Emergence of Metastable State Dynamics in Interconnected Cortical Networks with Propagation Delays

The importance of the large number of thin-diameter and unmyelinated axons that connect different cortical areas is unknown. The pronounced propagation delays in these axons may prevent synchronization of cortical networks and therefore hinder efficient information integration and processing. Yet, such global information integration across cortical areas is vital for higher cognitive function. We hypothesized that delays in communication between cortical areas can disrupt synchronization and therefore enhance the set of activity trajectories and computations interconnected networks can perform. To evaluate this hypothesis, we studied the effect of long-range cortical projections with propagation delays in interconnected large-scale cortical networks that exhibited spontaneous rhythmic activity. Long-range connections with delays caused the emergence of metastable, spatio-temporally distinct activity states between which the networks spontaneously transitioned. Interestingly, the observed activity patterns correspond to macroscopic network dynamics such as globally synchronized activity, propagating wave fronts, and spiral waves that have been previously observed in neurophysiological recordings from humans and animal models. Transient perturbations with simulated transcranial alternating current stimulation (tACS) confirmed the multistability of the interconnected networks by switching the networks between these metastable states. Our model thus proposes that slower long-range connections enrich the landscape of activity states and represent a parsimonious mechanism for the emergence of multistability in cortical networks. These results further provide a mechanistic link between the known deficits in connectivity and cortical state dynamics in neuropsychiatric illnesses such as schizophrenia and autism, as well as suggest non-invasive brain stimulation as an effective treatment for these illnesses.     

Molecular Dynamics Simulations and Classical Multidimensional Scaling Unveil New Metastable States in the Conformational Landscape of CDK2

Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS). The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the αC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms throughout the human kinome.     

Ion-Controlled Conformational Dynamics in the Outward-Open Transition from an Occluded State of LeuT

Neurotransmitter:sodium symporter (NSS) proteins are secondary Na⁺-driven active transporters that terminate neurotransmission by substrate uptake. Despite the availability of high-resolution crystal structures of a bacterial homolog of NSSs—Leucine Transporter (LeuT)—and extensive computational and experimental structure-function studies, unanswered questions remain regarding the transport mechanisms. We used microsecond atomistic molecular-dynamics (MD) simulations and free-energy computations to reveal ion-controlled conformational dynamics of LeuT in relation to binding affinity and selectivity of the more extracellularly positioned Na⁺ binding site (Na1 site). In the course of MD simulations starting from the occluded state with bound Na⁺, but in the absence of substrate, we find a spontaneous transition of the extracellular vestibule of LeuT into an outward-open conformation. The outward opening is enhanced by the absence of Na1 and modulated by the protonation state of the Na1-associated Glu-290. Consistently, the Na⁺ affinity for the Na1 site is inversely correlated with the extent of outward-open character and is lower than in the occluded state with bound substrate; however, the Na1 site retains its selectivity for Na⁺ over K⁺ in such conformational transitions. To the best of our knowledge, our findings shed new light on the Na⁺-driven transport cycle and on the symmetry in structural rearrangements for outward- and inward-open transitions.     

Population Dynamics of Metastable Growth-Rate Phenotypes

Neo-Darwinian evolution has presented a paradigm for population dynamics built on random mutations and selection with a clear separation of time-scales between single-cell mutation rates and the rate of reproduction. Laboratory experiments on evolving populations until now have concentrated on the fixation of beneficial mutations. Following the Darwinian paradigm, these experiments probed populations at low temporal resolution dictated by the rate of rare mutations, ignoring the intermediate evolving phenotypes. Selection however, works on phenotypes rather than genotypes. Research in recent years has uncovered the complexity of genotype-to-phenotype transformation and a wealth of intracellular processes including epigenetic inheritance, which operate on a wide range of time-scales. Here, by studying the adaptation dynamics of genetically rewired yeast cells, we show a novel type of population dynamics in which the intracellular processes intervene in shaping the population structure. Under constant environmental conditions, we measure a wide distribution of growth rates that coexist in the population for very long durations (>100 generations). Remarkably, the fastest growing cells do not take over the population on the time-scale dictated by the width of the growth-rate distributions and simple selection. Additionally, we measure significant fluctuations in the population distribution of various phenotypes: the fraction of exponentially-growing cells, the distributions of single-cell growth-rates and protein content. The observed fluctuations relax on time-scales of many generations and thus do not reflect noisy processes. Rather, our data show that the phenotypic state of the cells, including the growth-rate, for large populations in a constant environment is metastable and varies on time-scales that reflect the importance of long-term intracellular processes in shaping the population structure. This lack of time-scale separation between the intracellular and population processes calls for a new framework for population dynamics which is likely to be significant in a wide range of biological contexts, from evolution to cancer.     

Allostery and Conformational Dynamics in cAMP-binding Acyltransferases

Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner. These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. Here we provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases. Thus, we have identified mechanisms by which properties of similar proteins have diverged in two species of mycobacteria by modifications in amino acid sequence, which can dramatically alter the abundance of conformational states adopted by a protein.     

Redox-Linked Conformational Dynamics in Apoptosis-Inducing Factor

Apoptosis-inducing factor (AIF) is a bifunctional mitochondrial flavoprotein critical for energy metabolism and induction of caspase-independent apoptosis, whose exact role in normal mitochondria remains unknown. Upon reduction with NADH, AIF undergoes dimerization and forms tight, long-lived FADH₂-NAD charge-transfer complexes (CTC) that are proposed to be functionally important. To obtain a deeper insight into structure/function relations and redox mechanism of this vitally important protein, we determined the X-ray structures of oxidized and NADH-reduced forms of naturally folded recombinant murine AIF. Our structures reveal that CTC with the pyridine nucleotide is stabilized by (i) π-stacking interactions between coplanar nicotinamide, isoalloxazine, and Phe309 rings; (ii) rearrangement of multiple aromatic residues in the C-terminal domain, likely serving as an electron delocalization site; and (iii) an extensive hydrogen-bonding network involving His453, a key residue that undergoes a conformational switch to directly interact with and optimally orient the nicotinamide for charge transfer. Via the His453-containing peptide, redox changes in the active site are transmitted to the surface, promoting AIF dimerization and restricting access to a primary nuclear localization signal through which the apoptogenic form is transported to the nucleus. Structural findings agree with biochemical data and support the hypothesis that both normal and apoptogenic functions of AIF are controlled by NADH.     

Effects of Topology and Sequence in Protein Folding Linked via Conformational Fluctuations

Experiments have compared the folding of proteins with different amino acid sequences but the same basic structure, or fold. Results indicate that folding is robust to sequence variations for proteins with some nonlocal folds, such as all-β, whereas the folding of more local, all-α proteins typically exhibits a stronger sequence dependence. Here, we use a coarse-grained model to systematically study how variations in sequence perturb the folding energy landscapes of three model sequences with 3α, 4β + α, and β-barrel folds, respectively. These three proteins exhibit folding features in line with experiments, including expected rank order in the cooperativity of the folding transition and stability-dependent shifts in the location of the free-energy barrier to folding. Using a generalized-ensemble simulation approach, we determine the thermodynamics of around 2000 sequence variants representing all possible hydrophobic or polar single- and double-point mutations. From an analysis of the subset of stability-neutral mutations, we find that folding is perturbed in a topology-dependent manner, with the β-barrel protein being the most robust. Our analysis shows, in particular, that the magnitude of mutational perturbations of the transition state is controlled in part by the size or “width” of the underlying conformational ensemble. This result suggests that the mutational robustness of the folding of the β-barrel protein is underpinned by its conformationally restricted transition state ensemble, revealing a link between sequence and topological effects in protein folding.     

Thermal Adaptation of Conformational Dynamics in Ribonuclease H

The relationship between inherent internal conformational processes and enzymatic activity or thermodynamic stability of proteins has proven difficult to characterize. The study of homologous proteins with differing thermostabilities offers an especially useful approach for understanding the functional aspects of conformational dynamics. In particular, ribonuclease HI (RNase H), an 18 kD globular protein that hydrolyzes the RNA strand of RNA:DNA hybrid substrates, has been extensively studied by NMR spectroscopy to characterize the differences in dynamics between homologs from the mesophilic organism E. coli and the thermophilic organism T. thermophilus. Herein, molecular dynamics simulations are reported for five homologous RNase H proteins of varying thermostabilities and enzymatic activities from organisms of markedly different preferred growth temperatures. For the E. coli and T. thermophilus proteins, strong agreement is obtained between simulated and experimental values for NMR order parameters and for dynamically averaged chemical shifts, suggesting that these simulations can be a productive platform for predicting the effects of individual amino acid residues on dynamic behavior. Analyses of the simulations reveal that a single residue differentiates between two different and otherwise conserved dynamic processes in a region of the protein known to form part of the substrate-binding interface. Additional key residues within these two categories are identified through the temperature-dependence of these conformational processes.     

Nanosecond-Timescale Dynamics and Conformational Heterogeneity in Human GCK Regulation and Disease

Human glucokinase (GCK) is the prototypic example of an emerging class of proteins with allosteric-like behavior that originates from intrinsic polypeptide dynamics. High-resolution NMR investigations of GCK have elucidated millisecond-timescale dynamics underlying allostery. In contrast, faster motions have remained underexplored, hindering the development of a comprehensive model of cooperativity. Here, we map nanosecond-timescale dynamics and structural heterogeneity in GCK using a combination of unnatural amino acid incorporation, time-resolved fluorescence, and ¹⁹F nuclear magnetic resonance spectroscopy. We find that a probe inserted within the enzyme’s intrinsically disordered loop samples multiple conformations in the unliganded state. Glucose binding and disease-associated mutations that suppress cooperativity alter the number and/or relative population of these states. Together, the nanosecond kinetics characterized here and the millisecond motions known to be essential for cooperativity provide a dynamical framework with which we address the origins of cooperativity and the mechanism of activated, hyperinsulinemia-associated, noncooperative variants.     

Conformational Dynamics of a Ligand-Free Adenylate Kinase

Adenylate kinase (AdK) is a phosphoryl-transfer enzyme with important physiological functions. Based on a ligand-free open structure and a ligand-bound closed structure solved by crystallography, here we use molecular dynamics simulations to examine the stability and dynamics of AdK conformations in the absence of ligands. We first perform multiple simulations starting from the open or the closed structure, and observe their free evolutions during a simulation time of 100 or 200 nanoseconds. In all seven simulations starting from the open structure, AdK remained stable near the initial conformation. The eight simulations initiated from the closed structure, in contrast, exhibited large variation in the subsequent evolutions, with most (seven) undergoing large-scale spontaneous conformational changes and approaching or reaching the open state. To characterize the thermodynamics of the transition, we propose and apply a new sampling method that employs a series of restrained simulations to calculate a one-dimensional free energy along a curved pathway in the high-dimensional conformational space. Our calculated free energy profile features a single minimum at the open conformation, and indicates that the closed state, with a high (∼13 kcal/mol) free energy, is not metastable, consistent with the observed behaviors of the unrestrained simulations. Collectively, our simulations suggest that it is energetically unfavorable for the ligand-free AdK to access the closed conformation, and imply that ligand binding may precede the closure of the enzyme.     

Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 Å resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an “induced-fit” association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer''s and Parkinson''s, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications.     

A Physical Picture of Protein Dynamics and Conformational Changes

A physical model is reviewed which explains different aspects of protein dynamics consistently. At low temperatures, the molecules are frozen in conformational substates. Their average energy is 3/2RT. Solid-state vibrations occur on a time scale of femtoseconds to nanoseconds. Above a characteristic temperature, often called the dynamical transition temperature, slow modes of motions can be observed occurring on a time scale between about 140 and 1 ns. These motions are overdamped, quasidiffusive, and involve collective motions of segments of the size of an α-helix. Molecules performing these types of motion are in the “flexible state”. This state is reached by thermal activation. It is shown that these motions are essential for conformational relaxation. Based on this picture, a new approach is proposed to understand conformational changes. It connects structural fluctuations and conformational transitions.     

Conformational Dynamics of Protein Side Chains and Enzyme-Substrate Interaction

Abstract

The electric interaction of charged and polar protein side chains with dipole moments of the bonds of substrate molecules bound in enzyme active sites is considered. The conformational motion of the side chains leads to the fact that the electric interaction, besides a constant (electrostatic) part, contains a fluctuating one, which is a random force (noise) exerted on the substrate molecule. On the time scale of enzyme turnovers this noise can be considered as the white one with good approximation. The noise is external, and the explicit expression for its intensity has been obtained. The possible functional role of the noise as an activating factor in enzyme catalysis is discussed.     

Reconciling Mediating and Slaving Roles of Water in Protein Conformational Dynamics

Proteins accomplish their physiological functions with remarkably organized dynamic transitions among a hierarchical network of conformational substates. Despite the essential contribution of water molecules in shaping functionally important protein dynamics, their exact role is still controversial. Water molecules were reported either as mediators that facilitate or as masters that slave protein dynamics. Since dynamic behaviour of a given protein is ultimately determined by the underlying energy landscape, we systematically analysed protein self energies and protein-water interaction energies obtained from extensive molecular dynamics simulation trajectories of barstar. We found that protein-water interaction energy plays the dominant role when compared with protein self energy, and these two energy terms on average have negative correlation that increases with increasingly longer time scales ranging from 10 femtoseconds to 100 nanoseconds. Water molecules effectively roughen potential energy surface of proteins in the majority part of observed conformational space and smooth in the remaining part. These findings support a scenario wherein water on average slave protein conformational dynamics but facilitate a fraction of transitions among different conformational substates, and reconcile the controversy on the facilitating and slaving roles of water molecules in protein conformational dynamics.     

Conformational Dynamics and Ligand Binding in the Multi-Domain Protein PDC109

PDC109 is a modular multi-domain protein with two fibronectin type II (Fn2) repeats joined by a linker. It plays a major role in bull sperm binding to the oviductal epithelium through its interactions with phosphorylcholines (PhCs), a head group of sperm cell membrane lipids. The crystal structure of the PDC109-PhC complex shows that each PhC binds to the corresponding Fn2 domain, while the two domains are on the same face of the protein. Long timescale explicit solvent molecular dynamics (MD) simulations of PDC109, in the presence and absence of PhC, suggest that PhC binding strongly correlates with the relative orientation of choline-phospholipid binding sites of the two Fn2 domains; unless the two domains tightly bind PhCs, they tend to change their relative orientation by deforming the flexible linker. The effective PDC109-PhC association constant of 28 M, estimated from their potential of mean force is consistent with the experimental result. Principal component analysis of the long timescale MD simulations was compared to the significantly less expensive normal mode analysis of minimized structures. The comparison indicates that difference between relative domain motions of PDC109 with bound and unbound PhC is captured by the first principal component in the principal component analysis as well as the three lowest normal modes in the normal mode analysis. The present study illustrates the use of detailed MD simulations to clarify the energetics of specific ligand-domain interactions revealed by a static crystallographic model, as well as their influence on relative domain motions in a multi-domain protein.     

High-Sensitivity Fluorometry to Resolve Ion Channel Conformational Dynamics

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Functional Roles of Slow Enzyme Conformational Changes in Network Dynamics

Extensive studies from different fields reveal that many macromolecules, especially enzymes, show slow transitions among different conformations. This phenomenon is named such things as dynamic disorder, heterogeneity, hysteretic or mnemonic enzymes across these different fields, and has been directly demonstrated by single molecule enzymology and NMR studies recently. We analyzed enzyme slow conformational changes in the context of regulatory networks. A single enzymatic reaction with slow conformational changes can filter upstream network noises, and can either resonantly respond to the system stimulus at certain frequencies or respond adaptively for sustained input signals of the network fluctuations. It thus can serve as a basic functional motif with properties that are normally for larger intermolecular networks in the field of systems biology. We further analyzed examples including enzymes functioning against pH fluctuations, metabolic state change of Artemia embryos, and kinetic insulation of fluctuations in metabolic networks. The study also suggests that hysteretic enzymes may be building blocks of synthetic networks with various properties such as narrow-banded filtering. The work fills the missing gap between studies on enzyme biophysics and network level dynamics, and reveals that the coupling between the two is functionally important; it also suggests that the conformational dynamics of some enzymes may be evolutionally selected.