conformation visualized in presence of Arg-Gly-Asp (RGD) antagonists. In contrast to RGD antagonists, RUC-1 binds only to the α(IIb) subunit. Molecular dynamics revealed nearly identical binding. Two species-specific residues, α(IIb) Y190 and α(IIb) D232, in the RUC-1 binding site were confirmed as important by mutagenesis. In sharp contrast to RGD-based antagonists, RUC-1 did not induce α(IIb)β(3) to adopt an open conformation, as determined by gel filtration and dynamic light scattering. These studies provide insights into the factors that regulate integrin headpiece opening, and demonstrate the molecular basis for a novel mechanism of integrin antagonism.

Assessing the Relative Stability of Dimer Interfaces in G Protein-Coupled Receptors

Considerable evidence has accumulated in recent years suggesting that G protein-coupled receptors (GPCRs) associate in the plasma membrane to form homo- and/or heteromers. Nevertheless, the stoichiometry, fraction and lifetime of such receptor complexes in living cells remain topics of intense debate. Motivated by experimental data suggesting differing stabilities for homomers of the cognate human β1- and β2-adrenergic receptors, we have carried out approximately 160 microseconds of biased molecular dynamics simulations to calculate the dimerization free energy of crystal structure-based models of these receptors, interacting at two interfaces that have often been implicated in GPCR association under physiological conditions. Specifically, results are presented for simulations of coarse-grained (MARTINI-based) and atomistic representations of each receptor, in homodimeric configurations with either transmembrane helices TM1/H8 or TM4/3 at the interface, in an explicit lipid bilayer. Our results support a definite contribution to the relative stability of GPCR dimers from both interface sequence and configuration. We conclude that β1- and β2-adrenergic receptor homodimers with TM1/H8 at the interface are more stable than those involving TM4/3, and that this might be reconciled with experimental studies by considering a model of oligomerization in which more stable TM1 homodimers diffuse through the membrane, transiently interacting with other protomers at interfaces involving other TM helices.

Making Structural Sense of Dimerization Interfaces of Delta Opioid Receptor Homodimers

Opioid receptors, like other members of the G protein-coupled receptor (GPCR) family, have been shown to associate to form dimers and/or oligomers at the plasma membrane. Whether this association is stable or transient is not known. Recent compelling evidence suggests that at least some GPCRs rapidly associate and dissociate. We have recently calculated binding affinities from free energy estimates to predict transient association between mouse delta opioid receptor (DOR) protomers at a symmetric interface involving the fourth transmembrane (TM4) helix (herein termed “4” dimer). Here we present disulfide cross-linking experiments with DOR constructs with cysteines substituted at the extracellular ends of TM4 or TM5 that confirm the formation of DOR complexes involving these helices. Our results are consistent with the involvement of TM4 and/or TM5 at the DOR homodimer interface, but possibly with differing association propensities. Coarse-grained (CG) well-tempered metadynamics simulations of two different dimeric arrangements of DOR involving TM4 alone or with TM5 (herein termed “4/5” dimer) in an explicit lipid−water environment confirmed the presence of two structurally and energetically similar configurations of the 4 dimer, as previously assessed by umbrella sampling calculations, and revealed a single energetic minimum of the 4/5 dimer. Additional CG umbrella sampling simulations of the 4/5 dimer indicated that the strength of association between DOR protomers varies depending on the protein region at the interface, with the 4 dimer being more stable than the 4/5 dimer.

Exploring Molecular Mechanisms of Ligand Recognition by Opioid Receptors with Metadynamics

Opioid receptors are G protein-coupled receptors (GPCRs) of utmost significance in the development of potent analgesic drugs for the treatment of severe pain. An accurate evaluation at the molecular level of the ligand binding pathways into these receptors may play a key role in the design of new molecules with more desirable properties and reduced side effects. The recent characterization of high-resolution X-ray crystal structures of non-rhodopsin GPCRs for diffusible hormones and neurotransmitters presents an unprecedented opportunity to build improved homology models of opioid receptors, and to study in more detail their molecular mechanisms of ligand recognition. In this study, possible pathways for entry of the nonselective antagonist naloxone (NLX) from the water environment into the well-accepted alkaloid binding pocket of a delta opioid receptor (DOR) molecular model based on the beta2-adrenergic receptor crystal structure are explored using microsecond-scale well-tempered metadynamics simulations. Using as collective variables distances that account for the position of NLX and of the receptor extracellular loop 2 in relation to the DOR binding pocket, we were able to distinguish between the different states visited by the ligand (i.e., docked, undocked, and metastable bound intermediates) and to predict a free energy of binding close to experimental values after correcting for possible drawbacks of the sampling approach. The strategy employed herein holds promise for its application to the docking of diverse ligands to the opioid receptors as well as to other GPCRs.

Ligand-Induced Modulation of the Free-Energy Landscape of G Protein-Coupled Receptors Explored by Adaptive Biasing Techniques

Extensive experimental information supports the formation of ligand-specific conformations of G protein-coupled receptors (GPCRs) as a possible molecular basis for their functional selectivity for signaling pathways. Taking advantage of the recently published inactive and active crystal structures of GPCRs, we have implemented an all-atom computational strategy that combines different adaptive biasing techniques to identify ligand-specific conformations along pre-determined activation pathways. Using the prototypic GPCR β2-adrenergic receptor as a suitable test case for validation, we show that ligands with different efficacies (either inverse agonists, neutral antagonists, or agonists) modulate the free-energy landscape of the receptor by shifting the conformational equilibrium towards active or inactive conformations depending on their elicited physiological response. Notably, we provide for the first time a quantitative description of the thermodynamics of the receptor in an explicit atomistic environment, which accounts for the receptor basal activity and the stabilization of different active-like states by differently potent agonists. Structural inspection of these metastable states reveals unique conformations of the receptor that may have been difficult to retrieve experimentally.

Inward-facing conformation of the zinc transporter YiiP revealed by cryoelectron microscopy

YiiP is a dimeric Zn2+/H+ antiporter from Escherichia coli belonging to the cation diffusion facilitator family. We used cryoelectron microscopy to determine a 13-Å resolution structure of a YiiP homolog from Shewanella oneidensis within a lipid bilayer in the absence of Zn2+. Starting from the X-ray structure in the presence of Zn2+, we used molecular dynamics flexible fitting to build a model consistent with our map. Comparison of the structures suggests a conformational change that involves pivoting of a transmembrane, four-helix bundle (M1, M2, M4, and M5) relative to the M3-M6 helix pair. Although accessibility of transport sites in the X-ray model indicates that it represents an outward-facing state, our model is consistent with an inward-facing state, suggesting that the conformational change is relevant to the alternating access mechanism for transport. Molecular dynamics simulation of YiiP in a lipid environment was used to address the feasibility of this conformational change. Association of the C-terminal domains is the same in both states, and we speculate that this association is responsible for stabilizing the dimer that, in turn, may coordinate the rearrangement of the transmembrane helices.

Putative Active States of a Prototypic G-Protein-Coupled Receptor from Biased Molecular Dynamics

A major current focus of structural work on G-protein-coupled receptors (GPCRs) pertains to the investigation of their active states. However, for virtually all GPCRs, active agonist-bound intermediate states have been difficult to characterize experimentally owing to their higher conformational flexibility, and thus intrinsic instability, as compared to inactive inverse agonist-bound states. In this work, we explored possible activation pathways of the prototypic GPCR bovine rhodopsin by means of biased molecular dynamics simulations. Specifically, we used an explicit atomistic representation of the receptor and its environment, and sampled the conformational transition from the crystal structure of a photoactivated deprotonated state of rhodopsin to the low pH crystal structure of opsin in the presence of 11-trans-retinal, using adiabatic biased molecular dynamics simulations. We then reconstructed the system free-energy landscape along the predetermined transition trajectories using a path collective variable approach based on metadynamics. Our results suggest that the two experimental endpoints of rhodopsin/opsin are connected by at least two different pathways, and that the conformational transition is populated by at least four metastable states of the receptor, characterized by a different amplitude of the outward movement of transmembrane helix 6.

Exploring the protein G helix free-energy surface by solute tempering metadynamics.

The free‐energy landscape of the α‐helix of protein G is studied by means of metadynamics coupled with a solute tempering algorithm. Metadynamics allows to overcome large energy barriers, whereas solute tempering improves the sampling with an affordable computational effort. From the sampled free‐energy surface we are able to reproduce a number of experimental observations, such as the fact that the lowest minimum corresponds to a globular conformation displaying some degree of β‐structure, that the helical state is metastable and involves only 65% of the chain. The calculations also show that the system populates consistently a π‐helix state and that the hydrophobic staple motif is present only in the free‐energy minimum associated with the helices, and contributes to their stabilization. The use of metadynamics coupled with solute tempering results then particularly suitable to provide the thermodynamics of a short peptide, and its computational efficiency is promising to deal with larger proteins. Proteins 2008.

Lessons from Free Energy Simulations of δ-Opioid Receptor Homodimers Involving the Fourth Transmembrane Helix

Several G protein-coupled receptors (GPCRs), including opioid receptors δOR, μOR, and κOR, have been reported to form stable dimers or oligomers in lipid bilayers and cell membranes. This notion has been recently challenged by imaging data supporting a transient nature of GPCR association. Here we use umbrella sampling reconstructed free energies of δOR homodimers involving the fourth transmembrane helix to predict their association constant. The results of these simulations, combined with estimates of diffusion-limited association rates, suggest a short lifetime for δOR homodimers in the membrane, in agreement with recent trends.