Disruptive mRNA folding increases translational efficiency of catechol-O-methyltransferase variant.

Catechol-O-methyltransferase (COMT) is a major enzyme controlling catecholamine levels that plays a central role in cognition, affective mood and pain perception. There are three common COMT haplotypes in the human population reported to have functional effects, divergent in two synonymous and one nonsynonymous position. We demonstrate that one of the haplotypes, carrying the non-synonymous variation known to code for a less stable protein, exhibits increased protein expression in vitro. This increased protein expression, which would compensate for lower protein stability, is solely produced by a synonymous variation (C(166)T) situated within the haplotype and located in the 5' region of the RNA transcript. Based on mRNA secondary structure predictions, we suggest that structural destabilization near the start codon caused by the T allele could be related to the observed increase in COMT expression. Our folding simulations of the tertiary mRNA structures demonstrate that destabilization by the T allele lowers the folding transition barrier, thus decreasing the probability of occupying its native state. These data suggest a novel structural mechanism whereby functional synonymous variations near the translation initiation codon affect the translation efficiency via entropy-driven changes in mRNA dynamics and present another example of stable compensatory genetic variations in the human population.

Serotonin-induced hypersensitivity via inhibition of catechol O-methyltransferase activity.

The subcutaneous and systemic injection of serotonin reduces cutaneous and visceral pain thresholds and increases responses to noxious stimuli. Different subtypes of 5-hydroxytryptamine (5-HT) receptors are suggested to be associated with different types of pain responses. Here we show that serotonin also inhibits catechol O-methyltransferase (COMT), an enzyme that contributes to modultion the perception of pain, via non-competitive binding to the site bound by catechol substrates with a binding affinity comparable to the binding affinity of catechol itself (K(i) = 44 µM). Using computational modeling, biochemical tests and cellular assays we show that serotonin actively competes with the methyl donor S-adenosyl-L-methionine (SAM) within the catalytic site. Binding of serotonin to the catalytic site inhibits the access of SAM, thus preventing methylation of COMT substrates. The results of in vivo animal studies show that serotonin-induced pain hypersensitivity in mice is reduced by either SAM pretreatment or by the combined administration of selective antagonists for ß(2)- and ß(3)-adrenergic receptors, which have been previously shown to mediate COMT-dependent pain signaling. Our results suggest that inhibition of COMT via serotonin binding contributes to pain hypersensitivity, providing additional strategies for the treatment of clinical pain conditions.

Regioselectivity of Catechol O-Methyltransferase Confers Enhancement of Catalytic Activity.

Catechol O-methyltransferase (COMT) metabolizes catechol moieties by methylating a single hydroxyl group at the meta- or para- hydroxyl position. Hydrophobic amino acids near the active site of COMT influence the regioselectivity of this reaction. Our sequence analysis highlights their importance by showing that these residues are highly conserved throughout evolution. Reaction barriers calculated in the gas phase reveal a lower barrier during methylation at the meta- position, suggesting that the observed meta-regioselectivity of COMT can be attributed to the substrate itself, and that COMT has evolved residues to orient the substrate in a manner that increases the rate of catalysis.

Macromolecular crowding induces polypeptide compaction and decreases folding cooperativity.

A cell's interior is comprised of macromolecules that can occupy up to 40% of its available volume. Such crowded environments can influence the stability of proteins and their rates of reaction. Using discrete molecular dynamics simulations, we investigate how both the size and number of neighboring crowding reagents affect the thermodynamic and folding properties of structurally diverse proteins. We find that crowding induces higher compaction of proteins. We also find that folding becomes less cooperative with the introduction of crowders into the system. The crowders may induce alternative non-native protein conformations, thus creating barriers for protein folding in highly crowded media.

Ab initio folding of proteins with all-atom discrete molecular dynamics.

Discrete molecular dynamics (DMD) is a rapid sampling method used in protein folding and aggregation studies. Until now, DMD was used to perform simulations of simplified protein models in conjunction with structure-based force fields. Here, we develop an all-atom protein model and a transferable force field featuring packing, solvation, and environment-dependent hydrogen bond interactions. We performed folding simulations of six small proteins (20-60 residues) with distinct native structures by the replica exchange method. In all cases, native or near-native states were reached in simulations. For three small proteins, multiple folding transitions are observed, and the computationally characterized thermodynamics are in qualitative agreement with experiments. The predictive power of all-atom DMD highlights the importance of environment-dependent hydrogen bond interactions in modeling protein folding. The developed approach can be used for accurate and rapid sampling of conformational spaces of proteins and protein-protein complexes and applied to protein engineering and design of protein-protein interactions.

COMT gene locus: new functional variants.

Catechol-O-methyltransferase (COMT) metabolizes catecholaminergic neurotransmitters. Numerous studies have linked COMT to pivotal brain functions such as mood, cognition, response to stress, and pain. Both nociception and risk of clinical pain have been associated with COMT genetic variants, and this association was shown to be mediated through adrenergic pathways. Here, we show that association studies between COMT polymorphic markers and pain phenotypes in 2 independent cohorts identified a functional marker, rs165774, situated in the 3' untranslated region of a newfound splice variant, (a)-COMT. Sequence comparisons showed that the (a)-COMT transcript is highly conserved in primates, and deep sequencing data demonstrated that (a)-COMT is expressed across several human tissues, including the brain. In silico analyses showed that the (a)-COMT enzyme features a distinct C-terminus structure, capable of stabilizing substrates in its active site. In vitro experiments demonstrated not only that (a)-COMT is catalytically active but also that it displays unique substrate specificity, exhibiting enzymatic activity with dopamine but not epinephrine. They also established that the pain-protective A allele of rs165774 coincides with lower COMT activity, suggesting contribution to decreased pain sensitivity through increased dopaminergic rather than decreased adrenergic tone, characteristic of reference isoforms. Our results provide evidence for an essential role of the (a)-COMT isoform in nociceptive signaling and suggest that genetic variations in (a)-COMT isoforms may contribute to individual variability in pain phenotypes.

Discrete molecular dynamics: an efficient and versatile simulation method for fine protein characterization.

Until now it has been impractical to observe protein folding in silico for proteins larger than 50 residues. Limitations of both force field accuracy and computational efficiency make the folding problem very challenging. Here we employ discrete molecular dynamics (DMD) simulations with an all-atom force field to fold fast-folding proteins. We extend the DMD force field by introducing long-range electrostatic interactions to model salt-bridges and a sequence-dependent semiempirical potential accounting for natural tendencies of certain amino acid sequences to form specific secondary structures. We enhance the computational performance by parallelizing the DMD algorithm. Using a small number of commodity computers, we achieve sampling quality and folding accuracy comparable to the explicit-solvent simulations performed on high-end hardware. We demonstrate that DMD can be used to observe equilibrium folding of villin headpiece and WW domain, study two-state folding kinetics, and sample near-native states in ab initio folding of proteins of ∼100 residues.

Structural mechanism of S-adenosyl methionine binding to catechol O-methyltransferase.

Methyltransferases possess a homologous domain that requires both a divalent metal cation and S-adenosyl-L-methionine (SAM) to catalyze its reactions. The kinetics of several methyltransferases has been well characterized; however, the details regarding their structural mechanisms have remained unclear to date. Using catechol O-methyltransferase (COMT) as a model, we perform discrete molecular dynamics and computational docking simulations to elucidate the initial stages of cofactor binding. We find that COMT binds SAM via an induced-fit mechanism, where SAM adopts a different docking pose in the absence of metal and substrate in comparison to the holoenzyme. Flexible modeling of the active site side-chains is essential for observing the lowest energy state in the apoenzyme; rigid docking tools are unable to recapitulate the pose unless the appropriate side-chain conformations are given a priori. From our docking results, we hypothesize that the metal reorients SAM in a conformation suitable for donating its methyl substituent to the recipient ligand. The proposed mechanism enables a general understanding of how divalent metal cations contribute to methyltransferase function.

A didactic model of macromolecular crowding effects on protein folding.

A didactic model is presented to illustrate how the effect of macromolecular crowding on protein folding and association is modeled using current analytical theory and discrete molecular dynamics. While analytical treatments of crowding may consider the effect as a potential of average force acting to compress a polypeptide chain into a compact state, the use of simulations enables the presence of crowding reagents to be treated explicitly. Using an analytically solvable toy model for protein folding, an approximate statistical thermodynamic method is directly compared to simulation in order to gauge the effectiveness of current analytical crowding descriptions. Both methodologies are in quantitative agreement under most conditions, indication that both current theory and simulation methods are capable of recapitulating aspects of protein folding even by utilizing a simplistic protein model.