Conformational Motions Research Articles

Unexpected self-healing acceleration within urethane networks: lignosulfonate mediation of ionic and hydrogen bonds for ultra-strong recyclable elastomers

Stiff structures are generally considered detrimental to the materials’ self-healing efficiency because of the rigid conformation and restricted motion of the molecular chains. However, in this study, an unexpected acceleration in self-healing was observed in a urethane network when lignosulfonate, a bio-based stiff structure with stiff benzene rings, was introduced into the polyurethane matrix. An extraordinary increase in mechanical properties was achieved with just 3 wt% lignosulfonate added to the pristine sample, including a strength increase from 16.24 MPa to 42.89 MPa and elongation at break from 390% to 602%. Importantly, this significant enhancement in mechanical properties was accompanied by a notable acceleration in healing time efficiency, which increased by up to 44% compared to the pristine sample. This unique behavior can be attributed to the mediation of lignosulfonate on the ionic bonds between lignosulfonate and the urethane segment, as well as the hydrogen bonds formed within the urethane segments. This mediation ensures ultra-high strength without compromising the self-healing capacity and opens a way for accelerating elastomers self-healing efficiency through lignosulfonate, a readily available biomass-derived material. In addition, the addition of lignosulfonates gives the elastomer some Anti-UV performance.

Predicting protein conformational motions using energetic frustration analysis and AlphaFold2

Proteins perform their biological functions through motion. Although high throughput prediction of the three-dimensional static structures of proteins has proved feasible using deep-learning-based methods, predicting the conformational motions remains a challenge. Purely data-driven machine learning methods encounter difficulty for addressing such motions because available laboratory data on conformational motions are still limited. In this work, we develop a method for generating protein allosteric motions by integrating physical energy landscape information into deep-learning-based methods. We show that local energetic frustration, which represents a quantification of the local features of the energy landscape governing protein allosteric dynamics, can be utilized to empower AlphaFold2 (AF2) to predict protein conformational motions. Starting from ground state static structures, this integrative method generates alternative structures as well as pathways of protein conformational motions, using a progressive enhancement of the energetic frustration features in the input multiple sequence alignment sequences. For a model protein adenylate kinase, we show that the generated conformational motions are consistent with available experimental and molecular dynamics simulation data. Applying the method to another two proteins KaiB and ribose-binding protein, which involve large-amplitude conformational changes, can also successfully generate the alternative conformations. We also show how to extract overall features of the AF2 energy landscape topography, which has been considered by many to be black box. Incorporating physical knowledge into deep-learning-based structure prediction algorithms provides a useful strategy to address the challenges of dynamic structure prediction of allosteric proteins.

In Silico Analysis of Conformational Dynamics and Energetic Landscapes of Putative Insulinase PF11_0189 From the Plasmodium falciparum Genome With Insulin‐Derived Peptides: Approach to Rationale Design of Insulin Peptide‐Based Inhibitors

ABSTRACTGlucose abnormality is one of the atypical symptoms mostly overlooked during severe malaria. Screening for proteases involved in glucose abnormality suggests a potential new drug target. PF11_0189 is a putative insulin degrading enzyme found in the genome of Plasmodium falciparum. In silico approach revealed that PF11_0189 is similar to the metalloproteases exhibiting a zinc binding motif. The substrate binding region of PF11_0189 catalytic domain is lined by residues of a mixed nature, enabling the accommodation of both hydrophobic and hydrophilic residues from substrates. Analysis of each amino acid position from combinatorial peptide library suggests Thr and Ser to be the most crucial residues as upon its mutation significant improvement is observed in the binding. The peptides P‐21 (VPICSLY), P‐70 (TMICVLY), and P‐121 (AVICSLY) demonstrate significantly better interaction within the active site than a template peptide (TSICSLY). Molecular dynamic simulations confirm the complex's integrity, with all structures within the qualitative limit of compactness and stability during the simulation time. Principal component analysis (PCA) reveals major conformational motions and different energy landscapes, while dynamic cross‐correlation matrix (DCCM) highlights residue interactions of the complexes. The insights obtained through computational methods provide clues about substrate selectivity in PF11_0189, which can be utilized for peptide‐based therapeutics against severe malaria.

1,3,5–2,4,6‐Functionalized Benzene Molecular Cage: An Environmentally Responsive Scaffold that Supports Hierarchical Superstructures

AbstractNew stimulus‐responsive scaffolds are of interest as constituents of hierarchical supramolecular ensembles. 1,3,5–2,4,6‐Functionalized, facially segregated benzene moieties have a time‐honored role as building blocks for host molecules. However, their user as switchable motifs in the construction of multi‐component supramolecular structures remains poorly explored. Here, we report a molecular cage 1, which consists of a bent anthracene dimer 3 paired with 1,3,5‐tris(aminomethyl)‐2,4,6‐triethylbenzene 2. As the result of the pH‐induced ababab↔bababa isomerization of the constituent‐functionalized benzene units derived from 2, this cage can reversibly convert between an open state and a closed form, both in solution and in the solid state. Cage 1 was used to create stimuli‐responsive hierarchical superstructures, namely Russian doll‐like complexes with [K⊂18‐crown‐6⊂1]+ and [K⊂cryptand‐222⊂1]+. The reversible assembly and disassembly of these superstructures could be induced by switching cage 1 from its open to closed form. The present study thus provides an unusual example where pH‐triggered conformation motion within a cage‐like scaffold is used to control the formation and disassociation of hierarchical ensembles.

1,3,5-2,4,6-Functionalized Benzene Molecular Cage: An Environmentally Responsive Scaffold that Supports Hierarchical Superstructures.

New stimulus-responsive scaffolds are of interest as constituents of hierarchical supramolecular ensembles. 1,3,5-2,4,6-Functionalized, facially segregated benzene moieties have a time-honored role as building blocks for host molecules. However, their user as switchable motifs in the construction of multi-component supramolecular structures remains poorly explored. Here, we report a molecular cage 1, which consists of a bent anthracene dimer 3 paired with 1,3,5-tris(aminomethyl)-2,4,6-triethylbenzene 2. As the result of the pH-induced ababab↔bababa isomerization of the constituent-functionalized benzene units derived from 2, this cage can reversibly convert between an open state and a closed form, both in solution and in the solid state. Cage 1 was used to create stimuli-responsive hierarchical superstructures, namely Russian doll-like complexes with [K⊂18-crown-6⊂1]+ and [K⊂cryptand-222⊂1]+. The reversible assembly and disassembly of these superstructures could be induced by switching cage 1 from its open to closed form. The present study thus provides an unusual example where pH-triggered conformation motion within a cage-like scaffold is used to control the formation and disassociation of hierarchical ensembles.

They all rock: A systematic comparison of conformational movements in LeuT-fold transporters

Many membrane transporters share the LeuT fold-two five-helix repeats inverted across the membrane plane. Despite hundreds of structures, whether distinct conformational mechanisms are supported by the LeuT fold has not been systematically determined. After annotating published LeuT-fold structures, we analyzed distance difference matrices (DDMs) for nine proteins with multiple available conformations. We identified rigid bodies and relative movements of transmembrane helices (TMs) during distinct steps of the transport cycle. In all transporters, the bundle (first two TMs of each repeat) rotates relative to the hash (third and fourth TMs). Motions of the arms (fifth TM) to close or open the intracellular and outer vestibules are common, as is a TM1a swing, with notable variations in the opening-closing motions of the outer vestibule. Our analyses suggest that LeuT-fold transporters layer distinct motions on a common bundle-hash rock and demonstrate that systematic analyses can provide new insights into large structural datasets.

Explaining Conformational Diversity in Protein Families through Molecular Motions

Proteins play a central role in biological processes, and understanding their conformational variability is crucial for unraveling their functional mechanisms. Recent advancements in high-throughput technologies have enhanced our knowledge of protein structures, yet predicting their multiple conformational states and motions remains challenging. This study introduces Dimensionality Analysis for protein Conformational Exploration (DANCE) for a systematic and comprehensive description of protein families conformational variability. DANCE accommodates both experimental and predicted structures. It is suitable for analysing anything from single proteins to superfamilies. Employing it, we clustered all experimentally resolved protein structures available in the Protein Data Bank into conformational collections and characterized them as sets of linear motions. The resource facilitates access and exploitation of the multiple states adopted by a protein and its homologs. Beyond descriptive analysis, we assessed classical dimensionality reduction techniques for sampling unseen states on a representative benchmark. This work improves our understanding of how proteins deform to perform their functions and opens ways to a standardised evaluation of methods designed to sample and generate protein conformations.

Effects of Colored Noise in the Dynamic Motions and Conformational Exploration of Enzymes

The intracellular environment displays complex dynamics influenced by factors such as molecular crowding and the low Reynolds number of the cytoplasm. Enzymes exhibiting active matter properties further heighten this complexity which can lead to memory effects. Molecular simulations often neglect these factors, treating the environment as a “thermal bath” using the Langevin equation (LE) with white noise. One way to consider these factors is by using colored noise instead within the generalized Langevin equation (GLE) framework, which allows for the incorporation of memory effects that have been observed in experimental data. We investigated the structural and dynamic differences in Shikimate kinase (SK) using LE and GLE simulations. Our results suggest that GLE simulations, which reveal significant changes, could be utilized for assessing conformational motions’ impact on catalytic reactions.

D-Serine Inhibits Non-ionotropic NMDA Receptor Signaling.

NMDA-type glutamate receptors (NMDARs) are widely recognized as master regulators of synaptic plasticity, most notably for driving long-term changes in synapse size and strength that support learning. NMDARs are unique among neurotransmitter receptors in that they require binding of both neurotransmitter (glutamate) and co-agonist (e.g., d-serine) to open the receptor channel, which leads to the influx of calcium ions that drive synaptic plasticity. Over the past decade, evidence has accumulated that NMDARs also support synaptic plasticity via ion flux-independent (non-ionotropic) signaling upon the binding of glutamate in the absence of co-agonist, although conflicting results have led to significant controversy. Here, we hypothesized that a major source of contradictory results might be attributed to variable occupancy of the co-agonist binding site under different experimental conditions. To test this hypothesis, we manipulated co-agonist availability in acute hippocampal slices from mice of both sexes. We found that enzymatic scavenging of endogenous co-agonists enhanced the magnitude of long-term depression (LTD) induced by non-ionotropic NMDAR signaling in the presence of the NMDAR pore blocker MK801. Conversely, a saturating concentration of d-serine completely inhibited LTD and spine shrinkage induced by glutamate binding in the presence of MK801 or Mg2+ Using a Förster resonance energy transfer (FRET)-based assay in cultured neurons, we further found that d-serine completely blocked NMDA-induced conformational movements of the GluN1 cytoplasmic domains in the presence of MK801. Our results support a model in which d-serine availability serves to modulate NMDAR signaling and synaptic plasticity even when the NMDAR is blocked by magnesium.

Predicting FFAR4 agonists using structure-based machine learning approach based on molecular fingerprints

Free Fatty Acid Receptor 4 (FFAR4), a G-protein-coupled receptor, is responsible for triggering intracellular signaling pathways that regulate various physiological processes. FFAR4 agonists are associated with enhancing insulin release and mitigating the atherogenic, obesogenic, pro-carcinogenic, and pro-diabetogenic effects, normally associated with the free fatty acids bound to FFAR4. In this research, molecular structure-based machine-learning techniques were employed to evaluate compounds as potential agonists for FFAR4. Molecular structures were encoded into bit arrays, serving as molecular fingerprints, which were subsequently analyzed using the Bayesian network algorithm to identify patterns for screening the data. The shortlisted hits obtained via machine learning protocols were further validated by Molecular Docking and via ADME and Toxicity predictions. The shortlisted compounds were then subjected to MD Simulations of the membrane-bound FFAR4-ligand complexes for 100 ns each. Molecular analyses, encompassing binding interactions, RMSD, RMSF, RoG, PCA, and FEL, were conducted to scrutinize the protein–ligand complexes at the inter-atomic level. The analyses revealed significant interactions of the shortlisted compounds with the crucial residues of FFAR4 previously documented. FFAR4 as part of the complexes demonstrated consistent RMSDs, ranging from 3.57 to 3.64, with minimal residue fluctuations 5.27 to 6.03 nm, suggesting stable complexes. The gyration values fluctuated between 22.8 to 23.5 nm, indicating structural compactness and orderliness across the studied systems. Additionally, distinct conformational motions were observed in each complex, with energy contours shifting to broader energy basins throughout the simulation, suggesting thermodynamically stable protein–ligand complexes. The two compounds CHEMBL2012662 and CHEMBL64616 are presented as potential FFAR4 agonists, based on these insights and in-depth analyses. Collectively, these findings advance our comprehension of FFAR4’s functions and mechanisms, highlighting these compounds as potential FFAR4 agonists worthy of further exploration as innovative treatments for metabolic and immune-related conditions.

In Silico Investigation of Parkin-Activating Mutations Using Simulations and Network Modeling.

Complete loss-of-function mutations in the PRKN gene are a major cause of early-onset Parkinson's disease (PD). PRKN encodes the Parkin protein, an E3 ubiquitin ligase that works in conjunction with the ubiquitin kinase PINK1 in a distinct quality control pathway to tag damaged mitochondria for autophagic clearance, i.e., mitophagy. According to previous structural investigations, Parkin protein is typically kept in an inactive conformation via several intramolecular, auto-inhibitory interactions. Here, we performed molecular dynamics simulations (MDS) to provide insights into conformational changes occurring during the de-repression of Parkin and the gain of catalytic activity. We analyzed four different Parkin-activating mutations that are predicted to disrupt certain aspects of its auto-inhibition. All four variants showed greater conformational motions compared to wild-type protein, as well as differences in distances between domain interfaces and solvent-accessible surface area, which are thought to play critical roles as Parkin gains catalytic activity. Our findings reveal that the studied variants exert a notable influence on Parkin activation as they alter the opening of its closed inactive structure, a finding that is supported by recent structure- and cell-based studies. These findings not only helped further characterize the hyperactive variants but overall improved our understanding of Parkin's catalytic activity and nominated targets within Parkin's structure for potential therapeutic designs.

3D-QSAR and Molecular Dynamics Study of Isoxazole Derivatives to Identify the Structural Requirements for Farnesoid X Receptor (FXR) Agonists.

The farnesoid X receptor (FXR) has been recognized as a potential drug target for the treatment of non-alcoholic fatty liver disease (NAFLD). FXR agonists benefit NAFLD by modulating bile acid synthesis and transport, lipid metabolism, inflammation, and fibrosis pathways. However, there are still great challenges involved in developing safe and effective FXR agonists. To investigate the critical factors contributing to their activity on the FXR, 3D-QSAR molecular modeling was applied to a series of isoxazole derivatives, using comparative molecular field analysis (CoMFA (q2 = 0.664, r2 = 0.960, r2pred = 0.872)) and comparative molecular similarity indices analysis (CoMSIA (q2 = 0.706, r2 = 0.969, r2pred = 0.866)) models, which demonstrated strong predictive ability in our study. The contour maps generated from molecular modeling showed that the presence of hydrophobicity at the R2 group and electronegativity group at the R3 group in these compounds is crucial to their agonistic activity. A molecular dynamics (MD) simulation was carried out to further understand the binding modes and interactions between the FXR and its agonists in preclinical or clinical studies. The conformational motions of loops L: H1/H2 and L: H5/H6 in FXR-ligand binding domain (LBD) were crucial to the protein stability and agonistic activity of ligands. Hydrophobic interactions were formed between residues (such as LEU287, MET290, ALA291, HIS294, and VAL297) in helix H3 and ligands. In particular, our study found that residue ARG331 participated in salt bridges, and HIS447 participated in salt bridges and hydrogen bonds with ligands; these interactions were significant to protein-ligand binding. Eight new potent FXR agonists were designed according to our results, and their activities were predicted to be better than that of the first synthetic FXR agonist, GW4064.

On the origin of the electronic and magnetic circular dichroism of naphthyl C-glycosides: Anomeric configuration

Aryl C-glycosides, in which the glycosidic bond is changed to a carbon–carbon bond, are an important family of biologically-active compounds. They often serve as secondary metabolites or exhibit antibiotic and cytostatic activities. Their stability to hydrolysis has made them attractive targets for new drugs. Their conformational behavior often strongly influences the resulting function. Their detailed structural and conformational description is thus highly desirable.This work studies the structure of three different naphthyl C-glycosides using UV–vis absorption as well as electronic and magnetic circular dichroism. It also describes their conformational preferences using a combination of molecular dynamics and DFT calculations. The reliability of these preferences has been verified by simulations of spectral properties and a comparison with their measured spectra. In particular, ECD spectroscopy has been shown to distinguish easily between α- and β-pseudoanomers of aryl C-glycosides. Computer simulations and spectral decomposition have revealed how the resulting ECD patterns of the naphthyl glycosides studied are influenced by different conformer populations. In conclusion, reliable ECD patterns cannot be calculated by separating the naphthyl rotation from other conformational motions. MCD patterns have been similar for all the naphthyl C-glycosides studied. No clear diagnostic features have been found for either the pseudoanomeric configuration or the preferred hydroxymethyl rotamer. Nevertheless, the work has demonstrated the potential of MCD for the study of aryl glycosides interacting with proteins.

Cation Chloride Cotransporter NKCC1 Operates through a Rocking-Bundle Mechanism.

The sodium, potassium, and chloride cotransporter 1 (NKCC1) plays a key role in tightly regulating ion shuttling across cell membranes. Lately, its aberrant expression and function have been linked to numerous neurological disorders and cancers, making it a novel and highly promising pharmacological target for therapeutic interventions. A better understanding of how NKCC1 dynamically operates would therefore have broad implications for ongoing efforts toward its exploitation as a therapeutic target through its modulation. Based on recent structural data on NKCC1, we reveal conformational motions that are key to its function. Using extensive deep-learning-guided atomistic simulations of NKCC1 models embedded into the membrane, we captured complex dynamical transitions between alternate open conformations of the inner and outer vestibules of the cotransporter and demonstrated that NKCC1 has water-permeable states. We found that these previously undefined conformational transitions occur via a rocking-bundle mechanism characterized by the cooperative angular motion of transmembrane helices (TM) 4 and 9, with the contribution of the extracellular tip of TM 10. We found these motions to be critical in modulating ion transportation and in regulating NKCC1's water transporting capabilities. Specifically, we identified interhelical dynamical contacts between TM 10 and TM 6, which we functionally validated through mutagenesis experiments of 4 new targeted NKCC1 mutants. We conclude showing that those 4 residues are highly conserved in most Na+-dependent cation chloride cotransporters (CCCs), which highlights their critical mechanistic implications, opening the way to new strategies for NKCC1's function modulation and thus to potential drug action on selected CCCs.

Hydrodynamic Control of Alzheimer Aβ Fibrillation with Glucosaminic Acid Containing Click-Cyclized β-Bodies.

It is well established that the dynamic hydration shell plays a vital role in macromolecular functions such as protein-ligand, protein-protein, protein-DNA, and protein-lipid interactions. Here we investigate how the water modality affects conformational changes, solubility, and motion of fibrillar proteins. The hypothesis is that the introduction of a poly hydroxyl amino acid would increase solvation of the fibril forming peptides, preventing their misfolding and aggregation. For the amyloid β (Aβ) peptide, which is considered to be connected with nervous system diseases, including dementia and cognitive decline in Alzheimer's disease, the formation of β-sheet fibrils always occurs with a conformational change and a decrease in the dynamic hydration shell around Aβ(1-42). We present novel cyclic d-amino acid peptides that effectively inhibit fibrillation through affecting the dynamic hydration shell of Aβ(1-42) in vitro. Using de novo design within the software Molecular Operating Environment (MOE), five different peptides that recognize Alzheimer's fibrils were designed and synthesized. Three of them were cyclic all-d-amino acid peptides incorporating the same polyhydroxy building block derived from d-glucosaminic acid (GA). One peptide was the parent cyclic all d-amino acid inhibitor with no GA incorporated, and another was an all l-amino acid linear fibrillation inhibitor. The GA-containing peptides were found to show significantly improved inhibition of Aβ(1-42) aggregation. The inhibition was dramatically improved by the synergistic application of two GA peptides targeting each end of the growing fibril. The present study may facilitate future developments of intervention strategies for Alzheimer's disease and similar neurodegenerative diseases.

How does a biopolymer (protein) fold into a unique 3D structure?

The current state of the problem of folding proteins and other biopolymers is discussed. The concept of a multidimensional potential energy surface and a free energy surface for linear polymers is detailed, taking into account the topology of the configuration space and the presence of symmetry elements with respect to the permutation of identical monomer units. The presence of kinematic bonds for conformational movements in a viscous medium leads to a tendency to form helical structures of linear polymers. The dynamic effects of viscosity also lead to an almost uniform distribution of energy dissipation rates over the chain nodes. The combination of potential energy surface topography and viscosity effects provides a physical basis for the development of the theory of folding in the direction of interpreting various experimental observations and elucidating the principles for the formation of an amino acid code for 3D protein structures. The relationship between the denaturation temperature of the folded state of the biopolymer and the energy of non-valent interactions between monomers in the chain is analyzed.

Modifying the amino acids in conformational motion pathway of the α-amylase of Geobacillus stearothermophilus improved its activity and stability.

Amino acids along the conformational motion pathway of the enzyme molecule correlated to its flexibility and rigidity. To enhance the enzyme activity and thermal stability, the motion pathway of Geobacillus stearothermophilus α-amylase has been identified and molecularly modified by using the neural relational inference model and deep learning tool. The significant differences in substrate specificity, enzymatic kinetics, optimal temperature, and thermal stability were observed among the mutants with modified amino acids along the pathway. Mutants especially the P44E demonstrated enhanced hydrolytic activity and catalytic efficiency (kcat/KM) than the wild-type enzyme to 95.0% and 93.8% respectively, with the optimum temperature increased to 90°C. This mutation from proline to glutamic acid has increased the number and the radius of the bottleneck of the channels, which might facilitate transporting large starch substrates into the enzyme. The mutation could also optimize the hydrogen bonding network of the catalytic center, and diminish the spatial hindering to the substrate entry and exit from the catalytic center.

Reaction induced conformational change in polyindole: Polyindole/PVA film as biomimetic sensors of temperature and electrical energetic condition.

The influence of surrounding temperature and electrical energetic condition on the conformational movements (cooperative actuation) of polyindole is verified using a polyindole-coated polyvinyl alcohol (PIN/PVA) film. Chronopotentiometric studies revealsthat the consumed electrical energy during the reaction varies linearly with the change in working temperature. The influence of temperature on the reversible conformational movements of the polymer chain is related to the charge consumed during the reaction. The logarithmic dependence of reversible redox charge obtained from coulovoltammogram with inverse of temperature further proved the temperature sensing characteristics and the influence of temperature on the cooperative actuation of the PIN/PVA film. The conformational relaxation increases as the temperature increases through hosting higher number of counter anions with the solvent molecule. The extension of the redox reaction or charge consumption was found to decrease as the scan rate increases. The double logarithmic relation between the consumed redox charge and the scan rate has proved that the electrical energetic condition can influence the conformational movement or the extension of the redox reaction in a reversible manner. The results suggest that the PIN/PVA film can act as a biomimetic macro molecular sensor of working temperature and electrical energetic condition as biological muscles do.

Defining the Conformational Ensembles Associated with Ligand Binding to Cyclooxygenase-2.

Cyclooxygenases (COX) catalyze the committed step in the production of prostaglandins responsible for the maintenance of physiological homeostasis. While crystal structures of COX in complex with substrates and inhibitors have provided insight into the molecular interactions governing their binding, they have not uncovered specific details related to the protein conformational motions responsible for important aspects of the COX function. We created a cysteine-free COX-2 construct and introduced a free cysteine at position-122 to enable labeling with 3-bromo-1,1,1-trifluoroacetone (BTFA). Placement of the label adjacent to the cyclooxygenase channel entrance permitted the detection of alterations upon ligand binding. 19F-nuclear magnetic resonance spectroscopy (19F-NMR) was then used to probe the conformational ensembles arising from BTFA-labeled COX-2 constructs in the presence and absence of ligands known to allosterically activate or inhibit COX-2. 19F-NMR analyses performed in the presence of the time-dependent inhibitor flurbiprofen, as well as Arg-120, Tyr-355, and Glu-524 mutations, led to the classification of two ensembles as representing the relaxed and tightened states of the cyclooxygenase channel entrance. A third ensemble, generated in the presence of arachidonic acid and the Y355F mutant and modulated by the allosteric potentiators palmitic acid and oleic acid and the nonallosteric substrates 2-arachidonoyl glycerol ether and anandamide, was classified as being related to the allosteric regulation of COX activity. The ensemble-based insight into COX function demonstrated here complements the static information derived from crystal structure analyses, collectively providing a more detailed framework of the dynamics involved in the regulation of COX catalysis and inhibition.

Activation Loop Plasticity and Active Site Coupling in the MAP Kinase, ERK2

Previous studies of the protein kinase, ERK2, using NMR and hydrogen-exchange measurements have shown changes in dynamics accompanying its activation by phosphorylation. However, knowledge about the conformational motions involved is incomplete. Here, we examined ERK2 using long conventional molecular dynamics (MD) simulations starting from crystal structures of phosphorylated (2P) and unphosphorylated (0P) forms. Individual trajectories were run for (5 to 25) μs, totaling 727 μs. The results show unexpected flexibility of the A-loop, with multiple long-lived (>5 μs) conformational states in both 2P- and 0P-ERK2. Differential contact network and principal component analyses reveal coupling between the A-loop fold and active site dynamics, with evidence for conformational selection in the kinase core of 2P-ERK2 but not 0P-ERK2. Simulations of 2P-ERK2 show A-loop states corresponding to restrained dynamics within the N-lobe, including regions around catalytic residues. One A-loop conformer forms lasting interactions with the L16 segment, leading to reduced RMSF and greater compaction in the active site. By contrast, simulations of 0P-ERK2 reveal excursions of A-loop residues away from the C-lobe, leading to greater active site mobility. Thus, the A-loop in ERK2 switches between distinct conformations that reflect coupling with the active site, possibly via the L16 segment. Crystal packing interactions suggest that lattice contacts with the A-loop may restrain its structural variation in X-ray structures of ERK2. The novel conformational states identified by MD expand our understanding of ERK2 regulation, by linking the activated state of the kinase to reduced dynamics and greater compaction surrounding the catalytic site.