Session PA. There are 39 abstracts in this session.

Session: Biomolecules in Solution, poster number: 001
Comparison of DOSY-NMR and Dynamic Light Scattering for Measuring Diffusion Coefficients of Formulated Insulin: where the difference coming from
Sharadrao Patil; David Keire; Kang Chen
US FDA, Silver Spring, MD
Particle size distribution, is a critical quality attribute of liquid based drug products. The methods of Dynamic Light Scattering (DLS) and Diffusion Ordered SpectroscopY (DOSY) NMR can be used to measure diffusion and derive the hydrodynamic radius. Here, DLS and DOSY-NMR methods are shown to be orthogonal and yield identical results for protein standard, ribonuclease A. In contrast, different diffusion coefficients were observed for insulin drugs. The heterogeneity nature of insulin oligomers in formulation caused DOSY-NMR and DLS results to differ from each other. Each method is sensitive to a different range of particle size and complements each other; and, the application of both methods will increase the assurance of complex drug quality in similarity comparison.

Session: Biomolecules in Solution, poster number: 002
Structural Characterization of the Coiled-Coil Self-Association Domain of Swallow
Nikolaus M. Loening1; Elisar Barbar2
1Lewis & Clark College, Portland, OR; 2Oregon State University, Corvallis, OR
In Drosophila oocytes, the proper localization of several mRNAs involved in development depend on Swallow, a 62 kDA multidomain protein. Swallow also interacts with dynein light chain (LC8), a binding event that drives Swallow to form homodimers via a self-association domain in the center of the protein. Based on sequence analysis of Swallow, this domain is predicted to form a coiled-coil upon dimerization. Here, we detail the use of solution-state NMR spectroscopy to characterize the structure of this domain, work that has confirmed the formation of a parallel coiled-coil structure. This is the first reported structure of a coiled-coil domain from an LC8 binding partner and, to our knowledge, the largest coiled-coil structure determined by NMR to date.

Session: Biomolecules in Solution, poster number: 003
Resonance Assignment, Interdomain Orientation and Dynamics from Paramagnetic Effects in Robo1-Ig1-2
Robert Williams1, 2; Alexander Eletsky2; Yuejie Zhao1; Jeong Yeh Yang2; Pradeep Prabhakar2; Kelley Moremen2; I. Jonathan Amster1; James Prestegard1, 2
1Department of Chemistry, University of Georgia, Athens, GA; 2Complex Carbohydrate Research Center, Athens, GA
Robo1 is a cell surface receptor involved in angiogenesis, and midline crossing. Its function involves formation of a ternary complex with Slit2 and heparan sulfate (HS). Recent ion mobility-mass spectrometry data suggest an HS-induced conformation change. Solution NMR measurements are being undertaken to define the HS-bound geometry. Previous resonance assignments for lysine amide groups on a sparsely labeled Robo1-Ig1-2 construct with a lanthanide binding peptide insert have been extended to include those for valine amide and methyl groups. We anticipate that the additional sites and improved sensitivity of methyl-TROSY observation of valine methyl groups will allow the use of pseudocontact shifts (PCS) and paramagnetic relaxation enhancements from the inserted lanthanide binding loop in the determination of this geometry.

Session: Biomolecules in Solution, poster number: 004
Enzymatic methyl transfer catalysed by human S-catechol O-methyltransferase probed by heteronuclear NMR and crystallography
Sylwia Czarnota
Mrs, Manchester, United Kingdom
Our aim is to probe enzymatic methyl transfer (mechanism and structural biology) of human S-catechol O-methyltransferase (COMT) using nuclear magnetic resonance spectroscopy: backbone resonance assignments, high pressure and relaxation experiments. Conditions for protein expression, purification and NMR experiments were optimized. Backbone assignments of two different COMT complexes were completed and crystal structures of COMT complexed with SAM (S-adenosyl-L-methionine) or sinefungin, Mg2+ and inhibitor dinitrocatechol were solved to high resolution. Preliminary high-pressure data recorded between 1-2500 bars allows the identification of regions of local flexibility and rigidity. Relaxation measurements give information about ps-ns dynamics of the backbone. Comparison of the two complexes shows subtle rearrangement of the active site and repositioning of the catechol that is likely to be catalytically relevant.

Session: Biomolecules in Solution, poster number: 005
SHP-2/PD-1 interaction reveals unexpected features of SH2-domain functionality
Michelangelo Marasco1; Anna Berteotti2; Justyna Sikorska3; John Kirkpatrick1; Pablo Rios4; Joern Krausze1; Maja Koehn4; Teresa Carlomagno1, 3
1BMWZ, Hannover, DE; 2EMBL, Heidelberg, DE; 3HZI, Braunschweig, DE; 4BIOSS, Freiburg, DE
PD-1 has been intensely investigated since the discovery that PD-1/PD-L1 blockers are valuable anticancer agents. Its downstream effector, protein tyrosine phosphatase SHP-2, interacts with two conserved phosphotyrosyl motifs on PD-1, termed the Immune-Tyrosine Switch Motif (ITSM) and Immune-Tyrosine Inhibitory Motif (ITIM), and undergoes a structural rearrangement that stimulates its enzymatic activity. We have investigated this system by combining NMR and other structural biology approaches, revealing an intricate network of binding events, concerted motions and conformational rearrangements. In addition to providing structural and dynamical cues for the development of novel PD-1-pathway inhibitors, our data shed some light on how the physico-chemical characteristics of the binding partners allow to achieve optimal specificity and fine tunability in a signaling pathway.

Session: Biomolecules in Solution, poster number: 006
A Selective Double Resonance Technique for the Removal of Interfering Excipient Signals from 2D 1H-13C Methyl Spectra of Biopharmaceutical Products
Luke Arbogast; Frank Delaglio; John P. Marino
NIST-IBBR, Rockville, MD
Biotherapeutics are a clinically important, rapidly growing class of drugs. Their development and manufacture presents many challenges; in particular, characterization of higher order structure (HOS) is of critical importance, since misfolding or aggregation of biotherapeutics can lead to loss of efficacy or elicit immune responses. 2D 1H-13C-methyl NMR has proven to be a robust method to characterize biotherapeutic HOS at natural isotopic abundance. However, there remains a hindrance to recording 1H-13C-methyl spectra on biotherapeutics when formulation component resonances interfere with the analyte signal of interest. Here we detail modifications to the HSQC experiment including introducing a novel selective double resonance pulse scheme that allows for the attenuation of signals arising from formulation components while maintaining the sensitivity of the biotherapeutic.

Session: Biomolecules in Solution, poster number: 007
Pure Shift NMR with Water Suppression on A6DNA and R3230 Cancer Cells
Anthony A Ribeiro1; Andrea Sefler2
1Duke University, Durham, NC; 2Duke University/consultant, Cary, NC
Pure Shift (PS) (Zanger-Sterk, Psyche) NMR experiments simplify 1H spectra by yielding a homodecoupled spectrum of singlets at the true chemical shift of a multiplet. Here we report 1D and 2D PS spectra obtained with water suppression for A6DNA (24 bp) and R3230 cancer cells  A6DNA shows transient A*T and G*C Hoogsteen base pairs while retaining Watson–Crick base-pairing in a double helix. PS spectra simplify methyl, sugar and base signals. R3230 cancer cells allow study of lactate in tumors. High lactate in tumors is associated with poor survival in cancer patients. Lactate is taken up and converted to alanine and glutamate by R3230 cancer cells. PS spectra allow detection of lactate and alanine in a background of media signals.

Session: Biomolecules in Solution, poster number: 008
Novel Design of Triply-Compensated Pulses for Ultra High-Field Spectrometers
Manu Veliparambil Subrahmanian; Gianluigi Veglia
University of Minnesota, Minneapolis, MN
Using a novel optimization method – RF pulse design by Evolutionary Algorithm (REvolutionary or R pulses), we propose a new family of triply compensated RF pulses that cover larger bandwidth with superior compensation for experimental imperfections. In addition, heteronuclear dipolar coupling and weak J couplings are refocused during the execution of the R pulses. Here, we show the application of this pulse to the most common pulse sequences used for solution state NMR. Specifically, in the case of Nitrogen and Carbon HSQC experiments. We show a sensitivity improvement up to 100%. We anticipate that triply-compensated pulses will be crucial for improving the performance of multidimensional and multinuclear pulse sequences at ultra-high fields.

Session: Biomolecules in Solution, poster number: 009
Structural Study of Noncoding RNAs via NMR spectroscopy 
Hyeyeon Nam1; Andrew P. Longhini1; Regan M. LeBlanc2; Stuart LeGrice2; Theodore. K Dayie1
1University of Maryland, College park, MD; 2National Cancer Institude, Frederick, MD
Non-coding RNAs can undergo conformational transitions to regulate gene expression. Although X-ray crystallographic studies of such RNAs provide structural insight, NMR spectroscopy is the only high resolution technique capable of probing the highly dynamic state. However, NMR studies can be limited due to the fact that RNA incorporates only four bases leading to spectral crowding. Therefore, it is crucial to develop methods that can mitigate these problems for successful NMR studies. Here we present site-selective labeling strategies and NMR methodologies to study the structure and dynamics of non-coding RNAs. The combination of our site-selective labels with customized NMR experiments alleviates problems of rapid relaxation and spectral crowding, thereby allowing us to investigate the structure and dynamics of relatively large RNAs.

Session: Biomolecules in Solution, poster number: 010
Structural Characterization and Binding Studies of Polar Organizing Protein Z from Caulobacter crescentus using NMR Spectroscopy 
Christopher Nordyke1; Haibi Wang2; Shelby Follett2; Grant Bowman2; Krisztina Varga1
1University of New Hampshire, Dover, NH; 2University of Wyoming, Laramie, WY
Polar organizing protein Z (PopZ) is an intrinsically disordered protein from the bacterium Caulobacter crescentus. PopZ self-assembles into 3D polymeric superstructures and is necessary for the localization of other proteins at cell poles. PopZ has been shown to interact with at least eight different proteins. Current NMR work focuses on probing the binding of PopZ and its partners as well as characterizing potential structural changes of PopZ upon binding.

Session: Biomolecules in Solution, poster number: 011
NMR Studies of Structure and Function of AIPL1 FKBP Domain
Liping Yu
University of Iowa, Iowa City, IA
Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a specialized chaperone of phosphodiesterase 6 (PDE6), a key effector enzyme in the phototransduction cascade. AIPL1 contains a N-terminal FKBP-like domain and a C-terminal tetratricopeptide repeat domain. Mutations in AIPL1 FKBP domain have been associated with an early-onset inherited retinopathy that causes blindness in children. AIPL1 FKBP shares a high sequence homology with the FKBP domain of aryl hydrocarbon receptor-interacting protein (AIP). AIPLI FKBP binds isoprenyl moieties of PDE6, but AIP FKBP does not. Here we report the NMR studies of the AIPL1 FKBP domain in apo and in complexes with isoprenyl ligands. These results revealed the mechanism of isoprenyl binding and the function of AIPL1 FKBP.

Session: Biomolecules in Solution, poster number: 012
Breaking the Symmetry of Polyubiquitin Chains using Paramagnetic NMR and its Application to the Development of HIV-1 Budding Inhibitors
Madeleine Strickland1; Susan Watanabe2; Lorna S. Ehrlich2; Steven M. Bonn3; Christina M. Camara3; David Fushman3; Carol Carter2; Nico Tjandra1
1NHLBI, NIH, Bethesda, MD; 2Stony Brook University, Stony Brook, NY; 3University of Maryland, College Park, MD
Tsg101 is part of the human ESCRT machinery, which is involved in diverse membrane remodeling roles. Viral structural proteins such as HIV-1 Gag are capable of hijacking this system, diverting Tsg101 to the cell surface where viral budding occurs. Using NMR spectroscopy, we characterized the interaction of a new HIV-1 budding inhibitor and found that the drug interfered with the ability of Tsg101 to recognize ubiquitin chains. However, the study of ubiquitin chains is complicated by the fact that each ubiquitin monomer cannot be easily differentiated from one another due to resonance overlap. Using paramagnetic relaxation enhancement, we found that we could break the symmetry of diubiquitin, allowing us to probe its interaction with Tsg101.

Session: Biomolecules in Solution, poster number: 013
Use of high pressure NMR spectroscopy to rapidly identify internal ligand-binding cavities in proteins
Donald Gagne1; Bruce Johnson1; James Aramini1; Kazuyuki Akasaka2; Kevin Gardner1
1CUNY - Advanced Science Research Center, New York City, NY; 2Kyoto Prefectural University of Medicine, Kyoto, JP
5-10% of proteins are known to contain internal cavities large enough to accommodate a ligand. Of particular interest are systems which bind ligands to internal cavities to regulate their function, providing routes for biotechnological and therapeutic control. Having a way to identify such ligand-binding proteins more rapidly than the dominant method – X-ray diffraction – would significant speed up the discovery and validation of novel therapeutic targets. Here we report results using pressure NMR, building on the broad observation that pressure-induced effects on protein structure are often mediated by water insertion into cavities and defects. We validated this approach, offering a quick and sensitive way to screen for ligand-regulated proteins in the absence of structural information.

Session: Biomolecules in Solution, poster number: 014
Characterization of the Antifreeze Protein ApAFP752 
Korth Elliott1; Predrag Jevtic2; Daniel Levy2; Krisztina Varga1
1University of New Hampshire, Durham, NH; 2University of Wyoming, Laramie, WY
Antifreeze proteins (AFPs) are a unique class of protein characterized by their ability to depress the freezing point of water sufficient to prevent the growth of ice crystals by adsorbing to the surface of ice crystals. This property allows organisms which live in extremely cold climates to survive. Because of these proteins’ ability to prevent and slow the rate of ice crystal formation, they have great potential for application in the field of cryopreservation. We aim to characterize the antifreeze protein ApAFP752 from the beetle Anatolica polita to better understand its mechanism of action. NMR spectroscopy was utilized to investigate the structure and dynamics of this protein and to compare this protein to similar known AFPs.

Session: Biomolecules in Solution, poster number: 015
Paramagnetic Tags for Glycoproteins – Structural Restraints for Heparan Sulfate Binding to Robo1
Alexander Eletsky; Maria Moure; Qi Gao; You Zhuo; Laura Morris; Jeong-Yeh Yang; Digantkumar Chapla; Yuejie Zhao; Geert-Jan Boons; Kelley Moremen; James Prestegard
University of Georgia, Athens, GA
We report a novel chemoenzymatic method for site-specific paramagnetic spin labeling of glycoproteins. A single Asn-linked N-acetylglycosamine is used as the attachment point instead of a commonly used cysteine thiol group. We demonstrate the application of this method to studying the molecular mechanism of heparan sulfate (HS) binding to human Robo1 protein, a cell-surface glycoprotein involved in cellular signaling processes. A synthetic tetrasaccharide HS molecule with known high affinity for Robo1 was used as its binding partner. DOSY NMR spectroscopy was employed to filter out protein resonances allowing us to measure PREs for the tetrasaccharide HS in complex with Robo1. Distance restraints derived from these PREs were used to build a structural model of the Robo1/HS complex via molecular docking.

Session: Biomolecules in Solution, poster number: 016
Solution structure of MSI2-RRM1 and its interaction with RNA
Minli Xing1; Lan Lan2; Justin Douglas1; Philip Gao3; Robert Hanzlik4; Liang Xu2, 5
1University of Kansas Bio-NMR Core Facility, Lawrence, KS; 2University of Kansas Dept.of Molecular Biosciences, Lawrence, KS; 3University of Kansas Protein Production Group, Lawrence, KS; 4University of Kansas Dept. of Medicinal Chemistry, Lawrence, KS; 5University of Kansas Cancer Center, Kansas City, KS
Musashi2 (MSI2) is an RNA-binding protein that can bind to target mRNA and regulate translation of proteins that involve in oncogenic pathway, and participate in cancer stem cell populations maintenance, cancer invasion regulation, and metastasis and development of more aggressive cancer phenotype. MSI2 is a promising therapeutic target for cancer. MSI2 contains two N-terminal RRMs (RNA-recognition Motifs) RRM1 and RRM2, which mediate the binding to their target mRNAs. Here in this study, we determined the solution structure of MSI2-RRM1 by NMR, and characterized the direct interaction of MSI2-RRM1 with Numb RNA r(GUAGU) using 2D NMR titration experiment. Our work adds significant information to MSI2-RRM1 structure and RNA binding pocket, and contributes to the development of MSI2 inhibitors.

Session: Biomolecules in Solution, poster number: 017
Phosphorylated c-Met Recognition of SHIP2-SH2 Revealed by NMR Spectroscopy
Zi Wang; Yao Nie; Jiang Zhu; Rui Hu; Fan Luo; Bin Jiang; Xu Zhang; Maili Liu; Yunhuang Yang
Wuhan Institute of Physics and Mathematics, Wuhan, China
The SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) plays an essential role in PI3K-dependent insulin signaling, actin cytoskeleton remodeling and cell spreading. The role of SHIP2 in cell spreading is associated with the binding of SHIP2 to the hepatocyte growth factor (HGF/SF) receptor, c-Met, which is mediated by the SH2 domain of SHIP2 and required the phosphorylation of c-Met at Tyr1356. Here, we report the NMR structure of SHIP2-SH2, and the characterization of the binding interface and affinity of SHIP2-SH2 for a peptide derived from Tyr1356-phosphorylated c-MET through NMR titration experiments. Further validation for the roles of the binding interface residues using site-specific mutants of SHIP2-SH2 in Tyr1356-phosphorylated c-MET binding assay is under investigation.

Session: Biomolecules in Solution, poster number: 018
Structural characterization of Lys63-linked diphosphoUb in complex with the TAB2 NZF Ub binding domain
Fangling Ji1; Menglin Ran2; Mingyang Li1; Chun Tang2; Xu Dong2
1Dalian University of Technology, Dalian, China; 2Wuhan Institute of Physics and Mathematics of CAS, Wuhan, China
K63-Ub2 with Ser65 phosphorylated in the distal unit (Ser65-pK63-Ub2) interacting with the TAB2 NZF domain was characterized via NMR. A two-stage binding profile was observed with the TAB2 NZF domain titration into Ser65-pK63-Ub2, presenting different Kd values. This diverged with that unphosphorylated K63-Ub2 in the close state solely adopted one stage binding state with TAB2 NZF domain, indicating that phosphorylation modification alters the binding mode between them. To characterize the complex, the non-natural amino acid pAzF was introduced at the position of Asn25 site for tagging DTTA-C3 probe. With the PCS tensors determined for Tb3+- and Tm3+- chelated DTTA-C3 based on the intramolecular PCSs, we calculated the structure of Ser65-pK63-Ub2-TAB2 NZF complex by refining against the intermolecular PCSs.

Session: Biomolecules in Solution, poster number: 019
Bypassing second order effects for accurate homonuclear 1H,1H coupling constant determination

Marcos Battistel
Food and Drug Administration, Silver Spring, MD
Glycan 1H NMR signals are highly degenerate, where most ring 1H signals resonate between 3.5-4.5 ppm. Consequently, second order effects alter peak position and the frequency difference of split peaks of coupled 1H no longer reflect true coupling constant values. In this report we present three simple  pulse sequences to extract accurate homonuclear coupling constants (JHH) via scalable  splitting in the 13C dimension. The relatively narrow glycan 13C signal dispersion coupled with J-scaling and the utilization of NUS, affords extraction of JHH in a fast and sensitive manner. Importantly, we demonstrate that the extracted JHH are accurate, even when the 1H's are strongly coupled. We show how JHH can be extracted for sucrose and for the pentasaccharide sialyl Lewis X.

Session: Biomolecules in Solution, poster number: 020
An NMR Based Fragment Screen for Heterogenous Nuclear Ribonucleoprotein A18, A18
Katherine Coburn1; Braden Roth1; Paul Wilder1; Kristen Varney1; France Carrier1, 2; David Weber1
1University of Maryland School of Medicine, Baltimore, MD; 2Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD

Skin cancer is the most common form of all cancers and malignant melanoma (MM) accounts for over 72% of the skin cancer deaths each year. Heterogenous nuclear ribonucleoprotein A18 (A18) is an RNA binding protein differentially expressed between cancerous tissue and normal tissue in breast cancer, prostate cancer, and melanoma patient samples. Toward the development of small molecule inhibitors for A18, the x-ray crystal and NMR structures of the A18 RNA Recognition Motif (RRM) were solved, and were used for early-stage structure-based drug design via computer aided drug design (CADD) methods. These structural and dynamic data for the RRM of A18, the CADD screen, and results from a fragment-based screen completed by NMR will be presented.

Session: Biomolecules in Solution, poster number: 021
NMR Characterization of 15N-Labeled Therapeutic Antibodies Produced in Chinese Hamster Ovary (CHO) cells
Yan Hou
Merck, MRL, Kenilworth, NJ

Recent breakthroughs in cancer immunotherapy have been achieved by the development of monoclonal antibodies that modulate the interaction between immune cell receptors and ligands that are expressed on the surface of cancer cells. The discovery and development of these therapeutic proteins is often guided by biophysical characterization that includes X-ray crystallography, NMR spectroscopy and deuterium-exchange mass spectrometry.

We set out to explore 15N mAb labeling strategies to enable a wide range NMR structural studies with single-residue resolution. Large scale mAb protein production most often utilizes Chinese Hamster Ovary (CHO) cells,   which are the focus of this presentation.  The approach that we have developed achieves high yield and high level of stable-isotope enrichment of  (>95%) in stable cell lines

Session: Biomolecules in Solution, poster number: 022
Lanthanide-tagged U1A-RBD protein as a tool to yield NMR pseudocontact shifts on RNA for long-range structural information
Rohith Rajasekaran1; Madeleine Strickland1; Marie-Paule Strub1; Jan Marchant3; Jonathan Catazaro3; Guillermo Bermejo2; Michael Summers3; Nico Tjandra1
1NIH/NHLBI, Bethesda, MD; 2CIT/NIH, Bethesda , MD; 3University of Maryland Baltimore County, Baltimore, MD
Site-specific attachment of paramagnetic lanthanide metals can be used to yield unique structural information by NMR via pseudocontact shifts. Use of lanthanide tags with RNA is not readily accessible due to the complicated nature of chemical synthesis of isotopically-labeled RNA containing unnatural bases. Developing a practical method for incorporating lanthanide ions into RNA can serve to overcome intrinsic challenges of studying RNA by NMR, including resonance overlap and sparse restraints. We propose a novel technique that relies on lanthanide tags designed for site-specific tagging of proteins to study RNA. We propose to use the RNA-binding domain of U1A spliceosomal protein (U1A-RBD) as an intermediary between the lanthanide tag and the target RNA.

Session: Biomolecules in Solution, poster number: 023
NMR Line Shapes in Systems with Multiple Alternative Conformations
and Ligand Binding

Evgenii Kovrigin
Marquette University, Milwaukee, WI
In many proteins, ligand binding is coupled to complex structural transitions. One prominent example are intrinsically disordered proteins where multiple polypeptide conformations are in exchange, and the ligand selects the matching one. Another situation with multiple isomers arises when a protein undergoes local unfolding ("opening") to allow for the ligand binding, which will induce a new stable conformation. NMR spectra simulated for such models in a broad range of parameters revealed that multiplicity of isomers in exchange may lead to (expected) extreme broadening as well as (unexpected) transient narrowing phenomena. Reasonable estimates for distribution of the chemical shifts of isomers and kinetic rate constants will be required to allow for the meaningful fitting of such spectral data in practice.

Session: Biomolecules in Solution, poster number: 024
Translesion Synthesis DNA Polymerases: from NMR Structures to Anti-Cancer Drug Design
Dmitry Korzhnev
MBB, UCHC, Farmington, CT
Translesion synthesis (TLS) is a cellular pathway that enables replication of damaged DNA. TLS employs low-fidelity DNA polymerases that copy over DNA-lesions while leaving DNA-damage unrepaired. Replicative bypass of most DNA-lesions involves coordinated action of TLS enzymes organized in a complex assembled through protein-protein interactions mediated by the TLS polymerase Rev1. We determined NMR structures and characterized protein-protein interaction mediated by the Rev1-CT domain, which provides a binding platform for all major TLS enzymes. Furthermore, we identified small-molecule inhibitors disrupting the Rev1-CT interactions and demonstrated that these compounds sensitize cancer cells to killing by cisplatin and reduce mutagenesis preventing chemoresistance. These studies illustrate how structural knowledge of protein interactions underlying TLS regulation translate into the development of anti-cancer chemotherapeutics.

Session: Biomolecules in Solution, poster number: 025
Methyl-labeling assisted structure determination of a 66 kDa growth factor-receptor complex
Morkos A. Henen; Pardeep Mahlawat; Shahid N. Khan; Christian Zwieb; Ravi Kodali; Cynthia S. Hinck; Ramsey Hanna; Andrew P. Hinck
University of Pittsburgh, Pittsburgh, PA
TGF-β1, TGF-β2, and TGF-β3 are 26 kDa disulfide-linked homodimeric signaling proteins. They all signal through the TGF-β type I and II receptors, yet TGF-β2, which is well known to bind TβRII weakly has an additional requirement for the TGF-β type III receptor (TβRIII). The objective of this study was to determine the three-dimensional structure of the 66 kDa 2:1 complex formed between the TβRIII C-terminal domain (RIIIc) and TGF-β2, the structures of which are both known. To obtain the necessary experimental restraints to determine an accurate three-dimensional structure of the complex, we used methyl chemical shift perturbations to map the TGF-β2 and RIIIc binding surfaces and long-range distances between site-specifically attached MTSL on RIIIc and assigned methyl groups on TGF-β2.

Session: Biomolecules in Solution, poster number: 026
Solution NMR Structure of Melanoregulin Shows Cholesterol Recognition Motif Positioned Between Positive and Negatively Charged Patches on the Protein's Surface
Ashok Rout2; Xufeng Wu2; Mary Starich1; Marie-Paule Strub2; John Hammer III2; Nico Tjandra2
1National Institutes of Health (Contractor), Bethesda, MD; 2National Institutes of Health, Bethesda, MD
Skin and coat pigmentation arises from the production of melanosomes in specialized cells called melanocytes, followed by their transfer to keratinocytes.  Melanoregulin is a novel, 21 kDa protien implicated in this transfer process as a promoter of microtubule minus end-directed transport of organelles by the dynein-dynactin motor complex.  The 3D solution NMR structure of a 182 amino acid construct of melanoregulin is presented, revealing surface bands of positively and negatively charged residues on opposite sides of the structure which sandwich a tyrosine-based cholesterol recognition sequence (CRAC motrif).  We propose that the structural features observed, coupled with cholesterol recognition serve to orient melanoregulin on the membrane surface, allowing for interaction with other protein components involved in dynein motor recruitment (e.g. RILP).

Session: Biomolecules in Solution, poster number: 027
On-line high-pressure NMR technique applied to bacterial spores
Kazuyuki Akasaka1; Akihiro Maeno2; Shigeru Fujii2; Kenji Kanaori3
1Kyoto Prefectural University of Medicine, Kyoto, Japan; 2Kansai Medical University, Hirakata, Japan; 3Kyoto Institute of Technology, Kyoto, Japan
High-pressure NMR is a unique spectroscopic technique that allows, in principle, observation of molecular events in any system directly under pressure. Here we have applied the technique to investigate molecular events in aqueous suspensions of bacterial spore, a dormant state of bacteria, upon exposure to pressure perturbations. We have successfully detected 1H NMR signals, in real time, of the crucial spore-protecting natural agent, pyridine-2,6-dicarboxylic acid (dipicolinic acid, DPA), as it leaks from the spore of Bacillus subtilis natto into the bulk solution upon pressurization (up to 200 MPa) as well as upon de-pressurization. After this happening, the spore is easily inactivated at 80~100 °C, much below the autoclaving temperature (121 °C) normally required to inactivate spores.

Session: Biomolecules in Solution, poster number: 028

NMR studies of TREM2, a central sensor Linked to Late-onset Alzheimer’s Disease (LOAD)

Younghee Park; Charles R. Sanders
Vanderbilt University, Nashville, TN

TREM2 is an immunomodulatory receptor composed of an extracellular V-type Ig domain followed by a short stalk, a single transmembrane (TM), and a short cytoplasmic tail. TREM2 has been implicated in a wide array of functions related to microglial activation, survival, clustering, and phagocytosis. TREM2 variants have been identified as risk factors for Alzheimer’s disease (AD) and other neurodegenerative diseases. Although Ig domain structure was determined by x-ray crystallography, its engagement with ligands and TM domain interaction with DAP12, a co-receptor to mediate signaling functions, remain unclear. Here we describe purification of the TREM2 TM/cytosolic domain, its reconstitution into model membranes, and subsequent structural studies done by NMR.

Session: Biomolecules in Solution, poster number: 029
Application of DOSY NMR to Droplet Size Measurement for Emulsion Drug Products.
Sharadrao Patil; Jiangnan Peng; David Keire; Kang Chen
Division of Pharmaceutical Analysis, FDA, Silver Spring, Maryland
In a typical oil-in-water emulsion, oil droplets with varied sizes are dispersed in water phase and stabilized by surfactant. . More critically, analytical methods for oil droplet size determination need to introduce minimal perturbation to emulsion drug products. The classical methods of dynamic light scattering or electron microscopy can be used but require sample dilution or alteration. To better evaluate the correlation between droplet size and the manufacturing process, a simple solution NMR method was developed to non-invasively and directly assess the particle size using a two-dimensional (2D) Diffusion Ordered SpectroscopY (DOSY). The new method was applied to emulsion products with oil droplet size range from 30 to 140 nm in radius.

Session: Biomolecules in Solution, poster number: 030
Chain Selective Isotopic Labeling of Chlamydial Protein Complex Scc1-Scc4 and Investigation of the Binding Interface by Solution NMR Spectroscopy

Abigael Songok; Megan Macnaughtan
Louisiana State University, Baton Rouge, LA
Chlamydia trachomatis uses type three secretion system to translocate the effector proteins to the host cell during invasion. Chlamydia outer protein (CopN) serves as a plug and an effector in this system. Specific Chlamydia chaperones 1 and 4 (Scc1 and Scc4) stabilize and direct the secretion of CopN. One therapeutic approach for Chlamydia treatment is to interfere with the Scc1-Scc4 binding interface. We are therefore investigating the interface using isotope-filtered NMR experiments. We have developed a method for chain selective isotopic labeling. The overlay of 15NHisScc1-Scc4 and HisScc1-15NScc4 spectra with that of 15NHisScc1-15NScc4 spectrum has enabled us to assign the peaks to each protein. The one-protein labelled complex spectrum deconvolute the aggregated peaks in 15NHisScc1-15NScc4, thus simplifying backbone assignment process.

Session: Biomolecules in Solution, poster number: 031
Template Specific Fidelity of DNA Polymerase β Characterized by Interactions with the Templating Bases and a Base Specific Mutator
Kyle East; Cary Liptak; J. Patrick Loria
Yale University, New Haven, CT
DNA Polymerase β (pol β) is a 39 kDa polymerase that is the main polymerase in the Base Excision Repair (BER) Pathway and mutated in between 30 and 40 percent of cancers sequenced to date. In order to better understand the template specificity of pol β, amide and methyl resonances were used to analyze the structure of the apo, binary, and ternary complexes of the wild type enzyme with two of the four of the templating bases. To further determine the specificity of the enzyme, E288K a template A specific mutator was compared to the wild type enzyme in order to characterize the 6 fold loss of fidelity.

Session: Biomolecules in Solution, poster number: 032
Over-oxidation of Human DJ-1 Leads to Loss of Structural Integrity at Physiological Temperatures
Tessa Andrews; Jonathan Catazaro; Jiusheng Lin; Mark Wilson; Robert Powers
University of Nebraska-Lincoln, Lincoln, NE
Human DJ-1 has been shown to be up-regulated in multiple diseases, such as Parkinson’s disease, lung cancer and emphysema. Importantly, a highly conserved Cys106 residue is implicated as the site of DJ-1 activity by oxidation. Investigating the structural and dynamic perturbations of the reduced, oxidized and over-oxidized forms of DJ-1 may provide insight into DJ-1’s involvement in these diseases. By utilizing chemical exchange saturation transfer (CEST), sub-ns internal motions of certain residues of DJ-1 based on their sensitivity to the 1H-15N NOE experiment were observed. These results will be combined with structural data collected by NMR to compare key differences in the oxidation states and their roles in the mechanisms of disease.

Session: Biomolecules in Solution, poster number: 033


NMR characterization of the C3 domain from Streptococcus mutans adhesin P1

Gwladys Riviere; Jeanine Brady; Joanna Long
University of Florida, Gainesville, FL
Streptococcus mutans is a gram positive bacterium involve in dental caries. S. mutans adherence is mediated by both sucrose-dependent and independent mechanisms. In the absence of sucrose, the adhesin P1, localized on the surface of S. mutans, interacts with the glycoprotein salivary agglutinin complex (SAG).
Recently, our structural studies highlighted that the C-terminal region of adhesin P1 (the C123 domain) interacts with intact adhesin P1 on the cell surface. Additionally, the C123 domain is involved in amyloid formation within biofilms and is vital for S. mutans pathogenesis. However, with 519 amino acids long, C123 domain is too large to easily achieve high-resolution data through NMR analysis. Here, we focus on the NMR functional and structural characterization of the C3 domain.

Session: Biomolecules in Solution, poster number: 034
NMR Structure and Functional Implications of Plasminogen Binding Protein
Yue Yuan; Cunjia Qiu; Jaroslav Zajicek; Victoria A Ploplis; Shaun W. Lee; Francis J. Castellino
University of Notre Dame, Notre Dame, IN
Interactions of a streptococcal M-like plasminogen binding protein (PAM) with the isolated Kringle 2 domain of human plasminogen were studied by NMR spectroscopy to establish correlations between sequence, binding affinity, and structural alterations of PAM.Two truncated PAM mutants, VEK50RH1/AA and VEK50RH2/AA, have been designed by replacing two RH groups, which are critical to the binding affinity, with alanines, respectively. NMR-derived solution structures of VEKs in the apo- and bound-form were determined using a combined strategy of distance constraints and CS-ROSETTA. The VEK proteins undergo significant structural changes upon binding to Kringle 2. The HADDCOK-calculated structure models of the Kringle 2-VEK complexes provided the structural basis for the high-affinity and specificity of these VEK proteins.

Session: Biomolecules in Solution, poster number: 035
Structural and Functional Characterization of Atg3’s N-terminal Helix
Erin Tyndall; Yansheng Ye; Hong-gang Wang; Fang Tian
Pennsylvania State University, Hershey, PA
Autophagy is a key stress response pathway in cells. Atg3 is a protein that is required for autophagy, and the construction of the autophagosome. Atg3 is responsible for conjugating LC3 directly to the lipids in the membrane. This reaction is dependent on an N-terminal amphipathic helix (NAH), despite the fact that it is poorly conserved among isoforms. We used human and yeast orthologs to study the membrane binding, structure and membrane topology of the NAH and how binding to the membrane regulates the function and activity of Atg3 as a whole.

Session: Biomolecules in Solution, poster number: 036
Characterizing Dynamics Allosteric Changes of Antibody-Antigen Interactions
Gaohua Liu1; Alan Gibbs2; Ruth Steele2; Gaetano Montelione3; Rong Xiao1
1Nexomics, Bordentown, NJ; 2Janssen Research and Development LLC, Spring House, PA; 3Rutgers University, Piscataway, NJ
Nexomics Bioscience is leveraging advanced NMR technologies, including 19F-NMR, to study antibody-antigen interactions. Four humanized Fab’s were selected, and successfully expressed in E. coli with uniform 15N-enrichment and with 19F-Trp labeling. 2D 15N TROSY-HSQC and 1D 19F NMR spectra demonstrate that these isotope-specific labeled Fab are very well suited for antibody-antigen interaction studies by NMR. Significant chemical shift perturbations due to antigen binding were observed in both 2D 15N TROSY-HSQC and 1D 19F NMR spectra for all four Fab’s, which indicates structure/dynamics changes propagate throughout an allosteric network within the Fab structure. This study demonstrates that our strategy can become an extremely useful tool for the pharmaceutical and biotech communities for drug discovery, especially in the field of antibody engineering.

Session: Biomolecules in Solution, poster number: 037
NMR Characterization of Ligand Binding for the Endocannabinoid Carrier Protein hFABP7 and the Deactivation Enzyme hMGL
Xiaoyu Ma1; Sergiy Tyukhtenko1; Girija Rajarshi1; Jason Guo2; Alexandros Makriyannis1
1Center for Drug Discovery, Northeastern University, Boston, MA; 2Department of Chemistry, Northeastern University, Boston, MA
Human brain fatty acid binding protein (hFABP7) and monoacylglycerol lipase (hMGL) are two key endocannabinoid proteins involved in the intracellular trafficking and deactivation of the endocannabinoids such as anandamide and 2-arachidonoylglycerol. Both hFABP7 and hMGL have a lid domain which regulates ligand entry into the binding pocket. For hFABP7, we have identified key residues involved in the conformational changes as well as physicochemical conditions that can affect the conformational equilibrium. Our NMR results for hMGL revealed that single point mutations can either directly compromise the efficiency of the catalytic triad or induce significant conformational changes that affect the ligand gating. To further characterize ligand efficacy, we have developed an NMR-based approach to determine the residence time of potent hMGL inhibitors.

Session: Biomolecules in Solution, poster number: 038

Human Guanylate Kinase: NMR Structure Determination and Functional Investigation of a New Biomolecular Target for Lung Cancer

Nazimuddin Khan1; Parag Shah1; David Ban1; Pablo Trigo-Mourino2; Marta Carneiro3; Lynn DeLeeuw1; William Dean1; John Trent1; Levi Beverly1; Manfred Konrad2; Donghan Lee1; T. Michael Sabo1
1University of Louisville, Louisville, Kentucky; 2Max-Planck Institute for Biophysical Chemistry, Göttingen, Germany; 3ZoBio B.V., Leiden, The Netherlands

Human guanylate kinase (hGMPK) is the only identified enzyme responsible for cellular GDP production and it is essential for cellular viability and proliferation of cancer cells, yet, no three-dimensional structure of hGMPK exists. Here, we present the first structure of hGMPK, determined with NMR spectroscopy, which illustrates the dynamic nature of the two nucleotide-binding regions in the absence of substrates. Furthermore, we found that modulated functionality occurs through non-synonymous single-nucleotide variants (nsSNVs) of hGMPK while maintaining a similar structure to the wild-type. Finally, we show that knocking down the hGMPK gene in lung adenocarcinoma cell lines adversely affects cellular viability, while not altering the growth of immortalized human peripheral airway cells, suggesting that hGMPK is a promising new biomolecular target.

Session: Biomolecules in Solution, poster number: 039
A New Free Exchange Model for EmrE Transport
Katherine Henzler-Wildman1; Nathan Thomas1; Anne Robinson2; Emma Morrison2; Grant Hussey1; Bryan Balthazor2
1University of Wisconsin, Madison, WI; 2Washington University School of Medicine, St. Louis, MO
EmrE is small multidrug transporter in the inner membrane of E. coli. It uses the proton motive force to confer resistance to a wide range of toxic lipophilic cations. EmrE is an asymmetric, antiparallel homodimer with a single substrate binding site defined by a pair of glutamates in the dimer interface. It undergoes conformational exchange to allow alternating access of the binding site to either side of the membrane, thereby transporting bound substrate across the membrane. We have used NMR to directly monitor proton binding and alternating access of EmrE. Our NMR data revealed additional states and conformational transitions that must be included in the transport model and has fundamentally changed our understanding of the requirements for efficient proton-coupled transport.