Chia-en Chang - Academia.edu (original) (raw)

Papers by Chia-en Chang

BMC Biophysics, 2011

Background: Phosphopeptide-binding domains mediate many vital cellular processes such as signal t... more Background: Phosphopeptide-binding domains mediate many vital cellular processes such as signal transduction and protein recognition. We studied three well-known domains important for signal transduction: BRCT repeats, WW domain and forkhead-associated (FHA) domain. The first two recognize both phosphothreonine (pThr) and phosphoserine (pSer) residues, but FHA has high specificity for pThr residues. Here we used molecular dynamics (MD) simulations to reveal how FHA exclusively chooses pThr and how BRCT and WW recognize both pThr/pSer. The work also investigated the energies and thermodynamic information of intermolecular interactions. Results: Simulations carried out included wide-type and mutated systems. Through analysis of MD simulations, we found that the conserved His residue defines dual loops feature of the FHA domain, which creates a small cavity reserved for only the methyl group of pThr. These well-organized loop interactions directly response to the pThr binding selectivity, while single loop (the 2nd phosphobinding site of FHA) or in combination with α-helix (BRCT repeats) or β-sheet (WW domain) fail to differentiate pThr/pSer. Conclusions: Understanding the domain pre-organizations constructed by conserved residues and the driving force of domain-phosphopeptide recognition provides structural insight into pThr specific binding, which also helps in engineering proteins and designing peptide inhibitors.

Protein Science, 2015

The importance of protonation states and proton transfer in pyridoxal 5&a... more The importance of protonation states and proton transfer in pyridoxal 5'-phosphate (PLP)-chemistry can hardly be overstated. Although experimental approaches to investigate pKa values can provide general guidance for assigning proton locations, only static pictures of the chemical species are available. To obtain the overall protein dynamics for the interpretation of detailed enzyme catalysis in this study, guided by information from solid-state NMR, we performed molecular dynamics (MD) simulations for the PLP-dependent enzyme tryptophan synthase (TRPS), whose catalytic mechanism features multiple quasi-stable intermediates. The primary objective of this work is to elucidate how the position of a single proton on the reacting substrate affects local and global protein dynamics during the catalytic cycle. In general, proteins create a chemical environment and an ensemble of conformational motions to recognize different substrates with different protonations. The study of these interactions in TRPS shows that functional groups on the reacting substrate, such as the phosphoryl group, pyridine nitrogen, phenolic oxygen and carboxyl group, of each PLP-bound intermediate play a crucial role in constructing an appropriate molecular interface with TRPS. In particular, the protonation states of the ionizable groups on the PLP cofactor may enhance or weaken the attractions between the enzyme and substrate. In addition, remodulation of the charge distribution for the intermediates may help generate a suitable environment for chemical reactions. The results of our study enhance knowledge of protonation states for several PLP intermediates and help to elucidate their effects on protein dynamics in the function of TRPS and other PLP-dependent enzymes. This article is protected by copyright. All rights reserved.

In silico pharmacology, 2013

The thermodynamically favored complex between the nuclear vitamin D receptor (VDR) and 1α,25(OH)2... more The thermodynamically favored complex between the nuclear vitamin D receptor (VDR) and 1α,25(OH)2-vitamin D3 (1,25D3) triggers a shift in equilibrium to favor VDR binding to DNA, heterodimerization with the nuclear retinoid x receptor (RXR) and subsequent regulation of gene transcription. The key amino acids and structural requirements governing VDR binding to nuclear coactivators (NCoA) are well defined. Yet very little is understood about the internal changes in amino acid flexibility underpinning the control of ligand affinity, helix 12 conformation and function. Herein, we use molecular dynamics (MD) to study how the backbone and side-chain flexibility of the VDR differs when a) complexed to 1α,25(OH)2-vitamin D3 (1,25D3, agonist) and (23S),25-dehydro-1α(OH)-vitamin D3-26,23-lactone (MK, antagonist); b) residues that form hydrogen bonds with the C25-OH (H305 and H397) of 1,25D3 are mutated to phenylalanine; c) helix 12 conformation is changed and ligand is removed; and d) x-ray ...

Journal of molecular recognition : JMR, 2014

Revealing the processes of ligand-protein associations deepens our understanding of molecular rec... more Revealing the processes of ligand-protein associations deepens our understanding of molecular recognition and binding kinetics. Hydrogen bonds (H-bonds) play a crucial role in optimizing ligand-protein interactions and ligand specificity. In addition to the formation of stable H-bonds in the final bound state, the formation of transient H-bonds during binding processes contributes binding kinetics that define a ligand as a fast or slow binder, which also affects drug action. However, the effect of forming the transient H-bonds on the kinetic properties is little understood. Guided by results from coarse-grained Brownian dynamics simulations, we used classical molecular dynamics simulations in an implicit solvent model and accelerated molecular dynamics simulations in explicit waters to show that the position and distribution of the H-bond donor or acceptor of a drug result in switching intermolecular and intramolecular H-bond pairs during ligand recognition processes. We studied two...

Proceedings of the National Academy of Sciences of the United States of America, Jan 30, 2007

The restriction of a small molecule's motion on binding to a protein causes a loss of configu... more The restriction of a small molecule's motion on binding to a protein causes a loss of configurational entropy, and thus a penalty in binding affinity. Some energy models used in computer-aided ligand design neglect this entropic penalty, whereas others account for it based on an expected drop in the number of accessible rotamers upon binding. However, the validity of the physical assumptions underlying the various approaches is largely unexamined. The present study addresses this issue by using Mining Minima calculations to analyze the association of amprenavir with HIV protease. The computed loss in ligand configurational entropy is large, contributing approximately 25 kcal/mol (4.184 kJ/kcal) to DeltaG degrees. Most of this loss results from narrower energy wells in the bound state, rather than a drop in the number of accessible rotamers. Coupling among rotation/translation and internal degrees of freedom complicates the decomposition of the entropy change into additive terms....

The Journal of Physical Chemistry C, 2014

Colloidal nanomaterials with well-defined shapes have wide applications in many fields. However, ... more Colloidal nanomaterials with well-defined shapes have wide applications in many fields. However, the exact role of capping ligands, which often dictates the shape of products in colloidal syntheses, is often unclear. Here we use a classical molecular-mechanics force-field method, mining minima (M2), to compute the binding free energy of the ligands such as citrate, monocarboxylates, dicarboxylates, and tricarboxylates to both and facets of silver and to investigate the mechanisms of the anisotropic growth of silver nanoplates. The distribution of partial charges on a ligand, the geometry complementation in the complex, and the entropic penalty on binding played crucial roles in discriminating the two facets and determining a good or poor ligand. Our finding allows rational design of capping ligands that may perform as well as citrate in promoting the anisotropic growth of nanoplates; however, designing a compound that outperforms citrate is found to be challenging.

Methods in Molecular Biology, 2011

Cannabinoids represent a promising class of compounds for developing novel therapeutic agents. Si... more Cannabinoids represent a promising class of compounds for developing novel therapeutic agents. Since the isolation and identification of the major psychoactive component Δ(9)-THC in Cannabis sativa in the 1960s, numerous analogues of the classical plant cannabinoids have been synthesized and tested for their biological activity. These compounds primarily target the cannabinoid receptors 1 (CB1) and Cannabinoid receptors 2 (CB2). This chapter focuses on CB1. Despite the lack of crystal structures for CB1, protein-based homology modeling approaches and molecular docking methods can be used in the design and discovery of cannabinoid analogues. Efficient synthetic approaches for therapeutically interesting cannabinoid analogues have been developed to further facilitate the drug discovery process.

Journal of the American Chemical Society, Jan 17, 2014

The acid-base chemistry that drives catalysis in pyridoxal-5'-phosphate (PLP)-dependent enzym... more The acid-base chemistry that drives catalysis in pyridoxal-5'-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP's role as a coenzyme in this extensive class of enzymes. It was first proposed over 50 years ago that the initial step in the catalytic cycle is facilitated by a protonated Schiff base form of the holoenzyme in which the linking lysine ε-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first (15)N NMR chemical shift measurements of such a Schiff base linkage in the resting holoenzyme form, the internal aldimine state of tryptophan synthase. Double-resonance experiments confirm the assignment of the Schiff base nitrogen, and additional (13)C, (15)N, and (31)P chemical shift measurements of sites on the PLP coenzyme allow a detailed model of coenzyme protonation states to be established.

PLoS ONE, 2014

The regulation of a series of cellular events requires specific protein-protein interactions, whi... more The regulation of a series of cellular events requires specific protein-protein interactions, which are usually mediated by modular domains to precisely select a particular sequence from diverse partners. However, most signaling domains can bind to more than one peptide sequence. How do proteins create promiscuity from precision? Moreover, these complex interactions typically occur at the interface of a well-defined secondary structure, a helix and b sheet. However, the molecular recognition primarily controlled by loop architecture is not fully understood. To gain a deep understanding of binding selectivity and promiscuity by the conformation of loops, we chose the forkhead-associated (FHA) domain as our model system. The domain can bind to diverse peptides via various loops but only interact with sequences containing phosphothreonine (pThr). We applied molecular dynamics (MD) simulations for multiple free and bound FHA domains to study the changes in conformations and dynamics. Generally, FHA domains share a similar folding structure whereby the backbone holds the overall geometry and the variety of sidechain atoms of multiple loops creates a binding surface to target a specific partner. FHA domains determine the specificity of pThr by well-organized binding loops, which are rigid to define a phospho recognition site. The broad range of peptide recognition can be attributed to different arrangements of the loop interaction network. The moderate flexibility of the loop conformation can help access or exclude binding partners. Our work provides insights into molecular recognition in terms of binding specificity and promiscuity and helpful clues for further peptide design.

Nat Commun, 2014

Small RNAs (sRNAs) are loaded into ARGONAUTE (AGO) proteins to induce gene silencing. In plants, ... more Small RNAs (sRNAs) are loaded into ARGONAUTE (AGO) proteins to induce gene silencing. In plants, the 5 0 -terminal nucleotide is important for sRNA sorting into different AGOs. Here we show that microRNA (miRNA) duplex structure also contributes to miRNA sorting. Base pairing at the 15th nucleotide of a miRNA duplex is important for miRNA sorting in both Arabidopsis AGO1 and AGO2. AGO2 favours miRNA duplexes with no middle mismatches, whereas AGO1 tolerates, or prefers, duplexes with central mismatches. AGO structure modelling and mutational analyses reveal that the QF-V motif within the conserved PIWI domain contributes to recognition of base pairing at the 15th nucleotide of a duplex, while the DDDE catalytic core of AtAGO2 is important for recognition of the central nucleotides. Finally, we rescued the adaxialized phenotype of ago1-12, which is largely due to miR165 loss-of-function, by changing miR165 duplex structure which we predict redirects it to AGO2.

The Journal of Physical Chemistry B, 2012

Promiscuous proteins are commonly observed in biological systems, for example, in modular domains... more Promiscuous proteins are commonly observed in biological systems, for example, in modular domains that recognize phosphopeptides during signal transduction. This promiscuous recognition is of fundamental interest in chemistry and biology but is challenging when designing phosphopeptides in silico for cell biology studies. To investigate promiscuous recognition and binding processes of phosphopeptides and the modular domain, we selected a domain essential in breast cancerthe breast-cancer− associated protein 1 (BRCA1) C-terminal (BRCT) repeats as our model system. We performed molecular dynamics simulations and detailed analyses of the dihedral space to study protein fluctuation and conformational changes with phosphopeptide binding. We also studied the association processes of phosphorylated and unphosphorylated peptides using Brownian dynamics with a coarse-grained model. We found that the BRCT domain is preorganized for phosphopeptide binding but has a moderate arrangement of side chains to form complexes with various types of phosphopeptides. Phosphopeptide binding restricts the system motion in general, while the nonpolar phosphopeptide becomes more flexible in the bound state. Our analysis found that the BRCT domain utilizes different mechanisms, usually termed lock and key, induced-fit, and population-shift/conformational-selection models, to recognize peptides with different features. Brownian dynamics simulations revealed that the charged phosphate group may not always accelerate peptide association processes, but it helps the phosphopeptide orient into binding pockets accurately and stabilizes the complex. This work provides insights into molecular recognition in the promiscuous protein system. a The major binding residues, pSer and Phe+3, are in bold. Other residues with non-polar contributions are underlined.

The Journal of Physical Chemistry B, 2006

A method is presented for calculating the conformational free energy of a molecule in all degrees... more A method is presented for calculating the conformational free energy of a molecule in all degrees of freedom. The method uses the harmonic approximation with finite integration ranges, along with Mode Scanning, a fast correction for anharmonicity based upon internal bond-angle-torsion coordinates. Mode Scanning accounts for local anharmonicity without the need for expensive Monte Carlo integration. The method is efficient, and comparisons with analytic or highly detailed numerical calculations show excellent accuracy. Similar comparisons for the previously described Mode Integration method show that, although it is computationally demanding, it can be less accurate than the pure harmonic approximation. The inaccuracy of Mode Integration is traceable to its use of a Cartesian coordinate basis set; much more accurate results are obtained when the basis set consists of bond-angle-torsion coordinates.

The Analyst, 2014

Protein adsorption on nanoparticles is closely associated with the physicochemical properties of ... more Protein adsorption on nanoparticles is closely associated with the physicochemical properties of particles, in particular, their surface properties. We synthesized two batches of polyacrylic acid-coated nanoparticles under almost identical conditions except for the heating duration and found differences in the head-group structure of the polyacrylic acid. The structure change was confirmed by NMR and MS. The two batches of particles had varied binding affinities to a selected group of proteins. Computational work confirmed that the head group of the polymer on the surface of a nanoparticle could directly interact with a protein, and small structural changes in the head group were sufficient to result in a significant difference in the free energy of binding. Our results demonstrate that protein adsorption is so sensitive to the surface properties of particles that it can reveal even small variations in the structure of a nanoparticle surface ligand, and should be useful for quick assessment of nanoparticle properties. † Electronic supplementary information (ESI) available: It includes the electropherograms obtained by CE which were used for calculation of the zeta-potential of the NPs ; the mass spectrum for the standard PAA obtained under the same experimental conditions as in ; the plot showing gradual increase of the aPAA in the heated PAA sample with longer heating duration and the corresponding mass spectra ; comparison of the 1 H NMR spectrum of the PAA sample heated for 12 h with those obtained at other heating durations ; the HSQC and COSY NMR spectra for standard PAA ; the electropherograms and the affinity tting curve for K D measurement ; the electropherograms of the two types of Au-NPs coated with different ratios of His-tagged peptides on the surface and their electropherograms when incubating with b-casein . Two supporting tables (Tables S1 and S2) are also included, and they display the calculated binding energy with calmodulin, myoglobin, cytochrome c, and HSA. See

Proceedings of the National Academy of Sciences, 2008

The mechanisms by which a promiscuous protein can strongly interact with several different protei... more The mechanisms by which a promiscuous protein can strongly interact with several different proteins using the same binding interface are not completely understood. An example is protein kinase A (PKA), which uses a single face on its docking/dimerization domain to interact with multiple A-kinase anchoring proteins (AKAP) that localize it to different parts of the cell. In the current study, the configurational entropy contributions to the binding between the AKAP protein HT31 with the D/D domain of RII ␣-regulatory subunit of PKA were examined. The results show that the majority of configurational entropy loss for the interaction was due to decreased fluctuations within rotamer states of the side chains. The result is in contrast to the widely held approximation that the decrease in the number of rotamer states available to the side chains forms the major component. Further analysis showed that there was a direct linear relationship between total configurational entropy and the number of favorable, alternative contacts available within hydrophobic environments. The hydrophobic binding pocket of the D/D domain provides alternative contact points for the side chains of AKAP peptides that allow them to adopt different binding conformations. The increase in binding conformations provides an increase in binding entropy and hence binding affinity. We infer that a general strategy for a promiscuous protein is to provide alternative contact points at its interface to increase binding affinity while the plasticity required for binding to multiple partners is retained. Implications are discussed for understanding and treating diseases in which promiscuous protein interactions are used. energy well ͉ molecular recognition ͉ peptide-protein binding ͉ quasiharmonic approximation ͉ vibrational entropy Author contributions: C.-e.A.C and W.A.M contributed equally to this work; C.-e.A.C., W.A.M., W.W., and J.A.M. designed research; C.-e.A.C. and W.A.M. performed research; C.-e.A.C. and W.A.M. contributed new reagents/analytic tools; C.-e.A.C., W.A.M., and R.B. analyzed data; and C.-e.A.C., W.A.M., and J.A.M. wrote the paper.

PLoS Computational Biology, 2010

The oligomerization/co-localization of protein complexes and their cooperative regulation in prot... more The oligomerization/co-localization of protein complexes and their cooperative regulation in protein function is a key feature in many biological systems. The synergistic regulation in different subunits often enhances the functional properties of the multi-enzyme complex. The present study used molecular dynamics and Brownian dynamics simulations to study the effects of allostery, oligomerization and intermediate channeling on enhancing the protein function of tryptophan synthase (TRPS). TRPS uses a set of a/b-dimeric units to catalyze the last two steps of L-tryptophan biosynthesis, and the rate is remarkably slower in the isolated monomers. Our work shows that without their binding partner, the isolated monomers are stable and more rigid. The substrates can form fairly stable interactions with the protein in both forms when the protein reaches the final ligand-bound conformations. Our simulations also revealed that the a/b-dimeric unit stabilizes the substrate-protein conformation in the ligand binding process, which lowers the conformation transition barrier and helps the protein conformations shift from an open/inactive form to a closed/active form. Brownian dynamics simulations with a coarse-grained model illustrate how protein conformations affect substrate channeling. The results highlight the complex roles of protein oligomerization and the fine balance between rigidity and dynamics in protein function.

Nature Structural & Molecular Biology, 2010

Changing environmental conditions and lessening fresh water supplies have sparked intense interes... more Changing environmental conditions and lessening fresh water supplies have sparked intense interest in understanding and manipulating abscisic acid signaling, which controls adaptive responses to drought and other abiotic stressors. We recently discovered a selective ABA agonist, pyrabactin, and used it to discover its primary target PYR1, the founding member of the PYR/PYL family of soluble ABA receptors. To understand pyrabactin's selectivity we have taken a combined structural, chemical and genetic approach. We show that subtle differences between receptor binding pockets control ligand orientation between productive and non-productive modes. Non-productive binding occurs without gate closure and prevents receptor activation. Observations in solution show that these orientations are in rapid equilibrium that can be shifted by mutations to control maximal agonist activity. Our results provide a robust framework for the design of new agonists and reveal a new mechanism for agonist selectivity.

Journal of the American Chemical Society, 2014

Proteins can be viewed as small-world networks of amino acid residues connected through noncovale... more Proteins can be viewed as small-world networks of amino acid residues connected through noncovalent interactions. Nuclear magnetic resonance chemical shift covariance analyses were used to identify long-range amino acid networks in the α subunit of tryptophan synthase both for the resting state (in the absence of substrate and product) and for the working state (during catalytic turnover). The amino acid networks observed stretch from the surface of the protein into the active site and are different between the resting and working states. Modification of surface residues on the network alters the structural dynamics of active-site residues over 25 Å away and leads to changes in catalytic rates. These findings demonstrate that amino acid networks, similar to those studied here, are likely important for coordinating structural changes necessary for enzyme function and regulation.

Journal of the American Chemical Society, 2006

High macromolecular concentrations, or crowded conditions, have been shown to affect a wide varie... more High macromolecular concentrations, or crowded conditions, have been shown to affect a wide variety of molecular processes, including diffusion, association and dissociation, and protein folding and stability. Here, we model the effect of macromolecular crowding on the internal dynamics of a protein, HIV-1 protease, using Brownian dynamics simulations. HIV-1 protease possesses a pair of flaps which are postulated to open in the early stages of its catalytic mechanism. Compared to low concentrations, close packed concentrations of repulsive crowding agents are found to significantly reduce the fraction of time that the protease is open. Macromolecular crowding is likely to have a major effect on in vivo enzyme activity, and may play an important regulatory role in the viral life cycle.

Journal of the American Chemical Society, 2006

Synthetic receptors have a wide range of potential applications, but it has been difficult to des... more Synthetic receptors have a wide range of potential applications, but it has been difficult to design low molecular weight receptors that bind ligands with high, "proteinlike" affinities. This study uses novel computational methods to understand why it is hard to design a high-affinity receptor and to explore the limits of affinity, with the bioactive peptide RGD as a model ligand. The M2 modeling method is found to yield excellent agreement with experiment for a known RGD receptor and then is used to analyze a series of receptors generated in silico with a de novo design algorithm. Forces driving binding are found to be systematically opposed by proportionate repulsions due to desolvation and entropy. In particular, strong correlations are found between Coulombic attractions and the electrostatic desolvation penalty and between the mean energy change on binding and the cost in configurational entropy. These correlations help explain why it is hard to achieve high affinity. The change in surface area upon binding is found to correlate poorly with affinity within this series. Measures of receptor efficiency are formulated that summarize how effectively a receptor uses surface area, total energy, and Coulombic energy to achieve affinity. Analysis of the computed efficiencies suggests that a low molecular weight receptor can achieve proteinlike affinity. It is also found that macrocyclization of a receptor can, unexpectedly, increase the entropy cost of binding because the macrocyclic structure further restricts ligand motion.

Journal of the American Chemical Society, 2004

This study applies a novel computational method to study molecular recognition for three sets of ... more This study applies a novel computational method to study molecular recognition for three sets of synthetic hosts: molecular clips, molecular tweezers, and a synthetic barbiturate receptor. The computed standard free energies of binding for the 12 binding reactions agree closely with experiment and provide insight into the roles of configurational entropy, preorganization, and induced fit in the systems studied. The computed changes in configurational entropy are comparable in magnitude to the changes in mean potential plus solvation energy, and they result primarily from changes in the average width of the energy wells upon binding. A strong correlation is observed between the changes in configurational energy and configurational entropy upon binding, resulting in near-linear compensation analogous to classical entropyenthalpy compensation.

BMC Biophysics, 2011

Background: Phosphopeptide-binding domains mediate many vital cellular processes such as signal t... more Background: Phosphopeptide-binding domains mediate many vital cellular processes such as signal transduction and protein recognition. We studied three well-known domains important for signal transduction: BRCT repeats, WW domain and forkhead-associated (FHA) domain. The first two recognize both phosphothreonine (pThr) and phosphoserine (pSer) residues, but FHA has high specificity for pThr residues. Here we used molecular dynamics (MD) simulations to reveal how FHA exclusively chooses pThr and how BRCT and WW recognize both pThr/pSer. The work also investigated the energies and thermodynamic information of intermolecular interactions. Results: Simulations carried out included wide-type and mutated systems. Through analysis of MD simulations, we found that the conserved His residue defines dual loops feature of the FHA domain, which creates a small cavity reserved for only the methyl group of pThr. These well-organized loop interactions directly response to the pThr binding selectivity, while single loop (the 2nd phosphobinding site of FHA) or in combination with α-helix (BRCT repeats) or β-sheet (WW domain) fail to differentiate pThr/pSer. Conclusions: Understanding the domain pre-organizations constructed by conserved residues and the driving force of domain-phosphopeptide recognition provides structural insight into pThr specific binding, which also helps in engineering proteins and designing peptide inhibitors.

Protein Science, 2015

The importance of protonation states and proton transfer in pyridoxal 5&a... more The importance of protonation states and proton transfer in pyridoxal 5'-phosphate (PLP)-chemistry can hardly be overstated. Although experimental approaches to investigate pKa values can provide general guidance for assigning proton locations, only static pictures of the chemical species are available. To obtain the overall protein dynamics for the interpretation of detailed enzyme catalysis in this study, guided by information from solid-state NMR, we performed molecular dynamics (MD) simulations for the PLP-dependent enzyme tryptophan synthase (TRPS), whose catalytic mechanism features multiple quasi-stable intermediates. The primary objective of this work is to elucidate how the position of a single proton on the reacting substrate affects local and global protein dynamics during the catalytic cycle. In general, proteins create a chemical environment and an ensemble of conformational motions to recognize different substrates with different protonations. The study of these interactions in TRPS shows that functional groups on the reacting substrate, such as the phosphoryl group, pyridine nitrogen, phenolic oxygen and carboxyl group, of each PLP-bound intermediate play a crucial role in constructing an appropriate molecular interface with TRPS. In particular, the protonation states of the ionizable groups on the PLP cofactor may enhance or weaken the attractions between the enzyme and substrate. In addition, remodulation of the charge distribution for the intermediates may help generate a suitable environment for chemical reactions. The results of our study enhance knowledge of protonation states for several PLP intermediates and help to elucidate their effects on protein dynamics in the function of TRPS and other PLP-dependent enzymes. This article is protected by copyright. All rights reserved.

In silico pharmacology, 2013

The thermodynamically favored complex between the nuclear vitamin D receptor (VDR) and 1α,25(OH)2... more The thermodynamically favored complex between the nuclear vitamin D receptor (VDR) and 1α,25(OH)2-vitamin D3 (1,25D3) triggers a shift in equilibrium to favor VDR binding to DNA, heterodimerization with the nuclear retinoid x receptor (RXR) and subsequent regulation of gene transcription. The key amino acids and structural requirements governing VDR binding to nuclear coactivators (NCoA) are well defined. Yet very little is understood about the internal changes in amino acid flexibility underpinning the control of ligand affinity, helix 12 conformation and function. Herein, we use molecular dynamics (MD) to study how the backbone and side-chain flexibility of the VDR differs when a) complexed to 1α,25(OH)2-vitamin D3 (1,25D3, agonist) and (23S),25-dehydro-1α(OH)-vitamin D3-26,23-lactone (MK, antagonist); b) residues that form hydrogen bonds with the C25-OH (H305 and H397) of 1,25D3 are mutated to phenylalanine; c) helix 12 conformation is changed and ligand is removed; and d) x-ray ...

Journal of molecular recognition : JMR, 2014

Revealing the processes of ligand-protein associations deepens our understanding of molecular rec... more Revealing the processes of ligand-protein associations deepens our understanding of molecular recognition and binding kinetics. Hydrogen bonds (H-bonds) play a crucial role in optimizing ligand-protein interactions and ligand specificity. In addition to the formation of stable H-bonds in the final bound state, the formation of transient H-bonds during binding processes contributes binding kinetics that define a ligand as a fast or slow binder, which also affects drug action. However, the effect of forming the transient H-bonds on the kinetic properties is little understood. Guided by results from coarse-grained Brownian dynamics simulations, we used classical molecular dynamics simulations in an implicit solvent model and accelerated molecular dynamics simulations in explicit waters to show that the position and distribution of the H-bond donor or acceptor of a drug result in switching intermolecular and intramolecular H-bond pairs during ligand recognition processes. We studied two...

Proceedings of the National Academy of Sciences of the United States of America, Jan 30, 2007

The restriction of a small molecule's motion on binding to a protein causes a loss of configu... more The restriction of a small molecule's motion on binding to a protein causes a loss of configurational entropy, and thus a penalty in binding affinity. Some energy models used in computer-aided ligand design neglect this entropic penalty, whereas others account for it based on an expected drop in the number of accessible rotamers upon binding. However, the validity of the physical assumptions underlying the various approaches is largely unexamined. The present study addresses this issue by using Mining Minima calculations to analyze the association of amprenavir with HIV protease. The computed loss in ligand configurational entropy is large, contributing approximately 25 kcal/mol (4.184 kJ/kcal) to DeltaG degrees. Most of this loss results from narrower energy wells in the bound state, rather than a drop in the number of accessible rotamers. Coupling among rotation/translation and internal degrees of freedom complicates the decomposition of the entropy change into additive terms....

The Journal of Physical Chemistry C, 2014

Colloidal nanomaterials with well-defined shapes have wide applications in many fields. However, ... more Colloidal nanomaterials with well-defined shapes have wide applications in many fields. However, the exact role of capping ligands, which often dictates the shape of products in colloidal syntheses, is often unclear. Here we use a classical molecular-mechanics force-field method, mining minima (M2), to compute the binding free energy of the ligands such as citrate, monocarboxylates, dicarboxylates, and tricarboxylates to both and facets of silver and to investigate the mechanisms of the anisotropic growth of silver nanoplates. The distribution of partial charges on a ligand, the geometry complementation in the complex, and the entropic penalty on binding played crucial roles in discriminating the two facets and determining a good or poor ligand. Our finding allows rational design of capping ligands that may perform as well as citrate in promoting the anisotropic growth of nanoplates; however, designing a compound that outperforms citrate is found to be challenging.

Methods in Molecular Biology, 2011

Cannabinoids represent a promising class of compounds for developing novel therapeutic agents. Si... more Cannabinoids represent a promising class of compounds for developing novel therapeutic agents. Since the isolation and identification of the major psychoactive component Δ(9)-THC in Cannabis sativa in the 1960s, numerous analogues of the classical plant cannabinoids have been synthesized and tested for their biological activity. These compounds primarily target the cannabinoid receptors 1 (CB1) and Cannabinoid receptors 2 (CB2). This chapter focuses on CB1. Despite the lack of crystal structures for CB1, protein-based homology modeling approaches and molecular docking methods can be used in the design and discovery of cannabinoid analogues. Efficient synthetic approaches for therapeutically interesting cannabinoid analogues have been developed to further facilitate the drug discovery process.

Journal of the American Chemical Society, Jan 17, 2014

The acid-base chemistry that drives catalysis in pyridoxal-5'-phosphate (PLP)-dependent enzym... more The acid-base chemistry that drives catalysis in pyridoxal-5'-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP's role as a coenzyme in this extensive class of enzymes. It was first proposed over 50 years ago that the initial step in the catalytic cycle is facilitated by a protonated Schiff base form of the holoenzyme in which the linking lysine ε-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first (15)N NMR chemical shift measurements of such a Schiff base linkage in the resting holoenzyme form, the internal aldimine state of tryptophan synthase. Double-resonance experiments confirm the assignment of the Schiff base nitrogen, and additional (13)C, (15)N, and (31)P chemical shift measurements of sites on the PLP coenzyme allow a detailed model of coenzyme protonation states to be established.

PLoS ONE, 2014

The regulation of a series of cellular events requires specific protein-protein interactions, whi... more The regulation of a series of cellular events requires specific protein-protein interactions, which are usually mediated by modular domains to precisely select a particular sequence from diverse partners. However, most signaling domains can bind to more than one peptide sequence. How do proteins create promiscuity from precision? Moreover, these complex interactions typically occur at the interface of a well-defined secondary structure, a helix and b sheet. However, the molecular recognition primarily controlled by loop architecture is not fully understood. To gain a deep understanding of binding selectivity and promiscuity by the conformation of loops, we chose the forkhead-associated (FHA) domain as our model system. The domain can bind to diverse peptides via various loops but only interact with sequences containing phosphothreonine (pThr). We applied molecular dynamics (MD) simulations for multiple free and bound FHA domains to study the changes in conformations and dynamics. Generally, FHA domains share a similar folding structure whereby the backbone holds the overall geometry and the variety of sidechain atoms of multiple loops creates a binding surface to target a specific partner. FHA domains determine the specificity of pThr by well-organized binding loops, which are rigid to define a phospho recognition site. The broad range of peptide recognition can be attributed to different arrangements of the loop interaction network. The moderate flexibility of the loop conformation can help access or exclude binding partners. Our work provides insights into molecular recognition in terms of binding specificity and promiscuity and helpful clues for further peptide design.

Nat Commun, 2014

Small RNAs (sRNAs) are loaded into ARGONAUTE (AGO) proteins to induce gene silencing. In plants, ... more Small RNAs (sRNAs) are loaded into ARGONAUTE (AGO) proteins to induce gene silencing. In plants, the 5 0 -terminal nucleotide is important for sRNA sorting into different AGOs. Here we show that microRNA (miRNA) duplex structure also contributes to miRNA sorting. Base pairing at the 15th nucleotide of a miRNA duplex is important for miRNA sorting in both Arabidopsis AGO1 and AGO2. AGO2 favours miRNA duplexes with no middle mismatches, whereas AGO1 tolerates, or prefers, duplexes with central mismatches. AGO structure modelling and mutational analyses reveal that the QF-V motif within the conserved PIWI domain contributes to recognition of base pairing at the 15th nucleotide of a duplex, while the DDDE catalytic core of AtAGO2 is important for recognition of the central nucleotides. Finally, we rescued the adaxialized phenotype of ago1-12, which is largely due to miR165 loss-of-function, by changing miR165 duplex structure which we predict redirects it to AGO2.

The Journal of Physical Chemistry B, 2012

Promiscuous proteins are commonly observed in biological systems, for example, in modular domains... more Promiscuous proteins are commonly observed in biological systems, for example, in modular domains that recognize phosphopeptides during signal transduction. This promiscuous recognition is of fundamental interest in chemistry and biology but is challenging when designing phosphopeptides in silico for cell biology studies. To investigate promiscuous recognition and binding processes of phosphopeptides and the modular domain, we selected a domain essential in breast cancerthe breast-cancer− associated protein 1 (BRCA1) C-terminal (BRCT) repeats as our model system. We performed molecular dynamics simulations and detailed analyses of the dihedral space to study protein fluctuation and conformational changes with phosphopeptide binding. We also studied the association processes of phosphorylated and unphosphorylated peptides using Brownian dynamics with a coarse-grained model. We found that the BRCT domain is preorganized for phosphopeptide binding but has a moderate arrangement of side chains to form complexes with various types of phosphopeptides. Phosphopeptide binding restricts the system motion in general, while the nonpolar phosphopeptide becomes more flexible in the bound state. Our analysis found that the BRCT domain utilizes different mechanisms, usually termed lock and key, induced-fit, and population-shift/conformational-selection models, to recognize peptides with different features. Brownian dynamics simulations revealed that the charged phosphate group may not always accelerate peptide association processes, but it helps the phosphopeptide orient into binding pockets accurately and stabilizes the complex. This work provides insights into molecular recognition in the promiscuous protein system. a The major binding residues, pSer and Phe+3, are in bold. Other residues with non-polar contributions are underlined.

The Journal of Physical Chemistry B, 2006

A method is presented for calculating the conformational free energy of a molecule in all degrees... more A method is presented for calculating the conformational free energy of a molecule in all degrees of freedom. The method uses the harmonic approximation with finite integration ranges, along with Mode Scanning, a fast correction for anharmonicity based upon internal bond-angle-torsion coordinates. Mode Scanning accounts for local anharmonicity without the need for expensive Monte Carlo integration. The method is efficient, and comparisons with analytic or highly detailed numerical calculations show excellent accuracy. Similar comparisons for the previously described Mode Integration method show that, although it is computationally demanding, it can be less accurate than the pure harmonic approximation. The inaccuracy of Mode Integration is traceable to its use of a Cartesian coordinate basis set; much more accurate results are obtained when the basis set consists of bond-angle-torsion coordinates.

The Analyst, 2014

Protein adsorption on nanoparticles is closely associated with the physicochemical properties of ... more Protein adsorption on nanoparticles is closely associated with the physicochemical properties of particles, in particular, their surface properties. We synthesized two batches of polyacrylic acid-coated nanoparticles under almost identical conditions except for the heating duration and found differences in the head-group structure of the polyacrylic acid. The structure change was confirmed by NMR and MS. The two batches of particles had varied binding affinities to a selected group of proteins. Computational work confirmed that the head group of the polymer on the surface of a nanoparticle could directly interact with a protein, and small structural changes in the head group were sufficient to result in a significant difference in the free energy of binding. Our results demonstrate that protein adsorption is so sensitive to the surface properties of particles that it can reveal even small variations in the structure of a nanoparticle surface ligand, and should be useful for quick assessment of nanoparticle properties. † Electronic supplementary information (ESI) available: It includes the electropherograms obtained by CE which were used for calculation of the zeta-potential of the NPs ; the mass spectrum for the standard PAA obtained under the same experimental conditions as in ; the plot showing gradual increase of the aPAA in the heated PAA sample with longer heating duration and the corresponding mass spectra ; comparison of the 1 H NMR spectrum of the PAA sample heated for 12 h with those obtained at other heating durations ; the HSQC and COSY NMR spectra for standard PAA ; the electropherograms and the affinity tting curve for K D measurement ; the electropherograms of the two types of Au-NPs coated with different ratios of His-tagged peptides on the surface and their electropherograms when incubating with b-casein . Two supporting tables (Tables S1 and S2) are also included, and they display the calculated binding energy with calmodulin, myoglobin, cytochrome c, and HSA. See

Proceedings of the National Academy of Sciences, 2008

The mechanisms by which a promiscuous protein can strongly interact with several different protei... more The mechanisms by which a promiscuous protein can strongly interact with several different proteins using the same binding interface are not completely understood. An example is protein kinase A (PKA), which uses a single face on its docking/dimerization domain to interact with multiple A-kinase anchoring proteins (AKAP) that localize it to different parts of the cell. In the current study, the configurational entropy contributions to the binding between the AKAP protein HT31 with the D/D domain of RII ␣-regulatory subunit of PKA were examined. The results show that the majority of configurational entropy loss for the interaction was due to decreased fluctuations within rotamer states of the side chains. The result is in contrast to the widely held approximation that the decrease in the number of rotamer states available to the side chains forms the major component. Further analysis showed that there was a direct linear relationship between total configurational entropy and the number of favorable, alternative contacts available within hydrophobic environments. The hydrophobic binding pocket of the D/D domain provides alternative contact points for the side chains of AKAP peptides that allow them to adopt different binding conformations. The increase in binding conformations provides an increase in binding entropy and hence binding affinity. We infer that a general strategy for a promiscuous protein is to provide alternative contact points at its interface to increase binding affinity while the plasticity required for binding to multiple partners is retained. Implications are discussed for understanding and treating diseases in which promiscuous protein interactions are used. energy well ͉ molecular recognition ͉ peptide-protein binding ͉ quasiharmonic approximation ͉ vibrational entropy Author contributions: C.-e.A.C and W.A.M contributed equally to this work; C.-e.A.C., W.A.M., W.W., and J.A.M. designed research; C.-e.A.C. and W.A.M. performed research; C.-e.A.C. and W.A.M. contributed new reagents/analytic tools; C.-e.A.C., W.A.M., and R.B. analyzed data; and C.-e.A.C., W.A.M., and J.A.M. wrote the paper.

PLoS Computational Biology, 2010

The oligomerization/co-localization of protein complexes and their cooperative regulation in prot... more The oligomerization/co-localization of protein complexes and their cooperative regulation in protein function is a key feature in many biological systems. The synergistic regulation in different subunits often enhances the functional properties of the multi-enzyme complex. The present study used molecular dynamics and Brownian dynamics simulations to study the effects of allostery, oligomerization and intermediate channeling on enhancing the protein function of tryptophan synthase (TRPS). TRPS uses a set of a/b-dimeric units to catalyze the last two steps of L-tryptophan biosynthesis, and the rate is remarkably slower in the isolated monomers. Our work shows that without their binding partner, the isolated monomers are stable and more rigid. The substrates can form fairly stable interactions with the protein in both forms when the protein reaches the final ligand-bound conformations. Our simulations also revealed that the a/b-dimeric unit stabilizes the substrate-protein conformation in the ligand binding process, which lowers the conformation transition barrier and helps the protein conformations shift from an open/inactive form to a closed/active form. Brownian dynamics simulations with a coarse-grained model illustrate how protein conformations affect substrate channeling. The results highlight the complex roles of protein oligomerization and the fine balance between rigidity and dynamics in protein function.

Nature Structural & Molecular Biology, 2010

Changing environmental conditions and lessening fresh water supplies have sparked intense interes... more Changing environmental conditions and lessening fresh water supplies have sparked intense interest in understanding and manipulating abscisic acid signaling, which controls adaptive responses to drought and other abiotic stressors. We recently discovered a selective ABA agonist, pyrabactin, and used it to discover its primary target PYR1, the founding member of the PYR/PYL family of soluble ABA receptors. To understand pyrabactin's selectivity we have taken a combined structural, chemical and genetic approach. We show that subtle differences between receptor binding pockets control ligand orientation between productive and non-productive modes. Non-productive binding occurs without gate closure and prevents receptor activation. Observations in solution show that these orientations are in rapid equilibrium that can be shifted by mutations to control maximal agonist activity. Our results provide a robust framework for the design of new agonists and reveal a new mechanism for agonist selectivity.

Journal of the American Chemical Society, 2014

Proteins can be viewed as small-world networks of amino acid residues connected through noncovale... more Proteins can be viewed as small-world networks of amino acid residues connected through noncovalent interactions. Nuclear magnetic resonance chemical shift covariance analyses were used to identify long-range amino acid networks in the α subunit of tryptophan synthase both for the resting state (in the absence of substrate and product) and for the working state (during catalytic turnover). The amino acid networks observed stretch from the surface of the protein into the active site and are different between the resting and working states. Modification of surface residues on the network alters the structural dynamics of active-site residues over 25 Å away and leads to changes in catalytic rates. These findings demonstrate that amino acid networks, similar to those studied here, are likely important for coordinating structural changes necessary for enzyme function and regulation.

Journal of the American Chemical Society, 2006

High macromolecular concentrations, or crowded conditions, have been shown to affect a wide varie... more High macromolecular concentrations, or crowded conditions, have been shown to affect a wide variety of molecular processes, including diffusion, association and dissociation, and protein folding and stability. Here, we model the effect of macromolecular crowding on the internal dynamics of a protein, HIV-1 protease, using Brownian dynamics simulations. HIV-1 protease possesses a pair of flaps which are postulated to open in the early stages of its catalytic mechanism. Compared to low concentrations, close packed concentrations of repulsive crowding agents are found to significantly reduce the fraction of time that the protease is open. Macromolecular crowding is likely to have a major effect on in vivo enzyme activity, and may play an important regulatory role in the viral life cycle.

Journal of the American Chemical Society, 2006

Synthetic receptors have a wide range of potential applications, but it has been difficult to des... more Synthetic receptors have a wide range of potential applications, but it has been difficult to design low molecular weight receptors that bind ligands with high, "proteinlike" affinities. This study uses novel computational methods to understand why it is hard to design a high-affinity receptor and to explore the limits of affinity, with the bioactive peptide RGD as a model ligand. The M2 modeling method is found to yield excellent agreement with experiment for a known RGD receptor and then is used to analyze a series of receptors generated in silico with a de novo design algorithm. Forces driving binding are found to be systematically opposed by proportionate repulsions due to desolvation and entropy. In particular, strong correlations are found between Coulombic attractions and the electrostatic desolvation penalty and between the mean energy change on binding and the cost in configurational entropy. These correlations help explain why it is hard to achieve high affinity. The change in surface area upon binding is found to correlate poorly with affinity within this series. Measures of receptor efficiency are formulated that summarize how effectively a receptor uses surface area, total energy, and Coulombic energy to achieve affinity. Analysis of the computed efficiencies suggests that a low molecular weight receptor can achieve proteinlike affinity. It is also found that macrocyclization of a receptor can, unexpectedly, increase the entropy cost of binding because the macrocyclic structure further restricts ligand motion.

Journal of the American Chemical Society, 2004

This study applies a novel computational method to study molecular recognition for three sets of ... more This study applies a novel computational method to study molecular recognition for three sets of synthetic hosts: molecular clips, molecular tweezers, and a synthetic barbiturate receptor. The computed standard free energies of binding for the 12 binding reactions agree closely with experiment and provide insight into the roles of configurational entropy, preorganization, and induced fit in the systems studied. The computed changes in configurational entropy are comparable in magnitude to the changes in mean potential plus solvation energy, and they result primarily from changes in the average width of the energy wells upon binding. A strong correlation is observed between the changes in configurational energy and configurational entropy upon binding, resulting in near-linear compensation analogous to classical entropyenthalpy compensation.