Role Of Conformational Dynamics Research Articles

Role of conformational dynamics in the catalytic mechanism of viral macrodomains

Role of conformational dynamics in the catalytic mechanism of viral macrodomains

Interaction of the Inhibitory Peptides ShK and HmK with the Voltage-Gated Potassium Channel KV1.3: Role of Conformational Dynamics.

Peptide toxins that adopt the ShK fold can inhibit the voltage-gated potassium channel KV1.3 with IC50 values in the pM range and are therefore potential leads for drugs targeting autoimmune and neuroinflammatory diseases. Nuclear magnetic resonance (NMR) relaxation measurements and pressure-dependent NMR have shown that, despite being cross-linked by disulfide bonds, ShK itself is flexible in solution. This flexibility affects the local structure around the pharmacophore for the KV1.3 channel blockade and, in particular, the relative orientation of the key Lys and Tyr side chains (Lys22 and Tyr23 in ShK) and has implications for the design of KV1.3 inhibitors. In this study, we have performed molecular dynamics (MD) simulations on ShK and a close homologue, HmK, to probe the conformational space occupied by the Lys and Tyr residues, and docked the different conformations with a recently determined cryo-EM structure of the KV1.3 channel. Although ShK and HmK have 60% sequence identity, their dynamic behaviors are quite different, with ShK sampling a broad range of conformations over the course of a 5 μs MD simulation, while HmK is relatively rigid. We also investigated the importance of conformational dynamics, in particular the distance between the side chains of the key dyad Lys22 and Tyr23, for binding to KV1.3. Although these peptides have quite different dynamics, the dyad in both adopts a similar configuration upon binding, revealing a conformational selection upon binding to KV1.3 in the case of ShK. Both peptides bind to KV1.3 with Lys22 occupying the pore of the channel. Intriguingly, the more flexible peptide, ShK, binds with significantly higher affinity than HmK.

The role of conformational dynamics in intrinsically disordered lipid anchors of peripheral membrane proteins in specifying interactions with membrane lipids.

Membrane binding and function of many small GTPases depends on a prenylated hypervariable region (HVR), roughly a 10-30 residue-long intrinsically disordered C-terminus that often contains a polybasic domain (PBD) adjacent to the prenyl group. A long held view was that the PBD non-specifically interacts with anionic membrane phospholipids. However, our previous work on the KRAS HVR showed that the PBD sorts anionic lipids with defined head group and acyl chain structure. Molecular dynamics (MD) simulations suggested that this sorting is realized by backbone conformational dynamics. In the current study, we asked whether this finding is generalizable to HVRs of other PBD-containing lipidated small GTPases. To answer this question, we used atomistic MD simulations to examine the dynamics and interaction patterns of PBD-containing HVRs with varying net charge, sequence length, and lipid-modification (farnesylation and geranylgeranylation). Our results show that the interaction of the PBD with anionic lipids indeed depends on the conformational dynamics and orientation of the backbone, which is primarily determined by the sequence of the PBD but less so on the length of HVR.

Loop dynamics and evolvability in the TIM-barrel proteins of histidine and tryptophan biosynthesis.

Recent years have witnessed increasing interest in the role of conformational dynamics in enzyme evolution and engineering. In this context, TIM-barrel proteins are important model systems, as the TIM-barrel is ubiquitous, highly evolvable, supports a diverse range of chemistry, and the active site is typically decorated by one or more mobile loops that play an important role in catalysis and/or regulation of these enzymes. Here, the TIM-barrel proteins of histidine and tryptophan biosynthesis (HisA, TrpF and PriA) stand out, as their active sites are decorated by up to three long mobile loops, that move independently of each other, and the conformations of which are critically important for catalysis. These motions appear to be ligand-gated and occur on sufficiently slow timescales (ms or slower) to be at least partially rate-limiting. In recent work, we combined conventional and enhanced molecular dynamics simulations with empirical valence bond simulations to investigate the conformational behavior of the catalytic loops in these enzymes, and the link between their dynamical behavior and substrate selectivity and catalysis. We have explored the role of loop dynamics in facilitating the bifunctionality of PriA towards substrates ProFAR and PRA (precursors for histidine and tryptophan biosynthesis, respectively), as well as during the real-time evolution of HisA to yield HisA-specific, TrpF-specific and bifunctional variants. Finally, the insights this provides emphasizes the potential of loop engineering as a powerful tool to manipulate the selectivity and substrate scope of the TIM-barrel scaffold, which has historically been one of the most important scaffolds for de novo enzyme design.

The Role of Conformational Dynamics and Allostery in the Control of Distinct Efficacies of Agonists to the Glucocorticoid Receptor.

Glucocorticoid receptor (GR) regulates various cellular functions. Given its broad influence on metabolic activities, it has been the target of drug discovery for decades. However, how drugs induce conformational changes in GR has remained elusive. Herein, we used five GR agonists (dex, AZ938, pred, cor, and dibC) with different efficacies to investigate which aspect of the ligand induced the differences in efficacy. We performed molecular dynamics simulations on the five systems (dex-, AZ938-, pred-, cor-, and dibC-bound systems) and observed a distinct discrepancy in the conformation of the cofactor TIF2. Moreover, we discovered ligand-induced differences regarding the level of conformational changes posed by the binding of cofactor TIF2 and identified a pair of essential residues D590 and T39. We further found a positive correlation between the efficacies of ligands and the interaction of the two binding pockets’ domains, where D590 and T739 were involved, implying their significance in the participation of allosteric communication. Using community network analysis, two essential communities containing D590 and T739 were identified with their connectivity correlating to the efficacy of ligands. The potential communication pathways between these two residues were revealed. These results revealed the underlying mechanism of allosteric communication between the ligand-binding and cofactor-binding pockets and identified a pair of important residues in the allosteric communication pathway, which can serve as a guide for future drug discovery.

Structural insights into choline-O-sulfatase reveal the molecular determinants for ligand binding.

Choline-O-sulfatase (COSe; EC 3.1.6.6) is a member of the alkaline phosphatase (AP) superfamily, and its natural function is to hydrolyze choline-O-sulfate into choline and sulfate. Despite its natural function, the major interest in this enzyme resides in the landmark catalytic/substrate promiscuity of sulfatases, which has led to attention in the biotechnological field due to their potential in protein engineering. In this work, an in-depth structural analysis of wild-type Sinorhizobium (Ensifer) meliloti COSe (SmeCOSe) and its C54S active-site mutant is reported. The binding mode of this AP superfamily member to both products of the reaction (sulfate and choline) and to a substrate-like compound are shown for the first time. The structures further confirm the importance of the C-terminal extension of the enzyme in becoming part of the active site and participating in enzyme activity through dynamic intra-subunit and inter-subunit hydrogen bonds (Asn146A-Asp500B-Asn498B). These residues act as the `gatekeeper' responsible for the open/closed conformations of the enzyme, in addition to assisting in ligand binding through the rearrangement of Leu499 (with a movement of approximately 5 Å). Trp129 and His145 clamp the quaternary ammonium moiety of choline and also connect the catalytic cleft to the C-terminus of an adjacent protomer. The structural information reported here contrasts with the proposed role of conformational dynamics in promoting the enzymatic catalytic proficiency of an enzyme.

The role of conformational dynamics in the activity of polymer-conjugated CalB in organic solvents

Perennial interest in enzyme catalysis has been expanding its applicability from aqueous phases where enzymes are naturally evolved to organic solvents in which the majority of industrial chemical syntheses are carried out. Although conjugating an enzyme with a soluble polymer has been attempted to enhance enzyme activity in organic solvents, the underlying mechanism remains poorly understood in terms of the conformational dynamics and enzyme activity. Herein, we combine LF-NMR measurements and MD simulations to investigate the effects of polymer grafting on the conformational dynamics of CalB in organic solvents and the consequential impacts on the catalytic kinetics, using the lipase-catalyzed transesterification reaction as a model system. LF-NMR measurements confirm that conjugation with a soluble polymer improves the enzyme flexibility in organic solvents, leading to an increase in the catalytic efficiency of up to two orders of magnitude. MD simulations suggest that the conjugated enzyme samples a larger conformational space, compared to its native counterpart, validating the hypothesis that polymer motion enhances enzyme dynamics. These experimental and simulation results provide new insights for enhancing enzyme conformational dynamics and thereby catalytic kinetics in organic solvents.

Stereochemistry, Conformational Dynamics, and the Stability of Liquid Crystal Phases

Although discotic liquid crystal dimers have been widely targeted as organic semiconductors and as LC-glass formers, the role of conformational dynamics on the self-assembly of these flexible mesogens remains poorly understood. In an effort to probe this effect, we investigated the impact of linker stereochemistry on the phase behavior of discotic liquid crystalline dimers. Diastereomeric dibenzo[a,c]phenazine diesters were prepared from (2R,3R)- and meso-2,3-butanediol. While both dimers form columnar phases, the meso-isomer had a clearing temperature (Tc) that was 31 °C higher than that of its chiral diastereomer. Conformational analysis via DFT calculations, 1H-NMR, and DOSY experiments indicated that both compounds adopt predominantly extended conformations but that the meso-dimer shows a stronger preference to unfold in solution. To probe how conformation alters phase stability, we prepared derivatives in which catechol and hydroquinone act as rigid linkers that lock the dimers in a folded or an extended conformation, respectively. The diester of hydroquinone possessed a Tc that was nearly 100 °C higher than the catechol derivative, consistent with a model where extended conformations stabilize the LC phase. Extended dimers also exhibited higher transition enthalpies at the Tc, an indication that their columnar phases are more ordered than folded structures.

Conserved Conformational Hierarchy across Functionally Divergent Glycosyltransferases of the GT-B Structural Superfamily as Determined from Microsecond Molecular Dynamics.

It has long been understood that some proteins undergo conformational transitions en route to the Michaelis Complex to allow chemistry. Examination of crystal structures of glycosyltransferase enzymes in the GT-B structural class reveals that the presence of ligand in the active site triggers an open-to-closed conformation transition, necessary for their catalytic functions. Herein, we describe microsecond molecular dynamics simulations of two distantly related glycosyltransferases that are part of the GT-B structural superfamily, HepI and GtfA. Simulations were performed using the open and closed conformations of these unbound proteins, respectively, and we sought to identify the major dynamical modes and communication networks that interconnect the open and closed structures. We provide the first reported evidence within the scope of our simulation parameters that the interconversion between open and closed conformations is a hierarchical multistep process which can be a conserved feature of enzymes of the same structural superfamily. Each of these motions involves of a collection of smaller molecular reorientations distributed across both domains, highlighting the complexities of protein dynamic involved in the interconversion process. Additionally, dynamic cross-correlation analysis was employed to explore the potential effect of distal residues on the catalytic efficiency of HepI. Multiple distal nonionizable residues of the C-terminal domain exhibit motions anticorrelated to positively charged residues in the active site in the N-terminal domain involved in substrate binding. Mutations of these residues resulted in a reduction in negatively correlated motions and an altered enzymatic efficiency that is dominated by lower Km values with kcat effectively unchanged. The findings suggest that residues with opposing conformational motions involved in the opening and closing of the bidomain HepI protein can allosterically alter the population and conformation of the “closed” state, essential to the formation of the Michaelis complex. The stabilization effects of these mutations likely equally influence the energetics of both the ground state and the transition state of the catalytic reaction, leading to the unaltered kcat. Our study provides new insights into the role of conformational dynamics in glycosyltransferase’s function and new modality to modulate enzymatic efficiency.

The Role of Conformational Dynamics in the Recognition and Regulation of Ubiquitination.

The ubiquitination pathway is central to many cell signaling and regulatory events. One of the intriguing aspects of the pathway is the combinatorial sophistication of substrate recognition and ubiquitin chain building determinations. The abundant structural and biological data portray several characteristic protein folds among E2 and E3 proteins, and the understanding of the combinatorial complexity that enables interaction with much of the human proteome is a major goal to developing targeted and selective manipulation of the pathway. With the commonality of some folds, there are likely other aspects that can provide differentiation and recognition. These aspects involve allosteric effects and conformational dynamics that can direct recognition and chain building processes. In this review, we will describe the current state of the knowledge for conformational dynamics across a wide timescale, address the limitations of present approaches, and illustrate the potential to make new advances in connecting dynamics with ubiquitination regulation.

The Role of Conformational Dynamics and Allostery in Modulating Protein Evolution.

Advances in sequencing techniques and statistical methods have made it possible not only to predict sequences of ancestral proteins but also to identify thousands of mutations in the human exome, some of which are disease associated. These developments have motivated numerous theories and raised many questions regarding the fundamental principles behind protein evolution, which have been traditionally investigated horizontally using the tip of the phylogenetic tree through comparative studies of extant proteins within a family. In this article, we review a vertical comparison of the modern and resurrected ancestral proteins. We focus mainly on the dynamical properties responsible for a protein's ability to adapt new functions in response to environmental changes. Using the Dynamic Flexibility Index and the Dynamic Coupling Index to quantify the relative flexibility and dynamic coupling at a site-specific, single-amino-acid level, we provide evidence that the migration of hinges, which are often functionally critical rigid sites, is a mechanism through which proteins can rapidly evolve. Additionally, we show that disease-associated mutations in proteins often result in flexibility changes even at positions distal from mutational sites, particularly in the modulation of active site dynamics.

Role of Conformational Dynamics in NMDA Receptor Negative Cooperativity

Role of Conformational Dynamics in NMDA Receptor Negative Cooperativity

The Role of Conformational Dynamics in Abacavir-Induced Hypersensitivity Syndrome

Abacavir is an antiretroviral drug used to reduce human immunodeficiency virus (HIV) replication and decrease the risk of developing acquired immune deficiency syndrome (AIDS). However, its therapeutic value is diminished by the fact that it is associated with drug hypersensitivity reactions in up to 8% of treated patients. This hypersensitivity is strongly associated with patients carrying human leukocyte antigen (HLA)-B*57:01, but not patients carrying closely related alleles. Abacavir’s specificity to HLA-B*57:01 is attributed to its binding site within the peptide-binding cleft and subsequent influence of the repertoire of peptides that can bind HLA-B*57:01. To further our understanding of abacavir-induced hypersensitivity we used molecular dynamics (MD) to analyze the dynamics of three different peptides bound to HLA-B*57:01 in the presence and absence of abacavir or abacavir analogues. We found that abacavir and associated peptides bind to HLA-B*57:01 in a highly diverse range of conformations that are not apparent from static crystallographic snapshots, but observed no difference in either the conformations, nor degree of flexibility when compared to abacavir-unbound systems. Our results support hypersensitivity models in which abacavir-binding alters the conformational ensemble of neopeptides, so as to favour exposed peptide surfaces that are no longer recognized as self by circulating CD8+ T cells, and are conducive to TCR binding. Our findings highlight the need to also consider the role of dynamics in understanding drug-induced hypersensitivities at the molecular and mechanistic level. This additional insight can help inform the chemical modification of abacavir to prevent hypersensitivity reactions in HLA-B*57:01+ HIV patients whilst retaining potent antiretroviral activity.

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.

The Role of Conformational Dynamics and Allostery in the Disease Development of Human Ferritin

Determining the three-dimensional structure of myoglobin, the first solved structure of a protein, fundamentally changed the way protein function was understood. Even more revolutionary was the information that came afterward: protein dynamics play a critical role in biological functions. Therefore, understanding conformational dynamics is crucial to obtaining a more complete picture of protein evolution. We recently analyzed the evolution of different protein families including green fluorescent proteins (GFPs), β-lactamase inhibitors, and nuclear receptors, and we observed that the alteration of conformational dynamics through allosteric regulation leads to functional changes. Moreover, proteome-wide conformational dynamics analysis of more than 100 human proteins showed that mutations occurring at rigid residue positions are more susceptible to disease than flexible residue positions. These studies suggest that disease-associated mutations may impair dynamic allosteric regulations, leading to loss of function. Thus, in this study, we analyzed the conformational dynamics of the wild-type light chain subunit of human ferritin protein along with the neutral and disease forms. We first performed replica exchange molecular dynamics simulations of wild-type and mutants to obtain equilibrated dynamics and then used perturbation response scanning (PRS), where we introduced a random Brownian kick to a position and computed the fluctuation response of the chain using linear response theory. Using this approach, we computed the dynamic flexibility index (DFI) for each position in the chain for the wild-type and the mutants. DFI quantifies the resilience of a position to a perturbation and provides a flexibility/rigidity measurement for a given position in the chain. The DFI analysis reveals that neutral variants and the wild-type exhibit similar flexibility profiles in which experimentally determined functionally critical sites act as hinges in controlling the overall motion. However, disease mutations alter the conformational dynamic profile, making hinges more loose (i.e., softening the hinges), thus impairing the allosterically regulated dynamics.

Regulation of symmetric bacterial cell division by MinE: What is the role of conformational dynamics?

Symmetric cell division in Gram-negative bacteria requires the concerted action of three Min proteins that together ensure exclusive formation of the cell division septum at the mid-point of the cell. We have recently described the structure and dynamic properties of MinE, the protein responsible for directing the cell division inhibitor complex formed by the MinC and MinD proteins away from the middle of the cell. An unexpected feature of this structure was the location of MinD-binding residues at buried, non-accessible sites in the dimeric interface. Here we elaborate on the potential role of conformational changes that might be involved to allow access to these residues, along with the interesting questions raised by these features of the MinE structure.

Conformational propensities and dynamics of a βγ-crystallin, an intrinsically disordered protein

The three-dimensional folded structure of a protein has been considered essential for its function. However, recently many proteins have been identified to function without having a definite structure and they have been classified as intrinsically disordered proteins (IDPs). Recently, we have identified a βγ-crystallin domain in the genome of a marine bacterium called Hahella chejuensis on the basis of known sequence signatures. This protein, called Hahellin, was characterized by NMR spectroscopy as an IDP, which upon Ca(2+)-binding was shown to undergo a large conformational transformation and acquires a typical βγ-crystallin fold. In this paper, we have characterized this IDP by a combined use of NMR and Replica Exchange Molecular Dynamics simulation and found it to be in a highly dynamic, inter-converting population having a molten globular state with the C-terminal Greek key motif relatively more flexible as compared to its N-terminal counterpart. Network analysis and clustering on the observed conformational ensemble showed a heterogeneous mixture of eleven distinct clusters, classified into near-native and far-native populations, which are not in equilibrium. Several conformational clusters display an increased propensity for helical conformation(s) and a decreased β-strand propensity, which is consistent with the NMR observations made on this protein. The negatively charged Ca(2+)-coordinating residues form parts of the highly flexible polypeptide stretches, and thus act as seeds for the origin of different conformational clusters observed. This study thus helps us to understand the relationship between the role of conformational dynamics and the structural propensities of the intrinsically disordered state of apo-Hahellin.

Role of Conformational Dynamics in α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) Receptor Partial Agonism

We have investigated the range of cleft closure conformational states that the agonist-binding domains of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors occupy when bound to a series of willardiine derivatives using single-molecule FRET. These studies show that the agonist-binding domain exhibits varying degrees of dynamics when bound to the different willardiines with differing efficacies. The chlorowillardiine- and nitrowillardiine-bound form of the agonist-binding domain probes a narrower range of cleft closure states relative to the iodowillardiine bound form of the protein, with the antagonist (αS)-α-amino-3-[(4-carboxyphenyl)methyl]-3,4-dihydro-2,4-dioxo-1(2H)-pyrimidinepropanoic acid (UBP-282)-bound form exhibiting the widest range of cleft closure states. Additionally, the average cleft closure follows the order UBP-282 > iodowillardiine > chlorowillardiine > nitrowillardiine-bound forms of agonist-binding domain. These single-molecule FRET data, along with our previously reported data for the glutamate-bound forms of wild type and T686S mutant proteins, show that the mean currents under nondesensitizing conditions can be directly correlated to the fraction of the agonist-binding domains in the "closed" cleft conformation. These results indicate that channel opening in the AMPA receptors is controlled by both the ability of the agonist to induce cleft closure and the dynamics of the agonist-binding domain when bound to the agonist.

Twisted Schiff base intermediates and substrate locale revise transaldolase mechanism

We examined the catalytic cycle of transaldolase (TAL) from Thermoplasma acidophilum by cryocrystallography and were able to structurally characterize--for the first time, to our knowledge--different genuine TAL reaction intermediates. These include the Schiff base adducts formed between the catalytic lysine and the donor ketose substrates fructose-6-phosphate and sedoheptulose-7-phosphate as well as the Michaelis complex with acceptor aldose erythrose-4-phosphate. These structural snapshots necessitate a revision of the accepted reaction mechanism with respect to functional roles of active site residues, and they further reveal fundamental insights into the general structural features of enzymatic Schiff base intermediates and the role of conformational dynamics in enzyme catalysis, substrate binding and discrimination. A nonplanar arrangement of the substituents around the Schiff base double bond was observed, suggesting that a structurally encoded reactant-state destabilization is a driving force of catalysis. Protein dynamics and the intrinsic hydrogen-bonding pattern appear to be crucial for selective recognition and binding of ketose as first substrate.

Enzyme dynamics point to stepwise conformational selection in catalysis.

Recent data increasingly reveal that conformational dynamics are indispensable to enzyme function throughout the catalytic cycle, in substrate recruiting, chemical transformation, and product release. Conformational transitions may involve conformational selection and induced fit, which can be viewed as a special case in the catalytic network. NMR, X-ray crystallography, single-molecule FRET, and simulations clearly demonstrate that the free enzyme dynamics already encompass all the conformations necessary for substrate binding, preorganization, transition-state stabilization, and product release. Conformational selection and substate population shift at each step of the catalytic turnover can accommodate enzyme specificity and efficiency. Within such a framework, entropy can have a larger role in conformational dynamics than in direct energy transfer in dynamically promoted catalysis.