Binding of Two Intrinsically Disordered Peptides to a Multi-Specific Protein: A Combined Monte Carlo and Molecular Dynamics Study

The unique ability of intrinsically disordered proteins (IDPs) to fold upon binding to partner molecules makes them functionally well-suited for cellular communication networks. For example, the folding-binding of different IDP sequences onto the same surface of an ordered protein provides a mechanism for signaling in a many-to-one manner. Here, we study the molecular details of this signaling mechanism by applying both Molecular Dynamics and Monte Carlo methods to S100B, a calcium-modulated homodimeric protein, and two of its IDP targets, p53 and TRTK-12. Despite adopting somewhat different conformations in complex with S100B and showing no apparent sequence similarity, the two IDP targets associate in virtually the same manner. As free chains, both target sequences remain flexible and sample their respective bound, natively -helical states to a small extent. Association occurs through an intermediate state in the periphery of the S100B binding pocket, stabilized by nonnative interactions which are either hydrophobic or electrostatic in nature. Our results highlight the importance of overall physical properties of IDP segments, such as net charge or presence of strongly hydrophobic amino acids, for molecular recognition via coupled folding-binding.     

Molecular Determinants of Epidermal Growth Factor Binding: A Molecular Dynamics Study

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF ligands or the receptor itself.     

GABA Binding to an Insect GABA Receptor: A Molecular Dynamics and Mutagenesis Study

RDL receptors are GABA-activated inhibitory Cys-loop receptors found throughout the insect CNS. They are a key target for insecticides. Here, we characterize the GABA binding site in RDL receptors using computational and electrophysiological techniques. A homology model of the extracellular domain of RDL was generated and GABA docked into the binding site. Molecular dynamics simulations predicted critical GABA binding interactions with aromatic residues F206, Y254, and Y109 and hydrophilic residues E204, S176, R111, R166, S176, and T251. These residues were mutated, expressed in Xenopus oocytes, and their functions assessed using electrophysiology. The data support the binding mechanism provided by the simulations, which predict that GABA forms many interactions with binding site residues, the most significant of which are cation-π interactions with F206 and Y254, H-bonds with E204, S205, R111, S176, T251, and ionic interactions with R111 and E204. These findings clarify the roles of a range of residues in binding GABA in the RDL receptor, and also show that molecular dynamics simulations are a useful tool to identify specific interactions in Cys-loop receptors.Abbreviations used: nACh, nicotinic acetylcholine; AChBP, acetylcholine binding protein; GABA, gamma-aminobutyric acid; MD, molecular dynamics; RDL, resistant to dieldrin; RMSD, root mean-square displacement; RMSF, root mean-square fluctuation     

The Possible Structural Models for Polyglutamine Aggregation: A Molecular Dynamics Simulations Study

Abstract

Huntington's disease is a neurodegenerative disorder caused by a polyglutamine (polyQ) expansion near the N-terminus of huntingtin. Previous studies have suggested that polyQ aggregation occurs only when the number of glutamine (Q) residues is more than 36-40, the disease threshold. However, the structural characteristics of polyQ nucleation in the very early stage of aggregation still remain elusive. In this study, we designed 18 simulation trials to determine the possible structural models for polyQ nucleation and aggregation with various shapes and sizes of initial β-helical structures, such as left-handed circular, right-handed rectangular, and left- and right-handed triangular. Our results show that the stability of these models significantly increases with increasing the number of rungs, while it is rather insensitive to the number of Qs in each rung. In particular, the 3-rung β-helical models are stable when they adopt the left-handed triangular and right-handed rectangular conformations due to the fact that they preserve high β-turn and β-sheet contents, respectively, during the simulation courses. Thus, we suggested that these two stable β-helical structures with at least 3 rungs might serve as the possible nucleation seeds for polyQ depending on how the structural elements of β-turn and β-sheet are sampled and preserved during the very early stage of aggregation.     

Engineering Strontium Binding Affinity in an EF-hand Motif: A Quantum Chemical and Molecular Dynamics Study

Abstract

Proteins with the ability to specifically bind strontium would potentially be of great use in the field of nuclear waste management. Unfortunately, no such peptides or proteins are known—indeed, it is uncertain whether they exist under natural conditions due to low environmental concentrations of strontium. To investigate the possibility of devising such molecules, one of us (CV), in a previous experimental study [J. Biol. Inorg. Chem. 8, 33440 (2003)], proposed starting from an EF-hand motif of the protein calmodulin and mutating some residues to change the motif's specificity for calcium into one for strontium. In this paper, which represents a theoretical complement to the experimental work, we analyzed small-molecule crystallographic structures and performed quantum chemical calculations to identify possible mutations. We then constructed seven mutant sequences of the EF-hand motif and analyzed their dynamical and binding behaviors using molecular dynamics simulations and free-energy calculations (using the MM/PBSA method). As a result of these analyzes we were able to isolate some characteristics that could lead to mutant peptides with enhanced strontium affinity.     

Binding and Channeling of Alternative Substrates in the Enzyme DmpFG: a Molecular Dynamics Study

DmpFG is a bifunctional enzyme comprised of an aldolase subunit, DmpG, and a dehydrogenase subunit, DmpF. The aldehyde intermediate produced by the aldolase is channeled directly through a buried molecular channel in the protein structure from the aldolase to the dehydrogenase active site. In this study, we have investigated the binding of a series of progressively larger substrates to the aldolase, DmpG, using molecular dynamics. All substrates investigated are easily accommodated within the active site, binding with free energy values comparable to the physiological substrate 4-hydroxy-2-ketovalerate. Subsequently, umbrella sampling was utilized to obtain free energy surfaces for the aldehyde intermediates (which would be generated from the aldolase reaction on each of these substrates) to move through the channel to the dehydrogenase DmpF. Small substrates were channeled with limited barriers in an energetically feasible process. We show that the barriers preventing bulky intermediates such as benzaldehyde from moving through the wild-type protein can be removed by selective mutation of channel-lining residues, demonstrating the potential for tailoring this enzyme to allow its use for the synthesis of specific chemical products. Furthermore, positions of transient escape routes in this flexible channel were determined.     

Structural Dynamics of the Cereblon Ligand Binding Domain

Cereblon, a primary target of thalidomide and its derivatives, has been characterized structurally from both bacteria and animals. Especially well studied is the thalidomide binding domain, CULT, which shows an invariable structure across different organisms and in complex with different ligands. Here, based on a series of crystal structures of a bacterial representative, we reveal the conformational flexibility and structural dynamics of this domain. In particular, we follow the unfolding of large fractions of the domain upon release of thalidomide in the crystalline state. Our results imply that a third of the domain, including the thalidomide binding pocket, only folds upon ligand binding. We further characterize the structural effect of the C-terminal truncation resulting from the mental-retardation linked R419X nonsense mutation in vitro and offer a mechanistic hypothesis for its irresponsiveness to thalidomide. At 1.2Å resolution, our data provide a view of thalidomide binding at atomic resolution.     

Long-range Electrostatic Interactions in Molecular Dynamics: An Endothelin-1 Case Study

Abstract

An extensive conformational search in explicit solvent was performed in order to compare the influence of different long-range electrostatic interaction treatments in molecular dynamics. The short peptide endothelin-1 was selected as the subject of molecular dynamics studies that started from both X-ray and NMR obtained structures. Electrostatic interactions were treated using two of the most common methods—residue-based cutoff and particle mesh Ewald (PME). Analyses of free energy calculations (MM-PBSA method used), secondary structure elements and hydrogen bonds were performed, and there suggested that there is no unambiguous conclusion about which of the two methods of long-range electrostatics treatment should be used in MD simulations in this case. The most reliable data was provided by a trajectory that started with the NMR structure and used the cutoff method to treat electrostatic interactions. This leads to a recommendation that the choice of electrostatics treatment should be made carefully and not automatically by choosing the PME method simply because it is the most widely used.     

Integrating Structural Information to Study the Dynamics of Protein-Protein Interactions in Cells

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神经元突触前可塑性的结构及分子基础

突触可塑性是神经元间信息传递的重要生理调控机制,它包括突触前可塑性和突触后可塑性.突触前可塑性是指通过对神经递质释放过程的干预、修饰,调节突触强度的过程.突触强度的变化,是通过影响量子的大小,活动区的个数和囊泡释放概率来实现的.而突触前囊泡活动尤为重要：从转运、搭靠、融合至内吞进入下一轮循环,每一步都是由一群互相作用的蛋白质共同完成的.     

Interplay of the Bacterial Ribosomal A-Site,S12 Protein Mutations and Paromomycin Binding: A Molecular Dynamics Study

The conformational properties of the aminoacyl-tRNA binding site (A-site), and its surroundings in the Escherichia coli 30S ribosomal subunit, are of great relevance in designing antibacterial agents. The 30S subunit A-site is near ribosomal protein S12, which neighbors helices h27 and H69; this latter helix, of the 50S subunit, is a functionally important component of an intersubunit bridge. Experimental work has shown that specific point mutations in S12 (K42A, R53A) yield hyper-accurate ribosomes, which in turn confers resistance to the antibiotic ‘paromomycin’ (even when this aminoglycoside is bound to the A-site). Suspecting that these effects can be elucidated in terms of the local atomic interactions and detailed dynamics of this region of the bacterial ribosome, we have used molecular dynamics simulations to explore the motion of a fragment of the E. coli ribosome, including the A-site. We found that the ribosomal regions surrounding the A-site modify the conformational space of the flexible A-site adenines 1492/93. Specifically, we found that A-site mobility is affected by stacking interactions between adenines A1493 and A1913, and by contacts between A1492 and a flexible side-chain (K43) from the S12 protein. In addition, our simulations reveal possible indirect pathways by which the R53A and K42A mutations in S12 are coupled to the dynamical properties of the A-site. Our work extends what is known about the atomistic dynamics of the A-site, and suggests possible links between the biological effects of hyper-accurate mutations in the S12 protein and conformational properties of the ribosome; the implications for S12 dynamics help elucidate how the miscoding effects of paromomycin may be evaded in antibiotic-resistant mutants of the bacterial ribosome.     

Docking Study of Ligands into the Colchicine Binding Site of Tubulin

Cancer is a major cause of mortality in developed countries, following only cardiovascular diseases. Death of cancerous cells can be achieved by stopping mitosis and the antimitotic class of drugs formed by the spindle poisons can be used for this purpose. Their role is to disorganize the mitotic spindle by targeting its main constituent, the microtubules, themselves made of heterodimers of α and β-tubulin. They disrupt the dynamics of the microtubules either by stabilizing them, as do paclitaxel or epothilones, or destabilizing them, as do colchicine. The binding site of colchicine seems to lie between the two units of the tubulin dimer. Here, we report on the characterization of this site by the docking of a series of reference compounds, and the subsequent docking of ligands prepared in our laboratory.     

Structural Basis for the Binding Affinity of a Homologous Series of Synthetic Phenoxazone Drugs with DNA: NMR and Molecular Mechanics Analysis

Abstract

The molecular basis of the marked structure-activity relationship for a homologous series of DNA-binding phenoxazone drugs (ActII-ActIV) has been investigated by NMR spectroscopy and molecular mechanics. The spatial structures of the complexes between the drugs and a model deoxytetranucleotide, 5′-d(TpGpCpA), have been determined by molecular mechanics methods using homonuclear ¹H-¹H 2D-NOESY and heteronuclear ¹H-³¹P (HMBC) NMR spectroscopic data. Observed intermolecular NOE contacts and equilibrium binding studies confirm that the binding affinity of the synthetic phenoxazone derivatives with d(TGCA) decreases with an increase in the number of CH₂ groups in the dimethylami- noalkyl side chains, i.e., ActII > ActIII > ActIV, in agreement with the observed biological activity of these compounds. Molecular mechanics calculations of the spatial structures of the intercalated complexes of ActII-ActIV with d(TGCA) indicate that the different binding constants of the phenoxazone derivatives with the DNA oligomer are due to the different degrees of intercalation of the chromophore and the different steric arrangements of aminoalkyl side chains in the minor groove of the tetramer duplex; this results in different distances between the negatively-charged phosphates of the DNA duplex and the terminal positively-charged N(CH₃)₂ groups of the side chains.     

Dimensionality of Carbon Nanomaterials Determines the Binding and Dynamics of Amyloidogenic Peptides: Multiscale Theoretical Simulations

Experimental studies have demonstrated that nanoparticles can affect the rate of protein self-assembly, possibly interfering with the development of protein misfolding diseases such as Alzheimer''s, Parkinson''s and prion disease caused by aggregation and fibril formation of amyloid-prone proteins. We employ classical molecular dynamics simulations and large-scale density functional theory calculations to investigate the effects of nanomaterials on the structure, dynamics and binding of an amyloidogenic peptide apoC-II(60-70). We show that the binding affinity of this peptide to carbonaceous nanomaterials such as C60, nanotubes and graphene decreases with increasing nanoparticle curvature. Strong binding is facilitated by the large contact area available for π-stacking between the aromatic residues of the peptide and the extended surfaces of graphene and the nanotube. The highly curved fullerene surface exhibits reduced efficiency for π-stacking but promotes increased peptide dynamics. We postulate that the increase in conformational dynamics of the amyloid peptide can be unfavorable for the formation of fibril competent structures. In contrast, extended fibril forming peptide conformations are promoted by the nanotube and graphene surfaces which can provide a template for fibril-growth.     

Structural Consequences of D481N/K483Q Mutation at Glycine Binding Site of NMDA Ionotropic Glutamate Receptors: A Molecular Dynamics Study

Abstract

N-Methyl-D-Aspartate (NMDA) receptors are the ligand gated as well as voltage sensitive ionotropic glutamate receptors, widely distributed in the vertebrate central nervous system and they play critical role in the pathogenesis of schizophrenia. Molecular dynamics simulations have been carried out on high resolution crystal structure of NR1 subunit of NMDA receptor ligand binding core (S1S2) in four different conformations. We have investigated consequence of D481N/K483Q double mutation of NR1 subunit from simulation results of (a) glycine bound form (WG), (b) unbound (closed-apo) form (WOG), (c) a double mutated form (DM), and (d) the antagonist (5,7-dichlorokynuric acid) bound form (DCKA). The MD simulations and simulated annealing for 4ns show a distinct conformation for the double mutated conformation that neither follows the antagonist nor apo conformation. There are two distinct sites, loop1 and loop2 where the double mutated structure in its glycine bound form shows significant RMSD deviations as compared to the wild-type. The interactions of glycine with the receptor remain theoretically unchanged in the double mutated structure and there is no detachment of S1S2 domains. The results suggest that separation of S1 and S2 domains may not be essential for channel inactivation. Therefore, it is hypothesized that hypoactivation of NMDA receptor channels may arise out of the conformational changes at non-conserved Loop1 and Loop2 regions observed in the mutated structure. The Loop1 and Loop2 regions responsible for inter-subunit interactions in a functional NMDA receptor, may therefore, render the ligand bound form defunct. This may account for behavioral anomalies due to receptor inactivation seen in grin1 mutated mice.     

Correlation Spectroscopy and Molecular Dynamics Simulations to Study the Structural Features of Proteins

In this work, we used a combination of fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulation methodologies to acquire structural information on pH-induced unfolding of the maltotriose-binding protein from Thermus thermophilus (MalE2). FCS has emerged as a powerful technique for characterizing the dynamics of molecules and it is, in fact, used to study molecular diffusion on timescale of microsecond and longer. Our results showed that keeping temperature constant, the protein diffusion coefficient decreased from 84±4 µm²/s to 44±3 µm²/s when pH was changed from 7.0 to 4.0. An even more marked decrease of the MalE2 diffusion coefficient (31±3 µm²/s) was registered when pH was raised from 7.0 to 10.0. According to the size of MalE2 (a monomeric protein with a molecular weight of 43 kDa) as well as of its globular native shape, the values of 44 µm²/s and 31 µm²/s could be ascribed to deformations of the protein structure, which enhances its propensity to form aggregates at extreme pH values. The obtained fluorescence correlation data, corroborated by circular dichroism, fluorescence emission and light-scattering experiments, are discussed together with the MD simulations results.     

Structure and Dynamics of Ligand-Template Interactions of Topoisomerase Inhibitory Analogs of Hoechst 33258: High Field 1H-NMR and Restrained Molecular Mechanics Studies

Abstract

The binding characteristics of Hoechst 33258 (1), a synthetic bis-benzimidazole, and its structural analog 2, with one of the benzimidazoles replaced by a pyridoimidazole, to the self-complementary decadeoxyribonucleotide sequences d(CGCAATTGCG)² (A) and d-(CATGGCCATG)₂ (B) respectively, were examined using high field ¹H-NMR techniques. Selective complexation induced chemical shift changes, the presence of exchange signals and intermolecular NOE contacts between the ligands and the minor groove protons of the oligonucleotides suggest the preferred binding sites as the centrally located AATT segment for complex Al, and the CCAT segment for complex B2. The B-type conformations of the two DNA duplexes are preserved upon complexation, as confirmed by the 2D-NOESY based sequential connectivities involving DNA base and sugar protons. Close intermolecular NOE based contacts between the ligands and their respective DNA sequences were further refined to model the ligand-DNA complexes starting from the computer generated B-type structures for the oligonucleotides. Force field calculations of ligand-DNA interaction energies indicate a more favorable contribution from the van der Waals energy component in the case of complex Al consistent with its stronger net binding compared with the complex B2.

Overall, the incorporation of a pyridinic nitrogen in Hoechst 33258 structure alters its selectivity for base pair recognition from AT to G.C, resulting largely from the formation of a hydrogen bond between the new basic center and the 2-NH₂ group of a guanosine moiety. The rates for the exchange of ligands between the two equivalent binding sites (AATT for 1, and CCAT for 2) of the self-complementary DNA sequences, are estimated from analyses of coalescence of NMR signals to be 189 _s ^?1 at 301K for A1, and 79_s ^?1 at 297 K for B2; which correspond to ΔG? of 13.8 and 18.6 kcal.mol^?1 respectively.     

Study of the Interactions between Neurophysin II and Dipeptide Ligand by Means of Molecular Dynamics

The nonapeptide hormones oxytocin (OT) and vasopressin (VP), while transported in the posterior pituitary, are packaged into neurosecretory granules (NSG) in the form of high associates with disulfide-rich proteins known as neurophysin I (NPI) and neurophysin II (NPII), respectively. In the NSG, neurophysins serve as carrier proteins to the hormones, until the latter are dissociated upon secretion into blood. To shed more light on molecular self-recognition between NPs, and between NPs and their ligands, we have studied their molecular association, using as a starting point the recently published solid-state structure (C^α-trace) of the neurophysin II-dipeptide complex. Another purpose of this work was the development of reliable strategies for molecular modeling, that would utilize minimal structural information (like C^α-trace and/or structural homology) yet be useful for studies of protein/ligand interactions. An initial all-atom representation of the protein-peptide complex (2:2) was obtained by the conversion of the C^α-carbon trace deposited in the Brookhaven Protein Data Bank (file 1BN2), using the InsightII/Biopolymer modules from the suite of programs supplied by Biosym Technologies, San Diego. The free NPII homodimer was obtained by removal of the dipeptide ligands from the starting structures. Both associates, after initial immersion in water, were submitted to gradual (side chains first then all atoms) minimization of energy. Subsequently, they were thermally equilibrated and submitted to the molecular dynamics (AMBER 4.0) at 300K, until the total energy was stabilized. The structures, averaged over the last 20 ps of the dynamics, were compared with the starting C^α-trace and among themselves. The protein/ligand complex, simulated in water, compares favorably with the solid-state reference. An allosteric mechanism for the NPII dimer/ligand interaction is proposed and discussed.