Publications by authors named "Donald Hamelberg"

87 Publications

Structural Mechanism of Cooperative Regulation of Calcium-Sensing Receptor-Mediated Cellular Signaling.

Curr Opin Physiol 2020 Oct 11;17:269-277. Epub 2020 Sep 11.

Department of Chemistry, Center for Diagnostics and Therapeutics and Advanced Translational Imaging Facility, Georgia State University, Atlanta, GA, 30303.

Calcaium sensing receptors (CaSRs) play a central role in regulating extracellular calcium (Ca) homeostasis and many (patho)physiological processes. This regulation is primarily orchestrated in response to extracellular stimuli via the extracellular domain (ECD). This paper first reviews the modeled structure of the CaSR ECD and the prediction and investigation of the Ca and amino acid binding sites. Several recently solved X-ray structures are then compared to support a proposed CaSR activation model involving functional cooperativity. The review also discusses recent implications for drug development. These studies provide new insights into the molecular basis of diseases and the design of therapeutic agents that target CaSR and other family C G protein-coupled receptors (cGPCRs).
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http://dx.doi.org/10.1016/j.cophys.2020.08.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7943033PMC
October 2020

A Single-Point Mutation in d-Arginine Dehydrogenase Unlocks a Transient Conformational State Resulting in Altered Cofactor Reactivity.

Biochemistry 2021 Mar 25;60(9):711-724. Epub 2021 Feb 25.

Department of Chemistry, Georgia State University, Atlanta, Georgia 30302, United States.

Proteins are inherently dynamic, and proper enzyme function relies on conformational flexibility. In this study, we demonstrated how an active site residue changes an enzyme's reactivity by modulating fluctuations between conformational states. Replacement of tyrosine 249 (Y249) with phenylalanine in the active site of the flavin-dependent d-arginine dehydrogenase yielded an enzyme with both an active yellow FAD (Y249F-y) and an inactive chemically modified green FAD, identified as 6-OH-FAD (Y249F-g) through various spectroscopic techniques. Structural investigation of Y249F-g and Y249F-y variants by comparison to the wild-type enzyme showed no differences in the overall protein structure and fold. A closer observation of the active site of the Y249F-y enzyme revealed an alternative conformation for some active site residues and the flavin cofactor. Molecular dynamics simulations probed the alternate conformations observed in the Y249F-y enzyme structure and showed that the enzyme variant with FAD samples a metastable conformational state, not available to the wild-type enzyme. Hybrid quantum/molecular mechanical calculations identified differences in flavin electronics between the wild type and the alternate conformation of the Y249F-y enzyme. The computational studies further indicated that the alternate conformation in the Y249F-y enzyme is responsible for the higher spin density at the C6 atom of flavin, which is consistent with the formation of 6-OH-FAD in the variant enzyme. The observations in this study are consistent with an alternate conformational space that results in fine-tuning the microenvironment around a versatile cofactor playing a critical role in enzyme function.
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http://dx.doi.org/10.1021/acs.biochem.1c00054DOI Listing
March 2021

p53 Is Potentially Regulated by Cyclophilin D in the Triple-Proline Loop of the DNA Binding Domain.

Biochemistry 2021 Mar 16;60(8):597-606. Epub 2021 Feb 16.

The multifunctional protein p53 is the central molecular sensor of cellular stresses. The canonical function of p53 is to transcriptionally activate target genes in response to, for example, DNA damage that may trigger apoptosis. Recently, p53 was also found to play a role in the regulation of necrosis, another type of cell death featured by the mitochondrial permeability transition (mPT). In this process, p53 directly interacts with the mPT regulator cyclophilin D, the detailed mechanism of which however remains poorly understood. Here, we report a comprehensive computational investigation of the p53-cyclophilin D interaction using molecular dynamics simulations and associated analyses. We have identified the specific cyclophilin D binding site on p53 that is located at proline 151 in the DNA binding domain. As a peptidyl-prolyl isomerase, cyclophilin D binds p53 and catalyzes the isomerization of the peptide bond preceding proline 151. We have also characterized the effect of such an isomerization and found that the p53 domain in the state is overall more rigid than the state except for the local region around proline 151. Dynamical changes upon isomerization occur in both local and distal regions, indicating an allosteric effect elicited by the isomerization. We present potential allosteric communication pathways between proline 151 and distal sites, including the DNA binding surface. Our work provides, for the first time, a model for how cyclophilin D binds p53 and regulates its activity by switching the configuration of a specific site.
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http://dx.doi.org/10.1021/acs.biochem.0c00946DOI Listing
March 2021

Donor acceptor fluorophores: synthesis, optical properties, TD-DFT and cytotoxicity studies.

Org Biomol Chem 2021 03;19(8):1835-1846

Department of Chemistry, Petit Science Center, Georgia State University, 100 Piedmont Avenue SE, Atlanta, Georgia 30303, USA. and Center of Diagnostics and Therapeutics, Petit Science Center, Georgia State University, 100 Piedmont Avenue SE, Atlanta, Georgia 30303, USA.

Donor-π-acceptor (D-π-A) fluorophores consisting of a donor unit, a π linker, and an acceptor moiety have attracted attention in the last decade. In this study, we report the synthesis, characterization, optical properties, TD-DFT, and cytotoxicity studies of 17 near infrared (NIR) D-π-A analogs which have not been reported so far to the best of our knowledge. These fluorophores have chloroacrylic acid as the acceptor unit and various donor units such as indole, benzothiazole, benzo[e]indole, and quinoline. The fluorophores showed strong absorption in the NIR (700-970 nm) region due to their enhanced intramolecular charge transfer (ICT) between chloroacrylic acid and the donor moieties connected with the Vilsmeier-Haack linker. The emission wavelength maxima of the fluorophores were in between 798 and 870 nm. Compound 20 with a 4-quinoline donor moiety showed an emission wavelength above 1000 nm in the NIR II window. The synthesized fluorophores were characterized by 1H NMR and 13C NMR, and their optical properties were studied. Time dependent density functional theory (TD-DFT) calculations showed that the charge transfer occurs from the donor groups (indole, benzothiazole, benzo[e]indole, and quinoline) to the acceptor chloroacrylic acid moiety. Fluorophores with [HOMO] to [LUMO+1] transitions were shown to possess a charge separation character. The cytotoxicity of selected fluorophores, 4, 7, 10 and 12 was investigated against breast cancer cell lines and they showed better activity than the anti-cancer agent docetaxel.
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http://dx.doi.org/10.1039/d0ob02313bDOI Listing
March 2021

Editorial.

Comput Biol Chem 2020 Dec 5;89:107373. Epub 2020 Oct 5.

Department of Chemistry, Georgia State University, USA.

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http://dx.doi.org/10.1016/j.compbiolchem.2020.107373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534834PMC
December 2020

Detecting Functional Dynamics in Proteins with Comparative Perturbed-Ensembles Analysis.

Acc Chem Res 2019 12 3;52(12):3455-3464. Epub 2019 Dec 3.

Department of Chemistry , Georgia State University , Atlanta , Georgia 30302-3965 , United States.

Recent advances have made all-atom molecular dynamics (MD) a powerful tool to sample the conformational energy landscape. There are still however three major challenges in the application of MD to biological systems: accuracy of force field, time scale, and the analysis of simulation trajectories. Significant progress in addressing the first two challenges has been made and extensively reviewed previously. This Account focuses on strategies of analyzing simulation data of biomolecules that also covers ways to properly design simulations and validate simulation results. In particular, we examine an approach named , which we developed to efficiently detect dynamics in protein MD simulations that can be linked to biological functions. In our recent studies, we implemented this approach to understand allosteric regulations in several disease-associated human proteins. The central task of a comparative perturbed-ensembles analysis is to compare two or more conformational ensembles of a system generated by MD simulations under distinct perturbation conditions. Perturbations can be different sequence variations, ligand-binding conditions, and other physical/chemical modifications of the system. Each simulation is long enough (e.g., microsecond-long) to ensure sufficient sampling of the local substate. Then, sophisticated bioinformatic and statistical tools are applied to extract function-related information from the simulation data, including principal component analysis, residue-residue contact analysis, difference contact network analysis (dCNA) based on the graph theory, and statistical analysis of side-chain conformations. Computational findings are further validated with experimental data. By comparing distinct conformational ensembles, functional micro- to millisecond dynamics can be inferred. In contrast, such a time scale is difficult to reach in a single simulation; even when reached for a single condition of a system, it is elusive as to what dynamical motions are related to functions without, for example, comparing free and substrate-bound proteins at the minimum. We illustrate our approach with three examples. First, we discuss using the approach to identify allosteric pathways in cyclophilin A (CypA), a member of a ubiquitous class of peptidyl-prolyl isomerase enzymes. By comparing side-chain torsion-angle distributions of CypA in wild-type and mutant forms, we identified three pathways: two are consistent with recent nuclear magnetic resonance experiments, whereas the third is a novel pathway. Second, we show how the approach enables a dynamical-evolution analysis of the human cyclophilin family. In the analysis, both conserved and divergent conformational dynamics across three cyclophilin isoforms (CypA, CypD, and CypE) were summarized. The conserved dynamics led to the discovery of allosteric networks resembling those found in CypA. A residue wise determinant underlying the unique dynamics in CypD was also detected and validated with additional mutational MD simulations. In the third example, we applied the approach to elucidate a peptide sequence-dependent allosteric mechanism in human Pin 1, a phosphorylation-dependent peptidyl-prolyl isomerase. We finally present our outlook of future directions. Especially, we envisage how the approach could help open a new avenue in drug discovery.
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http://dx.doi.org/10.1021/acs.accounts.9b00485DOI Listing
December 2019

Synergistic mutations in soluble guanylyl cyclase (sGC) reveal a key role for interfacial regions in the sGC activation mechanism.

J Biol Chem 2019 11 23;294(48):18451-18464. Epub 2019 Oct 23.

Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250. Electronic address:

Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) and a central component of the NO-cGMP pathway, critical to cardiovascular function. NO binding to the N-terminal sensor domain in sGC enhances the cyclase activity of the C-terminal catalytic domain. Our understanding of the structural elements regulating this signaling cascade is limited, hindering structure-based drug design efforts that target sGC to improve the management of cardiovascular diseases. Conformational changes are thought to propagate the NO-binding signal throughout the entire sGC heterodimer, via its coiled-coil domain, to reorient the catalytic domain into an active conformation. To identify the structural elements involved in this signal transduction cascade, here we optimized a cGMP-based luciferase assay that reports on heterologous sGC activity in and identified several mutations that activate sGC. These mutations resided in the dorsal flaps, dimer interface, and GTP-binding regions of the catalytic domain. Combinations of mutations from these different elements synergized, resulting in even greater activity and indicating a complex cross-talk among these regions. Molecular dynamics simulations further revealed conformational changes underlying the functional impact of these mutations. We propose that the interfacial residues play a central role in the sGC activation mechanism by coupling the coiled-coil domain to the active site via a series of hot spots. Our results provide new mechanistic insights not only into the molecular pathway for sGC activation but also for other members of the larger nucleotidyl cyclase family.
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http://dx.doi.org/10.1074/jbc.RA119.011010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885636PMC
November 2019

Second Generation G-Quadruplex Stabilizing Trimethine Cyanines.

Bioconjug Chem 2019 10 26;30(10):2647-2663. Epub 2019 Sep 26.

Department of Chemistry , Georgia State University , Petit Science Center, 100 Piedmont Ave SE. Atlanta Georgia 30303 , United States.

G-Quadruplex DNA has been recognized as a highly appealing target for the development of new selective chemotherapeutics, which could result in markedly reduced toxicity toward normal cells. In particular, the cyanine dyes that bind selectively to G-quadruplex structures without targeting duplex DNA have attracted attention due to their high amenability to structural modifications that allows fine-tuning of their biomolecular interactions. We have previously reported pentamethine and symmetric trimethine cyanines designed to effectively bind G-quadruplexes through end stacking interactions. Herein, we are reporting a second generation of drug candidates, the asymmetric trimethine cyanines. These have been synthesized and evaluated for their quadruplex binding properties. Incorporating a benz[,]indolenine heterocyclic unit increased overall quadruplex binding, and elongating the alkyl length increases the quadruplex-to-duplex binding specificity.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7032661PMC
October 2019

Establishing a Framework of Using Residue-Residue Interactions in Protein Difference Network Analysis.

J Chem Inf Model 2019 07 3;59(7):3222-3228. Epub 2019 Jul 3.

Department of Chemistry , Georgia State University , Atlanta , Georgia 30302-3965 , United States.

Detailed understanding of interactions between amino acid residues is critical in using promising difference network analysis approaches to map allosteric communication pathways. Using experimental data as benchmarks, we scan values of two essential residue-residue contact parameters: the distance cutoff () and the cutoff of residue separation in sequence (). The optimal = 4.5 Å is revealed, which defines the upper bound of the first shell of residue-residue packing in proteins, whereas is found to have little effects on performance. We also develop a new energy-based contact method for network analyses and find an equivalency between the energy network using the optimal energy cutoff = 1.0 and the structure network using = 4.5 Å. The simple 4.5-Å contact method is further shown to have comparable prediction accuracy to a contact method using amino acid type-specific distance cutoffs and chemical shift prediction-based methods. This study provides necessary tools in mapping dynamics to functions.
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http://dx.doi.org/10.1021/acs.jcim.9b00320DOI Listing
July 2019

Combinatorial Coarse-Graining of Molecular Dynamics Simulations for Detecting Relationships between Local Configurations and Overall Conformations.

J Chem Theory Comput 2018 Nov 29;14(11):6026-6034. Epub 2018 Oct 29.

Department of Chemistry and the Center for Diagnostics & Therapeutics , Georgia State University , Atlanta , Georgia 30302-3965 , United States.

An automatic, multiscale, and three-dimensional (3D) summary of local configurations of the dynamics of proteins can help to discover and describe the relationships between different parts of proteins across spatial scales, including the overall conformation and 3D configurations of side chains and domains. These discoveries can improve our understanding of the function and allosteric mechanism of proteins and could potentially provide an avenue to test and improve the molecular mechanics force fields at different spatial resolutions. Many of the current methods are unable to effectively summarize shapes of 3D local configurations across all spatial scales. Here, we propose frequent substructure clustering (FSC) to fill this gap. Frequent substructure clustering of the C atoms of the GB3 protein identifies six clusters of co-occurring local configurations. The clusters that are localized at different regions contribute to the overall conformation, and form two anticorrelating groups. The results suggest that FSC could describe dynamical relationships between different parts of proteins by providing a 3D description of the frequently occurring local configurations at different spatial resolutions. FSC could augment the use of other methods, such as Markov state models, to study the function of subcellular processes and highlight the role of local configurations in biomolecular systems.
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http://dx.doi.org/10.1021/acs.jctc.8b00333DOI Listing
November 2018

Elucidating Allosteric Communications in Proteins with Difference Contact Network Analysis.

J Chem Inf Model 2018 07 11;58(7):1325-1330. Epub 2018 Jul 11.

Department of Chemistry , Georgia State University , Atlanta , Georgia 30302-3965 , United States.

A difference contact network analysis (dCNA) method is developed for delineating allosteric mechanisms in proteins. The new method addresses limitations of conventional network analysis methods and is particularly suitable for allosteric systems undergoing large-amplitude conformational changes during function. Tests show that dCNA works well for proteins of varying sizes and functions. The design of dCNA is general enough to facilitate analyses of diverse dynamic data generated by molecular dynamics, crystallography, or nuclear magnetic resonance.
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http://dx.doi.org/10.1021/acs.jcim.8b00250DOI Listing
July 2018

Decoding Allosteric Communication Pathways in Cyclophilin A with a Comparative Analysis of Perturbed Conformational Ensembles.

J Phys Chem B 2018 06 13;122(25):6528-6535. Epub 2018 Jun 13.

Department of Chemistry , Georgia State University , Atlanta , Georgia 30302-3965 , United States.

Conformational dynamics plays the key role in allosteric regulation of enzymes. Despite numerous experimental and computational efforts, the mechanism of how dynamics couple enzymatic function is poorly understood. Here, we introduce a new approach to exploring the dynamics-function relationship combining computational mutagenesis, microsecond-long molecular dynamics simulations, and side-chain torsion angle analyses. We apply our approach to elucidate the allosteric mechanism in cyclophilin A (CypA), a peptidyl-prolyl cis-trans isomerase known to participate in diverse biological processes and be associated with many diseases including cancer. Multiple single mutations are performed in CypA at previously discovered hotspot residues distal from the active site, and residues displaying significant dynamical changes upon mutations are then identified. The mutation-responsive residues delineate three distinct pathways potentially mediating allosteric communications between distal sites: two pathways resemble the allosteric networks identified in a recent experimental study, whereas the third represents a novel pathway. A residue-residue contact analysis is also performed to complement the findings. Furthermore, a recently developed difference contact network analysis is employed to explain mutation-specific allosteric effects. Our results suggest that comparing multiple conformational ensembles generated under various mutational conditions is a powerful tool to gain novel insights into enzymatic functions that are difficult to obtain through examining a single system such as the wild-type. Our approach is easy to extend for other systems. The results can also be utilized to facilitate the design of potent therapeutics targeting CypA.
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http://dx.doi.org/10.1021/acs.jpcb.8b03824DOI Listing
June 2018

Substrate Sequence Determines Catalytic Activities, Domain-Binding Preferences, and Allosteric Mechanisms in Pin1.

J Phys Chem B 2018 06 13;122(25):6521-6527. Epub 2018 Jun 13.

Pin1 is a unique phosphorylation-dependent peptidyl-prolyl isomerase that regulates diverse subcellular processes and an important potential therapeutic target. Functional mechanisms of Pin1 are complicated because of the two-domain structural organization: the catalytic domain both binds the specific pSer/Thr-Pro motif and catalyzes the cis/trans isomerization, whereas the WW domain can only bind the trans configuration and is speculated to be responsible for substrate-binding specificity. Numerous studies of Pin1 have led to two divergent conclusions on the functional role of the WW domain. One opinion states that the WW domain is an allosteric effector, and substrate binding to this domain modulates the binding and catalysis in the distal catalytic domain. The other opinion, however, argues that the WW domain does not have any allosteric role. Here, using molecular dynamics and binding free-energy calculations, we examine catalysis and allosteric mechanisms in Pin1 under various substrate- and WW-binding conditions. Our results reveal a strong substrate sequence dependency of catalysis, domain-binding preferences, and allosteric outputs in Pin1. Importantly, we show that the different opinions about the WW domain can be unified in one framework, in which substrate sequences determine whether a positive, negative, or neural allosteric effect will be elicited. Our work further elucidates detailed mechanisms underlying the sequence-dependent allostery of Pin1 and finds that interdomain contacts are key mediators of intraprotein allosteric communications. Our findings collectively provide new insights into the function of Pin1, which may facilitate the development of novel therapeutic drugs targeting Pin1 in the future.
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http://dx.doi.org/10.1021/acs.jpcb.8b03819DOI Listing
June 2018

Redox-Specific Allosteric Modulation of the Conformational Dynamics of κB DNA by Pirin in the NF-κB Supramolecular Complex.

Biochemistry 2017 09 6;56(37):5002-5010. Epub 2017 Sep 6.

Department of Chemistry and the Center for Diagnostics & Therapeutics, Georgia State University , Atlanta, Georgia 30302-3965, United States.

The molecular basis of gene regulation by Nuclear Factor-κB (NF-κB) transcription factors and their coregulators is not well understood. This family of transcription factors controls a number of essential subcellular processes. Human Pirin, a nonheme iron (Fe) binding protein, has been shown to modulate the binding affinity between p65 homodimeric NF-κB and κB DNA. However, the allosteric effect of the active Fe(III) form of Pirin on the DNA has not been established. Here, we use multiple microsecond-long molecular dynamics simulations to explore the conformational dynamics of the free DNA, the p65-DNA complex, and the Pirin-p65-DNA supramolecular complex. We show that only the Fe(III) form of Pirin enhances the affinity between p65 and the DNA in the Pirin-p65-DNA supramolecular complex, in agreement with experiments. Additionally, the results provide atomistic details of the effect of the active Fe(III) form of Pirin on the DNA upon binding to the p65-DNA complex. In general, unlike the Fe(II) form of Pirin, binding of the Fe(III) form of Pirin to the p65-DNA complex significantly alters both the conformational dynamics of the DNA and the interactions between p65 and the DNA. The results provide atomic level understanding of the modulation of the DNA as a result of a redox-specific Fe(II)/Fe(III) coregulation of NF-κB by Pirin, knowledge that is necessary to fully understand normal and aberrant subcellular processes and the role of a subtle single electron redox process in gene regulation.
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http://dx.doi.org/10.1021/acs.biochem.7b00528DOI Listing
September 2017

Enzyme-Mediated Conversion of Flavin Adenine Dinucleotide (FAD) to 8-Formyl FAD in Formate Oxidase Results in a Modified Cofactor with Enhanced Catalytic Properties.

Biochemistry 2017 07 11;56(29):3800-3807. Epub 2017 Jul 11.

School of Chemical and Biomolecular Engineering, Georgia Institute of Technology , Atlanta, Georgia 30332-0100, United States.

Flavins, including flavin adenine dinucleotide (FAD), are fundamental catalytic cofactors that are responsible for the redox functionality of a diverse set of proteins. Alternatively, modified flavin analogues are rarely found in nature as their incorporation typically results in inactivation of flavoproteins, thus leading to the disruption of important cellular pathways. Here, we report that the fungal flavoenzyme formate oxidase (FOX) catalyzes the slow conversion of noncovalently bound FAD to 8-formyl FAD and that this conversion results in a nearly 10-fold increase in formate oxidase activity. Although the presence of an enzyme-bound 8-formyl FMN has been reported previously as a result of site-directed mutagenesis studies of lactate oxidase, FOX is the first reported case of 8-formyl FAD in a wild-type enzyme. Therefore, the formation of the 8-formyl FAD cofactor in formate oxidase was investigated using steady-state kinetics, site-directed mutagenesis, ultraviolet-visible, circular dichroism, and fluorescence spectroscopy, liquid chromatography with mass spectrometry, and computational analysis. Surprisingly, the results from these studies indicate not only that 8-formyl FAD forms spontaneously and results in the active form of FOX but also that its autocatalytic formation is dependent on a nearby arginine residue, R87. Thus, this work describes a new enzyme cofactor and provides insight into the little-understood mechanism of enzyme-mediated 8α-flavin modifications.
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http://dx.doi.org/10.1021/acs.biochem.7b00335DOI Listing
July 2017

Allosteric Fine-Tuning of the Binding Pocket Dynamics in the ITK SH2 Domain by a Distal Molecular Switch: An Atomistic Perspective.

J Phys Chem B 2017 06 15;121(25):6131-6138. Epub 2017 Jun 15.

Department of Chemistry and ‡Center for Diagnostics and Therapeutics, Georgia State University , Atlanta, Georgia 30302-3965, United States.

Although the regulation of function of proteins by allosteric interactions has been identified in many subcellular processes, molecular switches are also known to induce long-range conformational changes in proteins. A less well understood molecular switch involving cis-trans isomerization of a peptidyl-prolyl bond could induce a conformational change directly to the backbone that is propagated to other parts of the protein. However, these switches are elusive and hard to identify because they are intrinsic to biomolecules that are inherently dynamic. Here, we explore the conformational dynamics and free energy landscape of the SH2 domain of interleukin-2-inducible T-cell or tyrosine kinase (ITK) to fully understand the conformational coupling between the distal cis-trans molecular switch and its binding pocket of the phosphotyrosine motif. We use multiple microsecond-long all-atom molecular dynamics simulations in explicit water for over a total of 60 μs. We show that cis-trans isomerization of the Asn286-Pro287 peptidyl-prolyl bond is directly coupled to the dynamics of the binding pocket of the phosphotyrosine motif, in agreement with previous NMR experiments. Unlike the cis state that is localized and less dynamic in a single free energy basin, the trans state samples two distinct conformations of the binding pocket-one that recognizes the phosphotyrosine motif and the other that is somewhat similar to that of the cis state. The results provide an atomic-level description of a less well understood allosteric regulation by a peptidyl-prolyl cis-trans molecular switch that could aid in the understanding of normal and aberrant subcellular processes and the identification of these elusive molecular switches in other proteins.
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http://dx.doi.org/10.1021/acs.jpcb.7b03470DOI Listing
June 2017

Importance of Loop L1 Dynamics for Substrate Capture and Catalysis in Pseudomonas aeruginosa d-Arginine Dehydrogenase.

Biochemistry 2017 05 2;56(19):2477-2487. Epub 2017 May 2.

Department of Chemistry, ‡Department of Biology, §Center for Diagnostics and Therapeutics, and ∥Center for Biotechnology and Drug Design, Georgia State University , Atlanta, Georgia 30302, United States.

Mobile loops located at the active site entrance in enzymes often participate in conformational changes required to shield the reaction from bulk solvent, to control the access of the substrate to the active site, and to position residues for substrate binding and catalysis. In d-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH), previous crystallographic data suggested that residues 45-47 in the FAD-binding domain and residues 50-56 in the substrate-binding domain in loop L1 could adopt two distinct conformations. In this study, we have used molecular dynamics, kinetics, and fluorescence spectroscopy on the S45A and A46G enzyme variants of PaDADH to investigate the impact of mutations in loop L1 on the catalytic function of the enzyme. Molecular dynamics showed that the mutant enzymes have probabilities of being in open conformations that are higher than that of wild-type PaDADH of loop L1, yielding an increased level of solvent exposure of the active site. In agreement, the flavin fluorescence intensity was ∼2-fold higher in the S45A and A46G enzymes than in wild-type PaDADH, with a 9 nm bathochromic shift of the emission band. In the variant enzymes, the k/K values with d-arginine were ∼13-fold lower than in wild-type PaDADH. Moreover, the pH profiles for the k value with d-arginine showed a hollow, consistent with restricted proton movements in catalysis, and no saturation was achieved with the alternate substrate d-leucine in the reductive half-reaction of the variant enzymes. Taken together, the computational and experimental data are consistent with the dynamics of loop L1 being important for substrate capture and catalysis in PaDADH.
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http://dx.doi.org/10.1021/acs.biochem.7b00098DOI Listing
May 2017

Fe(II)/Fe(III) Redox Process Can Significantly Modulate the Conformational Dynamics and Electrostatics of Pirin in NF-κB Regulation.

ACS Omega 2016 Nov 7;1(5):837-842. Epub 2016 Nov 7.

Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, P.O. Box 3965, Atlanta, Georgia 30302-3965, United States.

Pirin is an iron (Fe)-dependent regulatory protein of nuclear factor κB (NF-κB) transcription factors. Binding studies have suggested that the oxidative state of iron plays a crucial role in modulating the binding of Pirin to NF-κB p65, in turn enhancing the binding of p65 to DNA. The Fe(III) form of Pirin is the active form and binds to NF-κB, whereas the Fe(II) form does not bind to NF-κB. However, the surprising consequence of a single charge perturbation in the functional modulation of NF-κB is not well understood. Here, we use quantum mechanical calculations and microsecond-long molecular dynamics simulations to explore the free-energy landscapes of the Fe(II) and Fe(III) forms of Pirin. We show that the restricted conformational space and electrostatic complementarity of the Fe(III) form of Pirin are crucial for binding and regulation of NF-κB. Our results suggest that a subtle single-electron redox trigger could significantly modulate the conformational dynamics and electrostatics of proteins in subcellular allosteric regulatory processes.
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http://dx.doi.org/10.1021/acsomega.6b00231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640773PMC
November 2016

Coupled Dynamics and Entropic Contribution to the Allosteric Mechanism of Pin1.

J Phys Chem B 2016 08 28;120(33):8405-15. Epub 2016 Apr 28.

Department of Chemistry and the Center for Biotechnology and Drug Design, Georgia State University , Atlanta, Georgia 30302-3965, United States.

Allosteric communication in proteins regulates a plethora of downstream processes in subcellular signaling pathways. Describing the effects of cooperative ligand binding on the atomic level is a key to understanding many regulatory processes involving biomolecules. Here, we use microsecond-long molecular dynamics simulations to investigate the allosteric mechanism of Pin1, a potential therapeutic target and a phosphorylated-Ser/Thr dependent peptidyl-prolyl cis-trans isomerase that regulates several subcellular processes and has been implicated in many diseases, including cancer and Alzheimer's. Experimental studies suggest that the catalytic domain and the noncatalytic WW domain are allosterically coupled; however, an atomic level description of the dynamics associated with the interdomain communication is lacking. We show that binding of the substrate to the WW domain is directly coupled to the dynamics of the catalytic domain, causing rearrangement of the residue-residue contact dynamics from the WW domain to the catalytic domain. The binding affinity of the substrate in the catalytic domain is also enhanced upon binding of the substrate to the WW domain. Modulation of the dynamics of the catalytic domain upon binding of the substrate to the WW domain leads to prepayment of the entropic cost of binding the substrate to the catalytic domain. This study shows that Ile 28 at the interfacial region between the catalytic and WW domains is certainly one of the residues responsible for bridging the communication between the two domains. The results complement previous experiments and provide valuable atomistic insights into the role of dynamics and possible entropic contribution to the allosteric mechanism of proteins.
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http://dx.doi.org/10.1021/acs.jpcb.6b02123DOI Listing
August 2016

Dynamical network of residue-residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation.

Proc Natl Acad Sci U S A 2016 Apr 11;113(17):4735-40. Epub 2016 Apr 11.

Department of Chemistry, Georgia State University, Atlanta, GA 30302;

Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue-residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general.
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http://dx.doi.org/10.1073/pnas.1523573113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855540PMC
April 2016

Role of F357 as an Oxygen Gate in the Oxidative Half-Reaction of Choline Oxidase.

Biochemistry 2016 Mar 3;55(10):1473-84. Epub 2016 Mar 3.

Department of Chemistry, ‡Department of Biology, §Center for Biotechnology and Drug Design, and ∥Center for Diagnostics and Therapeutics, Georgia State University , Atlanta, Georgia 30302, United States.

Choline oxidase from Arthrobacter globiformis catalyzes the oxidation of choline to glycine betaine by using oxygen as an electron acceptor. A partially rate limiting isomerization of the reduced wild-type enzyme during the reaction with oxygen was previously detected using solvent viscosity effects. In this study, we hypothesized that the side chains of M62 and F357, located at the entrance to the active site of choline oxidase, may be related to the slow isomerization detected. We engineered a double-variant enzyme M62A/F357A. The kinetic characterization of the double-variant enzyme showed a lack of the isomerization detected in wild-type choline oxidase, and a lack of saturation with an oxygen concentration as high as 1 mM, while most other kinetic parameters were similar to those of wild-type choline oxidase. The kinetic characterization of the single-variant enzymes established that only the side chain of F357 plays a role in the isomerization of choline oxidase in the oxidative half-reaction. Molecular dynamics studies suggest that the slow isomerization related to F357 is possibly due to the participation of the phenyl ring in a newly proposed gating mechanism for a narrow tunnel, assumed to regulate the access of oxygen to the reduced cofactor.
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http://dx.doi.org/10.1021/acs.biochem.5b01356DOI Listing
March 2016

Conserved Hydration Sites in Pin1 Reveal a Distinctive Water Recognition Motif in Proteins.

J Chem Inf Model 2016 Jan 23;56(1):139-47. Epub 2015 Dec 23.

Departments of Chemistry and ‡Biology and the §Centers for Diagnostics and Therapeutics and ∥Biotechnology and Drug Design, Georgia State University , Atlanta, Georgia 30302-3965, United States.

Structurally conserved water molecules are important for biomolecular stability, flexibility, and function. X-ray crystallographic studies of Pin1 have resolved a number of water molecules around the enzyme, including two highly conserved water molecules within the protein. The functional role of these localized water molecules remains unknown and unexplored. Pin1 catalyzes cis/trans isomerizations of peptidyl prolyl bonds that are preceded by a phosphorylated serine or threonine residue. Pin1 is involved in many subcellular signaling processes and is a potential therapeutic target for the treatment of several life threatening diseases. Here, we investigate the significance of these structurally conserved water molecules in the catalytic domain of Pin1 using molecular dynamics (MD) simulations, free energy calculations, analysis of X-ray crystal structures, and circular dichroism (CD) experiments. MD simulations and free energy calculations suggest the tighter binding water molecule plays a crucial role in maintaining the integrity and stability of a critical hydrogen-bonding network in the active site. The second water molecule is exchangeable with bulk solvent and is found in a distinctive helix-turn-coil motif. Structural bioinformatics analysis of nonredundant X-ray crystallographic protein structures in the Protein Data Bank (PDB) suggest this motif is present in several other proteins and can act as a water site, akin to the calcium EF hand. CD experiments suggest the isolated motif is in a distorted PII conformation and requires the protein environment to fully form the α-helix-turn-coil motif. This study provides valuable insights into the role of hydration in the structural integrity of Pin1 that can be exploited in protein engineering and drug design.
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http://dx.doi.org/10.1021/acs.jcim.5b00560DOI Listing
January 2016

Oscillatory Diffusion and Second-Order Cyclostationarity in Alanine Tripeptide from Molecular Dynamics Simulation.

J Chem Theory Comput 2016 Jan 16;12(1):372-82. Epub 2015 Dec 16.

Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University , Atlanta, Georgia 30302-3965, United States.

Molecular dynamics (MD) simulation distinctly describes motions of biomolecules at high resolution and can potentially be used to explain allosteric mechanism in subcellular processes. Statistical methods are necessary to realize this potential because MD simulations generate a large volume of data and because the analysis is never efficient, objective, or thorough without using appropriate statistical approaches. Tracing the flow of information within a biomolecule requires not only a description of an overall mechanism but also a multiscale statistical description from atomic interactions to the overall mechanism. The foundation of this multiscale description, in general, is a measure of correlation between motions of atoms or residues, as reflected by dynamic cross-correlation, Pearson correlation, or mutual information. However, these correlations can be inadequate because they assume wide sense stationarity, which means that the instantaneous average and correlation of a particular property are time-independent. Consequently, these measures of correlation cannot account for correlation between motions of different frequencies, since frequency implies oscillation and variation over time. Here, we characterize the nonstationarity in the form of pure oscillatory instantaneous variance in the signed dihedral angular accelerations (SDAA) along the main chain of alanine tripeptide in MD simulations by power spectrum, corrected squared envelope spectrum (CSES), and cross-CSES. This oscillation has a physical interpretation of an oscillatory diffusion. The fraction of this oscillation in all motions is as high as about 40% at some frequencies. This shows that oscillatory instantaneous variance exists in the SDAA and that significant correlation may not be accounted for in current correlation analysis. This oscillation is also found to transmit between dihedral angles. These results could have implications in the understanding of the dynamics of biomolecules.
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http://dx.doi.org/10.1021/acs.jctc.5b00876DOI Listing
January 2016

Pushing the Limits of a Molecular Mechanics Force Field To Probe Weak CH···π Interactions in Proteins.

J Chem Theory Comput 2015 Apr;11(4):1854-63

Department of Chemistry and the Center for Biotechnology and Drug Design, Georgia State University , Atlanta, Georgia 30302-3965, United States.

The relationship among biomolecular structure, dynamics, and function is far from being understood, and the role of subtle, weak interactions in stabilizing different conformational states is even less well-known. The cumulative effect of these interactions has broad implications for biomolecular stability and recognition and determines the equilibrium distribution of the ensemble of conformations that are critical for function. Here, we accurately capture the stabilizing effects of weak CH···π interaction using an empirical molecular mechanics force field in excellent agreement with experiments. We show that the side chain of flanking C-terminal aromatic residues preferentially stabilize the cis isomer of the peptidyl-prolyl bond of the protein backbone through this weak interaction. Cis-trans isomerization of peptidyl-prolyl protein bond plays a pivotal role in many cellular processes, including signal transduction, substrate recognition, and many diseases. Although the cis isomer is relatively less stable than the trans isomer, aromatic side chains of neighboring residues can play a significant role in stabilizing the cis relative to the trans isomer. We carry out extensive regular and accelerated molecular dynamics simulations and establish an approach to simulate the pH profile of the cis/trans ratio in order to probe the stabilizing role of the CH···π interaction. The results agree very well with NMR experiments, provide detailed atomistic description of this crucial biomolecular interaction, and underscore the importance of weak stabilizing interactions in protein function.
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http://dx.doi.org/10.1021/ct501036rDOI Listing
April 2015

Enhanced molecular dynamics sampling of drug target conformations.

Biopolymers 2016 Jan;105(1):35-42

Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, 30302-3965.

Computational docking and virtual screening are two main important methods employed in structure-based drug design. Unlike the traditional approach that allows docking of a flexible ligand against a handful of receptor structures, receptor flexibility has now been appreciated and increasingly incorporated in computer-aided docking. Using a diverse set of receptor conformations increases the chances of finding potential drugs and inhibitors. Molecular dynamics (MD) is greatly useful to generate various receptor conformations. However, the diversity of the structures of the receptor, which is usually much larger than the ligand, depends on the sampling efficiency of MD. Enhanced sampling methods based on accelerated molecular dynamics (aMD) can alleviate the sampling limitation of conventional MD and aid in representation of the phase space to a much greater extent. RaMD-db, a variant of aMD that applies boost potential to the rotatable dihedrals and non-bonded diffusive degrees of freedom has been proven to reproduce the equilibrium properties more accurately and efficiently than aMD. Here, we discuss recent advances in the aMD methodology and review the applicability of RaMD-db as an enhanced sampling method. RaMD-db is shown to be able to generate a broad distribution of structures of a drug target, Cyclophilin A. These structures that have never been observed previously in very long conventional MD can be further used for structure-based computer-aided drug discovery, and docking, and thus, in the identification and design of potential novel inhibitors.
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http://dx.doi.org/10.1002/bip.22740DOI Listing
January 2016

An Iron Reservoir to the Catalytic Metal: THE RUBREDOXIN IRON IN AN EXTRADIOL DIOXYGENASE.

J Biol Chem 2015 Jun 27;290(25):15621-15634. Epub 2015 Apr 27.

Department of Chemistry, Georgia State University, Atlanta, Georgia 30303; Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30303; Molecular Basis of Disease Program, Georgia State University, Atlanta, Georgia 30303. Electronic address:

The rubredoxin motif is present in over 74,000 protein sequences and 2,000 structures, but few have known functions. A secondary, non-catalytic, rubredoxin-like iron site is conserved in 3-hydroxyanthranilate 3,4-dioxygenase (HAO), from single cellular sources but not multicellular sources. Through the population of the two metal binding sites with various metals in bacterial HAO, the structural and functional relationship of the rubredoxin-like site was investigated using kinetic, spectroscopic, crystallographic, and computational approaches. It is shown that the first metal presented preferentially binds to the catalytic site rather than the rubredoxin-like site, which selectively binds iron when the catalytic site is occupied. Furthermore, an iron ion bound to the rubredoxin-like site is readily delivered to an empty catalytic site of metal-free HAO via an intermolecular transfer mechanism. Through the use of metal analysis and catalytic activity measurements, we show that a downstream metabolic intermediate can selectively remove the catalytic iron. As the prokaryotic HAO is often crucial for cell survival, there is a need for ensuring its activity. These results suggest that the rubredoxin-like site is a possible auxiliary iron source to the catalytic center when it is lost during catalysis in a pathway with metabolic intermediates of metal-chelating properties. A spare tire concept is proposed based on this biochemical study, and this concept opens up a potentially new functional paradigm for iron-sulfur centers in iron-dependent enzymes as transient iron binding and shuttling sites to ensure full metal loading of the catalytic site.
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http://dx.doi.org/10.1074/jbc.M115.650259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505474PMC
June 2015

Design, synthesis and biological evaluation of di-substituted noscapine analogs as potent and microtubule-targeted anticancer agents.

Bioorg Med Chem Lett 2015 31;25(10):2133-40. Epub 2015 Mar 31.

Department of Biology, Georgia State University, Atlanta, GA 30303, USA. Electronic address:

Noscapine is an opium-derived kinder-gentler microtubule-modulating drug, currently in Phase I/II clinical trials for cancer chemotherapy. Here, we report the synthesis of four more potent di-substituted brominated derivatives of noscapine, 9-Br-7-OH-NOS (2), 9-Br-7-OCONHEt-NOS (3), 9-Br-7-OCONHBn-NOS (4), and 9-Br-7-OAc-NOS (5) and their chemotherapeutic efficacy on PC-3 and MDA-MB-231 cells. The four derivatives were observed to have higher tubulin binding activity than noscapine and significantly affect tubulin polymerization. The equilibrium dissociation constant (KD) for the interaction between tubulin and 2, 3, 4, 5 was found to be, 55±6μM, 44±6μM, 26±3μM, and 21±1μM respectively, which is comparable to parent analog. The effects of these di-substituted noscapine analogs on cell cycle parameters indicate that the cells enter a quiescent phase without undergoing further cell division. The varying biological activity of these analogs and bulk of substituent at position-7 of the benzofuranone ring system of the parent molecule was rationalized utilizing predictive in silico molecular modeling. Furthermore, the immunoblot analysis of protein lysates from cells treated with 4 and 5, revealed the induction of apoptosis and down-regulation of survivin levels. This result was further supported by the enhanced activity of caspase-3/7 enzymes in treated samples compared to the controls. Hence, these compounds showed a great potential for studying microtubule-mediated processes and as chemotherapeutic agents for the management of human cancers.
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http://dx.doi.org/10.1016/j.bmcl.2015.03.076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4465564PMC
January 2016

Dynamical role of phosphorylation on serine/threonine-proline Pin1 substrates from constant force molecular dynamics simulations.

J Chem Phys 2015 Feb;142(7):075102

Department of Chemistry, Georgia State University, Atlanta, Georgia 30302-3965, USA.

Cis-trans isomerization of peptidyl-prolyl bonds of the protein backbone plays an important role in numerous biological processes. Cis-trans isomerization can be the rate-limiting step due its extremely slow dynamics, compared to the millisecond time scale of many processes, and is catalyzed by a widely studied family of peptidyl-prolyl cis-trans isomerase enzymes. Also, mechanical forces along the peptide chain can speed up the rate of isomerization, resulting in "mechanical catalysis," and have been used to study peptidyl-prolyl cis-trans isomerization and other mechanical properties of proteins. Here, we use constant force molecular dynamics simulations to study the dynamical effects of phosphorylation on serine/threonine-proline protein motifs that are involved in the function of many proteins and have been implicated in many aberrant biological processes. We show that the rate of cis-trans isomerization is slowed down by phosphorylation, in excellent agreement with experiments. We use a well-grounded theory to describe the force dependent rate of isomerization. The calculated rates at zero force are also in excellent agreement with experimentally measured rates, providing additional validation of the models and force field parameters. Our results suggest that the slowdown in the rate upon phosphorylation is mainly due to an increase in the friction along the peptidyl-prolyl bond angle during isomerization. Our results provide a microscopic description of the dynamical effects of post-translational phosphorylation on cis-trans isomerization and insights into the properties of proteins under tension.
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http://dx.doi.org/10.1063/1.4907884DOI Listing
February 2015

Computational perspective and evaluation of plausible catalytic mechanisms of peptidyl-prolyl cis-trans isomerases.

Biochim Biophys Acta 2015 Oct 10;1850(10):1994-2004. Epub 2015 Jan 10.

Department of Chemistry and the Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30302-3965, USA. Electronic address:

Background: Peptidyl prolyl cis-trans isomerization of the protein backbone is involved in the regulation of many biological processes. Cis-trans isomerization is notoriously slow and is catalyzed by a family of cis-trans peptidyl prolyl isomerases (PPIases) that have been implicated in many diseases. A general consensus on how these enzymes speed up prolyl isomerization has not been reached after decades of both experimental and computational studies.

Scope Of Review: Computational studies carried out to understand the catalytic mechanism of the prototypical FK506 binding protein 12, Cyclophilin A and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) are reviewed. A summary and an evaluation of the implications of the proposed mechanisms from computational studies are presented.

Major Conclusions: The analysis of computational studies and evaluation of the proposed mechanisms provide a general consensus and a better understanding of PPIase catalysis. The speedup of the rate of peptidyl-prolyl isomerization by PPIases can be best described by a catalytic mechanism in which the substrate in transition state configuration is stabilized. The enzymes preferentially bind the transition state configuration of the substrate relative to the cis conformation, which in most cases is bound better than the trans conformation of the substrate. Stabilization of the transition state configuration of the substrate leads to a lower free energy barrier and a faster rate of isomerization when compared to the uncatalyzed isomerization reaction.

General Significance: Fully understanding the catalytic mechanism of PPIases has broad implications for drug design, elucidation of the molecular basis of many diseases, protein engineering, and enzyme catalysis in general. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.
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http://dx.doi.org/10.1016/j.bbagen.2014.12.023DOI Listing
October 2015

Loss of intramolecular electrostatic interactions and limited conformational ensemble may promote self-association of cis-tau peptide.

Proteins 2015 Mar 21;83(3):436-44. Epub 2015 Jan 21.

Department of Chemistry and the Center for Biotechnology and Drug Design, Georgia State University, Atlanta, Georgia, 30302-4098.

Self-association of proteins can be triggered by a change in the distribution of the conformational ensemble. Posttranslational modification, such as phosphorylation, can induce a shift in the ensemble of conformations. In the brain of Alzheimer's disease patients, the formation of intra-cellular neurofibrillary tangles deposition is a result of self-aggregation of hyper-phosphorylated tau protein. Biochemical and NMR studies suggest that the cis peptidyl prolyl conformation of a phosphorylated threonine-proline motif in the tau protein renders tau more prone to aggregation than the trans isomer. However, little is known about the role of peptidyl prolyl cis/trans isomerization in tau aggregation. Here, we show that intra-molecular electrostatic interactions are better formed in the trans isomer. We explore the conformational landscape of the tau segment containing the phosphorylated-Thr(231)-Pro(232) motif using accelerated molecular dynamics and show that intra-molecular electrostatic interactions are coupled to the isomeric state of the peptidyl prolyl bond. Our results suggest that the loss of intra-molecular interactions and the more restricted conformational ensemble of the cis isomer could favor self-aggregation. The results are consistent with experiments, providing valuable complementary atomistic insights and a hypothetical model for isomer specific aggregation of the tau protein.
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http://dx.doi.org/10.1002/prot.24740DOI Listing
March 2015