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positive allosteric modulators by interacting with the Ca2þ binding site. In ad-dition, extracellular Ca2þ differentially reduces the inhibition of the receptor byantagonists and negative allosteric modulators. Our studies open a new avenuefor modulating drug effects and developing novel drugs against neurodegener-ative diseases.

2064-Pos Board B83Improved Quantitative Modeling of Ligand-Activated MacromolecularReceptors using Conditional BindingThomas R. Middendorf, Keegan E. Hines, Jennifer N. Greeson-Bernier,Richard W. Aldrich.The University of Texas at Austin, Austin, TX, USA.Experimental investigations of ligand-activated receptors are often based onbinding measurements that detect the total fraction of occupied binding sitesat various ligand concentrations. We use calmodulin, an important calcium sen-sor protein, as a test system to document the uncertainties in model parametersestimated by fitting various models to total binding data. Using nonlinearleast-squaresmethods, we obtain excellent fits (<1%RMSerror) to synthetic to-tal binding data with the same characteristics as the published binding data usingparameter sets with binding affinities varying by over four orders of magnitudefor each site. This result identifies a significant obstacle blocking progress to-ward the goal of developing accurate, quantitativemodels of receptor activation.The use of noiseless data in our analysis suggests that the large uncertainties inthe estimated parameters are not a problem of data quality, but rather reflect anintrinsic limitation of total binding data. Using analytical matrix algebra tech-niques and numerical simulations, we discover a fundamental relationship be-tween the mathematical structure of the equations describing various types ofbinding data and the number and type of parameters that may be determinedaccurately from regression analysis of that data. Ideas based on Boolean logicalprinciples are used to design a new type of binding experiment that significantlyimproves upon total binding data in its power to constrain physically realisticmodels of receptor activation. These experiments, which we call conditionalbinding, report on the simultaneous occupancies of two different receptor bind-ing sites. Our approach is general and the conclusions are applicable to the manymacromolecular systems that are activated or modulated by ligand binding.

2065-Pos Board B84Modeling Complex between FBA and TIM: Functional Motions of FBAand TIM are Preserved in their ComplexAtaur R. Katebi, Robert L. Jernigan.Iowa State University, Ames, IA, USA.Fructuose bisphostate aldolase (FBA) and triosephosphate isomerase (TIM) arethe fourth and fifth enzymes in the glycolysis pathway and they are known tobind. FBA cleaves the six-carbon fructose 1, 6-bisphosphate into two three-carbon components – dihydroxyacetone phosphate (DHAP) and glyceralde-hyde 3-phosphate (GAP). TIM converts DHAP into GAP, a substrate for thesubsequent synthetic step. These two alpha/beta barrel proteins have high struc-tural similarity – with a core RMSD 4.8A. These two enzymes have low activ-ity in the monomeric form with the functionally active structures present inhigher oligomeric states. By applying Elastic Network Model, we find themodes of motions that are functionally important for these proteins. We buildmodels for the complex between these two proteins to investigate their impor-tant motions in their complexes as well as for their different oligomeric statesincluding those that are different in different species to learn their importantmodes of motions for different functionalities. For each protein, by multiple se-quence alignment across the species, we predict the coevolving residues andcluster these residues along the structure. We build the information transferpathways from the important interface residues to the catalytic residues.Change in these pathways in different oligomeric states maybe be related tothe change of motions in the catalytic region in different oligomeric states.We use this knowledge about the changes in motions and the information trans-fer pathways within the structure of these interacting proteins as constraints forselecting the computational docked models of complexes between these twoproteins to preserve their functional motions.

2066-Pos Board B85How Can a Ligand be a Positive and Negative Allosteric Effector for theSame Protein?Hesam N. Motlagh1, Vincent J. Hilser1,2.1T.C. Jenkins Department of Biophysics, Johns Hopkins University,Baltimore, MD, USA, 2Department of Biology, Johns Hopkins University,Baltimore, MD, USA.For several transcription factors, the same ligand can act as a positive and neg-ative allosteric effector in a context dependent manner. The structural andmolecular bases of such effects are unknown. Here it is shown that modulationof conformational fluctuations within allosteric systems can be used as a mech-

anism to tune protein ensembles such that a given ligand can act as both a pos-itive and negative allosteric effector. Importantly, this mechanism can berobustly encoded in the ensemble, and does not require that the interactions be-tween the ligand and the protein differ when it is acting either as a positive ornegative effector. Instead, the effect is due to the relative probabilities of statesprior to the addition of the ligand and is encoded in the thermodynamic cou-pling architectures between protein domains. The ensemble view of allosterythat is illuminated by these studies suggests that rather than being seen asswitches with fixed responses to allosteric activation, ensembles can evolveto be ‘‘functionally pluripotent’’, with the capacity to up or down regulate ac-tivity in response to a stimulus. This result not only helps to explain the prev-alence of intrinsic disorder in transcription factors and other cell signalingproteins, it provides important insights about the energetic ground rules govern-ing site-to-site communication in all allosteric systems.Relevant citationMotlagh HN, Hilser VJ: Agonism/antagonism switching in allosteric ensem-bles. Proceedings of the National Academy of Sciences of the United States ofAmerica (2012) 109(11):4134-4139.

2067-Pos Board B86Allosteric Modulation of WT and H1047R Mutant PI3Ka Investigated byMD SimulationsParaskevi Gkeka, Thomas Evangelidis, Zoe Cournia.Biomedical Research Foundation, Academy of Athens, Athens, Greece.Kinases are one of the most intensively pursued drug targets investigated forthe treatment of cancer. Kinase inhibitors usually target the ATP bindingsite; however, the similarity of this site across many kinases often results tonon-selectivity. Therefore, allosteric modulation of kinases is of paramount im-portance as it may result in increased selectivity; many highly selective inhib-itors have been reported to inhibit kinases by allosteric mechanisms. PI3Ka isthe most frequently mutated kinase in human cancers with one of its most com-mon mutations being a histidine changed to arginine in exon 20 (H1047R).PIK-108, a known PI3Ka inhibitor, was recently found to occupy an allostericbinding pocket in the wild type (WT) murine protein, close to the H1047R mu-tation. In order to assess the interactions, stability and allosteric effects of theinhibitor on PI3Ka, MD simulations in aqueous solution were performed for130ns for the WT human, WT murine, and H1047R human mutant proteinswith PIK-108 placed in both catalytic and allosteric sites. Interestingly, PIK-108 remained stable in both sites in all three variants. While in both the WThuman and murine forms, the same ligand:protein interaction motifs are ob-served in the allosteric and catalytic pockets, these interactions are markedlydifferent in the mutant form. In the mutant form, the allosteric pocket opensup and forms an altered hydrogen bond network with the ligand compared totheWT. Additionally, in the catalytic pocket, significant differences are evidentin the interaction network formed between the inhibitor, P-loop, and the activa-tion loop between the two protein forms. Overall, the ligand:protein interactiondifferences between the mutant andWT PI3Ka proteins observed in the presentstudy provide a rich basis for the design of mutant-specific PI3Ka inhibitors.

2068-Pos Board B87Hetero Interaction with an Amino Acid Globally Enhances CooperativeActivation of CaSR in Response to Extracellular SignalingChen Zhang1, You Zhuo1, Jie Feng1, Heather Strachan2, Nagaraju Mulpuri1,Donald Hamelberg1, Kelley Moremen2, Edward M. Brown3, Jenny J. Yang1.1Georgia State University, Atlanta, GA, USA, 2University of Gerogia,Athens, GA, USA, 3Endocrine-Hypertension Division, Department ofMedicine, Brigham and Women’s Hospital, Boston, MA, USA.Calcium sensing receptor (CaSR), along other members of the family C Gprotein-coupled receptors (GPCRs), play very important roles in respondingto changes in the extracellular calcium concentrations and in circulating levelsof amino acids and integrating these extracellular signals into alterations in in-tracellular signaling pathways. However, detailed structure properties of theCaSR which are necessary to characterize the mechanism of its physiologicalfunction are still unrevealed. We have reported several potential calcium-binding sites located within the CaSR’s extracellular domain using ourdeveloped computational algorithms. In the present study, we first report thedifferential effects of several disease-related mutations located at the predictedcalcium binding sites on the inhibition and activation of intracellular calciumresponses using single cell imaging. Mutating to different residues at two lo-cations near the hinge region of the ECD could lead to either significantlylose of function of the receptor or gain of function (switch function mutations).Amino acid binding results in differential rescue effect in altering intracellularcalcium responses, especially calcium oscillations.We have further probe theeffect of mutation and amino acid binding on the correlation motion, cooper-ativity, and synergistic activation using mammalian expressed and purified

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extracellular domain (ECD) of CaSR with various spectroscopic methods in-cluding the tryptophan fluorescence titration, circular dichroism (CD) and nu-clear magnetic resonance (NMR) as well as molecular dynamic simulation.These results provide important implications for our understanding of howthe CaSR integrates information about these two completely different classesof agonists–an inorganic divalent cation, and another hand, a nutrient– how thereceptor senses these agonists in healthy and diseased states.

2069-Pos Board B88Synthetic Demethylwedelolactones Derivatives Inhibit Invasive Growth ofMda-Mb-231 Breast Cancer Cells In Vitro and In VivoYean-Jang Lee1, Tsui-Hwa Tseng2.1Changhua University of Education, Changhua, Taiwan, 2Chung ShanMedical University, Taichung, Taiwan.Combretastatins, which are an important group of anticancer drugs,were iso-lated by Pettit et al. from the African tree Combretum caffrum in 1989. Addi-tionally, Liang et al. have reported that ten coumestans were isolated fromtheroots of Hedysarum multijugum, which is a plant in Hedysarum Linn. of thefamily Leguminosae used as a folk herbal drug in northwest China. Coumestanscomprise a class of naturally occurring products with a variety of biological ac-tivities including phytoestrogenic, antibacterial, antifungal, antimyotoxic, andphytoalexine effects. The anticancer properties of demethylwedelolactone(DWEL) and wedelolactone (WEL), which are naturally occurring coumestans,have not been well characterized. Due to their biological activities, the synthe-sis of DWEL is achieved in which the longest linear sequence is only eightsteps in 38% overall yield from commercially available phloroglucinol. Furth-more, the molecular model was examined the interactions of proteins and li-gands as well. Finally, in this study, we investigated the anti-invasive effectsof synthetic WEL and DWEL on human MDA-MB-231 breast cancer cells.We found that WEL and DWEL inhibited the anchorage-independent growthand also suppressed cell motility and cell invasion of MDA-MB-231 cells. Inaddition, WEL and DWEL reduced the activity and expression of matrix metal-loproteinases (MMPs) involved in blocking the IkB-a/NFkB and MEK/ERKsignaling pathways in MDA-MB-231 cells. Furthermore, DWEL suppressedthe metastasis and lung colonization of the tumor cells in the nude mice. Alto-gether, these data suggest that DWEL derivatives exert anti-invasive growth ef-fect on breast cancer cells.

2070-Pos Board B89Molecular Dynamics of DOT1L and Modeling of EZH2Javier Pineda1, Jason Marineau2, Guille Estiu1, James Bradner2.1University of Notre Dame, Notre Dame, IN, USA, 2Dana Farber CancerInstitute, Boston, MA, USA.Histone methyltransferases are enzymes that modify histone proteins via meth-ylation of lysine or arginine residues. These epigenetic modifiers, such asDOT1L and EZH2, have been found to play important roles in leukemogenicprocesses.Crystallographic and docking methods studied interactions within the DOT1Lbinding site. Crystal structures of DOT1L also demonstrated variance in thebinding mode of ligands, possibly due to rearrangement in the DOT1L bindingsite. We investigated this possibility by molecular dynamics (MD) simulationsand confirmed significant rearrangement of the substrate binding and activationloops of DOT1L upon binding to competitive inhibitors. The druggability andvolume fluctuations of the binding pocket over time were determined.The EZH2 methyltransferase is of interest due to its aberrant activity in manycancers. Because there is no published crystal structure of EZH2, we used ho-mology modeling with homologous proteins as templates. This model provides

structural information regarding the bindingmodes of the S-adenosyl methionine (SAM)cofactor and potential inhibitors of EZH2.Through the synergistic combination ofin silico drug design, organic synthesisand biochemical assays, our modeling ef-forts for DOT1L and EZH2 will guide thechemical syntheses of potent and selectiveinhibitors of these enzymes.

2071-Pos Board B90T Cell Receptor Specificity, Cross-Reactivity, and MHC Restriction areInextricably Linked and Result from Cooperative Engagement of theComposite Peptide/MHC SurfaceBrian M. Baker, Kurt H. Piepenbrink, Sydney J. Blevins.University of Notre Dame, Notre Dame, IN, USA.T cell receptors (TCRs) recognize peptides bound and presented by major his-tocompatibility complex (MHC) proteins using multiple complementarity de-termining region (CDR) loops. While numerous analyses have illuminated

structural and biophysical aspects of TCR recognition, how the distributionof binding free energy within TCR-pMHC interfaces promotes unique TCRrecognition features, including MHC restriction and the apparent dichotomyof specificity and cross-reactivity, remains unclear. Utilizing double mutant cy-cles, here we performed a comprehensive structural and thermodynamic decon-struction of the interaction between the A6 TCR and the Tax peptide presentedby the class I MHC HLA-A2. In contrast with general expectations, we ob-served that the central regions of the peptide and its interactions with the hyper-variable CDR3 loops contribute little to specificity, instead promoting bydynamic effects the cross-reactivity that is a hallmark of TCR recognition.We also observed that TCR restriction towards HLA-A2 results from notfrom conserved interactions with the germline loops, but instead from stronginteractions with the hypervariable CDR3 loop of the a chain. Formation ofthese latter interactions, however, is dependent upon the unique structural prop-erties of the peptide, highlighting that TCR specificity towards peptide andMHC can emerge from the need to engage a unique, composite peptide/MHC interface with tightly coupled structural properties.

2072-Pos Board B91Dissecting Signal Control in the Multidrug Sensor, BMRRHerschel Wade.Johns Hopkins University SOM, Baltimore, MD, USA.Multidrug (MD) (or xenobiotic) e,ux actively removes cytotoxic chemicalsfrom the interiors of normal-functioning cells. However, high levels ofe,ux can render drug-targeted cells resistant to a broad-range of therapeuticagents, including those to which cells were never exposed. Key multidrugresistance (MDR) contributors include allosteric e,ux pumps, gene regulatorsand other sensory systems that mediate the detection and extrusion of diversedrugs from cellular environments. To date, MDR functions remain only par-tially understood. Ligand-dependent allosteric control in BmrR has been quan-titatively addressed using in vitro transcription experiments, dose-responsecurves and thermodynamic models that relate the observed transcriptionalresponses to ligand binding and changes in BmrR conformation. Preliminaryresults indicate that allosteric control in BmrR is sensitive to both energeticand structural aspects of ligand recognition. Importantly, increased coopera-tivity in signal control relative to recognition implicates a major allostericrole for the RNA polymerase.

2073-Pos Board B92Fragment-Based Approach Identifies a Novel Inhibitory Site on DHPSDalia Hammoudeh, Mi-Kyung Yun, Ariele Viacava Follis, Ying Zhao,Brett Waddell, Richard E. Lee, Stephen White.St. Jude Children’s Research Hospital, Memphis, TN, USA.Dihydropteroate synthase (DHPS) is an essential enzyme in the bacterial folatebiosynthetic pathway. It catalyzes the condensation of 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPP) with p-aminobenzoic acid (pABA) toform the folate intermediate, 7,8-dihydropteroate. DHPS is the target of thesulfonamide class of antibiotics. Widespread resistance to sulfonamides has de-creased their clinical use. The active site of DHPS is comprised of three sub-sites: the structured ‘‘pterin’’ site, the flexible pABA site, and the anion bindingpocket. Most of the drug resistant mutations have been mapped to the pABAsub-site of DHPS. using an NMR ligand-based screening approach, a numberof structurally unrelated fragment-like small molecules have been identifiedthat inhibit the enzymatic activity of DHPS from Bacillus anthracis (Ba), Yer-sinia pestis (Yp), and Staphylococcus aureus (Sa). Fragment hits were shown totarget the three sub-pockets of the active site and a novel site distinct from theactive site. The latter site potentially inhibits via an allosteric mechanism andhas been characterized by high resolution X-ray crystallography.We screened the Maybridge� Fragment library of 1,100 fragments using waterligand observed gradient spectroscopy (waterLOGSY) as a primary screenwhich resulted in a hit rate of 6.7 %. Of the 74 hits, 25 were shown to inhibitDHPS activity using two independent enzyme activity assays. A total of eightcompounds inhibited the activity of DHPS from three different species (Ba, Yp,and Sa). In addition to screening for inhibition, the fragment hits were validatedusing a number of biophysical techniques including 2D NMR, SPR, competi-tion waterLOGSY, and X-ray crystallography. Herein, we focus on two frag-ment hits for which high-resolution x-ray crystal structures are available

2074-Pos Board B93Interactomics of BlebbistatinMiklos Kepiro, Boglarka H. Varkuti, Gyorgy Hegyi, Mihaly Kovacs,Andras Malnasi-Csizmadia.Eotvos University, Budapest, Hungary.Photoreactive molecules like aryl azides play important role in life sciences,as practical tools to achieve precisely timed covalent cross-links between li-gands and targets. Since the azido group is small, stable and biologically inert,