Ligand-dependent activation and deactivation of the human adenosine A(2A) receptor.

G-protein-coupled receptors (GPCRs) are membrane proteins with critical functions in cellular signal transduction, representing a primary class of drug targets. Acting by direct binding, many drugs modulate GPCR activity and influence the signaling pathways associated with numerous diseases. However, complete details of ligand-dependent GPCR activation/deactivation are difficult to obtain from experiments. Therefore, it remains unclear how ligands modulate a GPCR's activity. To elucidate the ligand-dependent activation/deactivation mechanism of the human adenosine A2A receptor (AA2AR), a member of the class A GPCRs, we performed large-scale unbiased molecular dynamics and metadynamics simulations of the receptor embedded in a membrane. At the atomic level, we have observed distinct structural states that resemble the active and inactive states. In particular, we noted key structural elements changing in a highly concerted fashion during the conformational transitions, including six conformational states of a tryptophan (Trp246(6.48)). Our findings agree with a previously proposed view that, during activation, this tryptophan residue undergoes a rotameric transition that may be coupled to a series of coherent conformational changes, resulting in the opening of the G-protein binding site. Further, metadynamics simulations provide quantitative evidence for this mechanism, suggesting how ligand binding shifts the equilibrium between the active and inactive states. Our analysis also proposes that a few specific residues are associated with agonism/antagonism, affinity, and selectivity, and suggests that the ligand-binding pocket can be thought of as having three distinct regions, providing dynamic features for structure-based design. Additional simulations with AA2AR bound to a novel ligand are consistent with our proposed mechanism. Generally, our study provides insights into the ligand-dependent AA2AR activation/deactivation in addition to what has been found in crystal structures. These results should aid in the discovery of more effective and selective GPCR ligands.

Generation of a consensus protein domain dictionary.

MOTIVATION: The discovery of new protein folds is a relatively rare occurrence even as the rate of protein structure determination increases. This rarity reinforces the concept of folds as reusable units of structure and function shared by diverse proteins. If the folding mechanism of proteins is largely determined by their topology, then the folding pathways of members of existing folds could encompass the full set used by globular protein domains. RESULTS: We have used recent versions of three common protein domain dictionaries (SCOP, CATH and Dali) to generate a consensus domain dictionary (CDD). Surprisingly, 40% of the metafolds in the CDD are not composed of autonomous structural domains, i.e. they are not plausible independent folding units. This finding has serious ramifications for bioinformatics studies mining these domain dictionaries for globular protein properties. However, our main purpose in deriving this CDD was to generate an updated CDD to choose targets for MD simulation as part of our dynameomics effort, which aims to simulate the native and unfolding pathways of representatives of all globular protein consensus folds (metafolds). Consequently, we also compiled a list of representative protein targets of each metafold in the CDD. AVAILABILITY AND IMPLEMENTATION: This domain dictionary is available at www.dynameomics.org.

The effect of context on the folding of ß-hairpins.

Small ß-hairpin peptides have been widely used as models for the folding of ß-sheets. But how applicable is the folding of such models to ß-structure in larger proteins with conventional hydrophobic cores? Here we present multiple unfolding simulations of three such proteins that contain the WW domain double hairpin ß-sheet motif: cold shock protein A (CspA), cold shock protein B (CspB) and glucose permease IIA domain. We compare the behavior of the free motif in solution and in the context of proteins of different size and architecture. Both Csp proteins lost contacts between the double-hairpin motif and the protein core as the first step of unfolding and proceeded to unfold with loss of the third ß-strand, similar to the isolated WW domain. The glucose permease IIA domain is a larger protein and the contacts between the motif and the core were not lost as quickly. Instead the unfolding pathway of glucose permease IIA followed a different pathway with ß1 pulling away from the sheet first. Interestingly, when the double hairpin motif was excised from the glucose permease IIA domain and simulated in isolation in water it unfolded by the same pathway as the WW domain, indicating that it is tertiary interactions with the protein that alter the motif's unfolding not a sequence dependent effect on its intrinsic unfolding behavior. With respect to the unfolding of the hairpins, there was no consistent order to the loss of hydrogen bonds between the ß-strands in the hairpins in any of the systems. Our results show that while the folding behavior of the isolated WW domain is generally consistent with the double hairpin motif's behavior in the cold shock proteins, it is not the case for the glucose permease IIA domain. So, one must be cautious in extrapolating findings from model systems to larger more complicated proteins where tertiary interactions can overwhelm intrinsic behavior.

A comprehensive multidimensional-embedded, one-dimensional reaction coordinate for protein unfolding/folding.

The goal of the Dynameomics project is to perform, store, and analyze molecular dynamics simulations of representative proteins, of all known globular folds, in their native state and along their unfolding pathways. To analyze unfolding simulations, the location of the protein along the unfolding reaction coordinate (RXN) must be determined. Properties such as the fraction of native contacts and radius of gyration are often used; however, there is an issue regarding degeneracy with these properties, as native and nonnative species can overlap. Here, we used 15 physical properties of the protein to construct a multidimensional-embedded, one-dimensional RXN coordinate that faithfully captures the complex nature of unfolding. The unfolding RXN coordinates for 188 proteins (1534 simulations and 22.9 mus in explicit water) were calculated. Native, transition, intermediate, and denatured states were readily identified with the use of this RXN coordinate. A global native ensemble based on the native-state properties of the 188 proteins was created. This ensemble was shown to be effective for calculating RXN coordinates for folds outside the initial 188 targets. These RXN coordinates enable, high-throughput assignment of conformational states, which represents an important step in comparing protein properties across fold space as well as characterizing the unfolding of individual proteins.

Dynameomics: a consensus view of the protein unfolding/folding transition state ensemble across a diverse set of protein folds.

The Dynameomics project aims to simulate a representative sample of all globular protein metafolds under both native and unfolding conditions. We have identified protein unfolding transition state (TS) ensembles from multiple molecular dynamics simulations of high-temperature unfolding in 183 structurally distinct proteins. These data can be used to study individual proteins and individual protein metafolds and to mine for TS structural features common across all proteins. Separating the TS structures into four different fold classes (all proteins, all-alpha, all-beta, and mixed alpha/beta and alpha +beta) resulted in no significant difference in the overall protein properties. The residues with the most contacts in the native state lost the most contacts in the TS ensemble. On average, residues beginning in an alpha-helix maintained more structure in the TS ensemble than did residues starting in beta-strands or any other conformation. The metafolds studied here represent 67% of all known protein structures, and this is, to our knowledge, the largest, most comprehensive study of the protein folding/unfolding TS ensemble to date. One might have expected broad distributions in the average global properties of the TS relative to the native state, indicating variability in the amount of structure present in the TS. Instead, the average global properties converged with low standard deviations across metafolds, suggesting that there are general rules governing the structure and properties of the TS.

Shared unfolding pathways of unrelated immunoglobulin-like ß-sandwich proteins.

The Dynameomics project contains native state and unfolding simulations of 807 protein domains, where each domain is representative of a different metafold; these metafolds encompass ~97% of protein fold space. There is a long-standing question in structural biology as to whether proteins in the same fold family share the same folding/unfolding characteristics. Using molecular dynamics simulations from the Dynameomics project, we conducted a detailed study of protein unfolding/folding pathways for 5 protein domains from the immunoglobulin (Ig)-like ß-sandwich metafold (the highest ranked metafold in our database). The domains have sequence similarities ranging from 4 to 15% and are all from different SCOP superfamilies, yet they share the same overall Ig-like topology. Despite having very different amino acid sequences, the dominant unfolding pathway is very similar for the 5 proteins, and the secondary structures that are peripheral to the aligned, shared core domain add variability to the unfolding pathway. Aligned residues in the core domain display consensus structure in the transition state primarily through conservation of hydrophobic positions. Commonalities in the obligate folding nucleus indicate that insights into the major events in the folding/unfolding of other domains from this metafold may be obtainable from unfolding simulations of a few representative proteins.

Dynameomics: mass annotation of protein dynamics and unfolding in water by high-throughput atomistic molecular dynamics simulations.

The goal of Dynameomics is to perform atomistic molecular dynamics (MD) simulations of representative proteins from all known folds in explicit water in their native state and along their thermal unfolding pathways. Here we present 188-fold representatives and their native state simulations and analyses. These 188 targets represent 67% of all the structures in the Protein Data Bank. The behavior of several specific targets is highlighted to illustrate general properties in the full dataset and to demonstrate the role of MD in understanding protein function and stability. As an example of what can be learned from mining the Dynameomics database, we identified a protein fold with heightened localized dynamics. In one member of this fold family, the motion affects the exposure of its phosphorylation site and acts as an entropy sink to offset another portion of the protein that is relatively immobile in order to present a consistent interface for protein docking. In another member of this family, a polymorphism in the highly mobile region leads to a host of disease phenotypes. We have constructed a web site to provide access to a novel hybrid relational/multidimensional database (described in the succeeding two papers) to view and interrogate simulations of the top 30 targets: http://www.dynameomics.org. The Dynameomics database, currently the largest collection of protein simulations and protein structures in the world, should also be useful for determining the rules governing protein folding and kinetic stability, which should aid in deciphering genomic information and for protein engineering and design.

The role of the turn in beta-hairpin formation during WW domain folding.

The folding of WW domains is rate limited by formation of a beta-hairpin comprising residues from strands 1 and 2. Residues in the turn of this hairpin have reported Phi-values for folding close to 1 and have been proposed to nucleate folding. High Phi-values do not necessarily imply that the energetics of formation are a driving force for initiating folding. We demonstrate by NMR studies and molecular dynamics simulations that the first turn of the hYAP, FBP28, and PIN1 WW domains is structurally dynamic and solvent exposed in the native and folding transition states. It is, therefore, unlikely that the formation of the beta-turn per se provides the energetic driving force for hairpin folding. It is more likely that the turn acts as an easily formed hinge that facilitates the formation of the hairpin; it is a nucleus as defined by the nucleation-condensation mechanism whereby a diffuse nucleus is stabilized by associated interactions.

Phi-analysis at the experimental limits: mechanism of beta-hairpin formation.

The 37-residue Formin-binding protein, FBP28, is a canonical three-stranded beta-sheet WW domain. Because of its small size, it is so insensitive to chemical denaturation that it is barely possible to determine accurately a denaturation curve, as the transition spans 0-7 M guanidinium hydrochloride (GdmCl). It is also only marginally stable, with a free energy of denaturation of just 2.3 kcal/mol at 10 degrees Celsius so only small changes in energy upon mutation can be tolerated. But these properties and relaxation times for folding of 25 micros-400 micros conspire to allow the rapid acquisition of accurate and reproducible kinetic data for Phi-analysis using classical temperature-jump methods. The transition state for folding is highly polarized with some regions having Phi-values of 0 and others 1, as readily seen in chevron plots, with Phi-values of 0 having the refolding arms overlaying and those of 1 the unfolding arms superimposable. Good agreement is seen with transition state structures identified from independent molecular dynamics (MD) simulations at 60, 75, and 100 degrees Celsius, which allows us to explore further the details of the folding and unfolding pathway of FBP28. The first beta-turn is near native-like in the transition state for folding (experimental) and unfolding (MD and experiment). The simulations show that there are transient contacts between the aromatic side-chains of the beta-strands in the denatured state and that these interactions provide the driving force for folding of the first beta-hairpin of this three-stranded sheet. Only after the backbone hydrogen bonds are formed between beta1 and beta2 does a hydrogen bond form to stabilize the intervening turn, or the first beta-turn.

Essential chemistry for biochemists.

Within every living organism, countless reactions occur every second. These reactions typically occur more rapidly and with greater efficiency than would be possible under the same conditions in the chemical laboratory, and while using only the subset of elements that are readily available in nature. Despite these apparent differences between life and the laboratory, biological reactions are governed by the same rules as any other chemical reaction. Thus, a firm understanding of the fundamentals of chemistry is invaluable in biochemistry. There are entire textbooks devoted to the application of chemical principles in biological systems and so it is not possible to cover all of the relevant topics in depth in this short article. The aim is instead to provide a brief overview of those areas in chemistry that are most relevant to biochemistry. We summarize the basic principles, give examples of how these principles are applied in biological systems and suggest further reading on individual topics.

Early steps in thermal unfolding of superoxide dismutase 1 are similar to the conformational changes associated with the ALS-associated A4V mutation.

There are over 100 mutations in Cu/Zn superoxide dismutase (SOD1) that result in a subset of familial amyotrophic lateral sclerosis (fALS) cases. The hypothesis that dissociation of the dimer, misfolding of the monomer and subsequent aggregation of mutant SOD1 leads to fALS has been gaining support as an explanation for how these disparate missense mutations cause the same disease. These forms are only responsible for a fraction of the ALS cases; however, the rest are sporadic. Starting with a folded apo monomer, the species considered most likely to be involved in misfolding, we used high-temperature all-atom molecular dynamics simulations to explore the events of the wild-type protein unfolding through the denatured state. All simulations showed early loss of structure along the ß5-ß6 edge of the ß-sandwich, supporting earlier findings of instability in this region. Transition state structures identified from the simulations are in good agreement with experiment, providing detailed, validated molecular models for this elusive state. Furthermore, we compare the process of thermal unfolding investigated here to that of the lethal A4V mutant-induced unfolding at physiological temperature and find that the pathways are very similar.

Dynameomics: protein dynamics and unfolding across fold space.

All currently known structures of proteins together define 'protein fold space'. To increase the general understanding of protein dynamics and protein folding, we selected a set of 807 proteins and protein domains that represent 95% of the currently known autonomous folded domains present in globular proteins. Native state and unfolding simulations of these representatives are now complete and accessible via a novel database containing over 11 000 simulations. Because protein folding is a microscopically reversible process, these simulations effectively sample protein folding across all of protein fold space. Here, we give an overview of how the representative proteins were selected and how the simulations were performed and validated. We then provide examples of different types of analyses that can be performed across our large set of simulations, made possible by the database approach. We further show how the unfolding simulations can be used to compare unfolding of structural elements in isolation and in different structural contexts, using as an example a short, triple stranded ß-sheet that forms the WW domain and is present in several larger unrelated proteins.

Dynameomics: a comprehensive database of protein dynamics.

The dynamic behavior of proteins is important for an understanding of their function and folding. We have performed molecular dynamics simulations of the native state and unfolding pathways of over 2000 protein/peptide systems (approximately 11,000 independent simulations) representing the majority of folds in globular proteins. These data are stored and organized using an innovative database approach, which can be mined to obtain both general and specific information about the dynamics and folding/unfolding of proteins, relevant subsets thereof, and individual proteins. Here we describe the project in general terms and the type of information contained in the database. Then we provide examples of mining the database for information relevant to protein folding, structure building, the effect of single-nucleotide polymorphisms, and drug design. The native state simulation data and corresponding analyses for the 100 most populated metafolds, together with related resources, are publicly accessible through http://www.dynameomics.org.