Gateway NMR 2020 Speaker Abstracts

Keynote Speaker

Richard W. Kriwacki

St. JudeChildren’s Research Hospital, Memphis TN, Structural Biology

Proteins are now viewed as being comprised of both folded domains and intrinsically disordered regions (IDRs) that synergistically contribute to function. However, how IDRs contribute to protein function and how synergy with folded domains is achieved is often poorly understood. We will discuss several recent protein systems in which detailed structural, biochemical and cell biological investigations have revealed the molecular basis for functional synergy between IDRs and folded protein domains. These systems to be discussed address biological process ranging from regulation of cell division and apoptosis to ribosome biogenesis in the nucleolus.

The latter process involves the topic of liquid-liquid phase separation (LLPS), which has emerged in the last decade as a ubiquitous mechanism for organizing biomolecules within membraneless compartments in cells referred to as biomolecular condensates. Condensates mediate diverse biological processes, including many aspects of RNA metabolism. However, in many cases, knowledge gaps exist regarding how LLPS occurs and how it contributes to biomolecular function. Proteins that undergo LLPS often exhibit one or more IDRs that mediate interactions with themselves, with other proteins, and with RNA. The nucleolus is the largest biomolecular condensate and the pentameric phosphoprotein, Nucleophosmin (NPM1), which displays both folded domains and an IDR, is highly abundant within the nucleolus, where it functions in ribosome biogenesis and cellular stress sensing. We will discuss recent findings on how multi-component LLPS enables dynamic responsiveness of the nucleolus to variable levels of NPM1 and other components, which underlies ribosome subunit assembly and stress responses. The Arf tumor suppressor is a small, R-motif-rich disordered protein that is expressed under conditions of oncogenic stress, including Myc overexpression. Arf integrates within and modulates the liquid nature of the nucleolus, inhibiting ribosome biogenesis and activating p53. In contrast, Arginine-rich dipeptide repeat (DPR) polypeptides, expressed in certain forms of amyotrophic lateral sclerosis (ALS), also integrate within the nucleolus through interactions with NPM1; however, these interactions disrupt the nucleolus and cause cell death.

We will discuss how synergy between folded protein domains and IDRs mediates the diverse biological functions of the systems mentioned above based on findings from methods that probe molecular assemblies from the Ångstrom and nanometer (NMR, SANS) to micron (microscopy) length scales.

Session 1 – Tutorial Session, Friday afternoon

Clemens Anklin

Vice President of NMR Applications and Training, Bruker BioSpin – Billerica, Massachusetts, USA

Advanced 2D and 3D processing, tips tricks and unknown features

This course will introduce the audience to advanced processing options in TopSpin for 2 and higher dimensional datasets. Many of the processing commands have unknown but useful options.

Amy Freund

Senior Applications Scientist II, Bruker BioSpin – Billerica, Massachusetts, USA

1D quantitative NMR acquisition parameters, experiment options, and processing parameters

Introduction to quantitative NMR. This webinar will focus on 1D quantitative NMR acquisition parameters, experiment options, and processing parameters. We will also review Topspin Tools for data analysis.

Session 2, Saturday Morning

Leah B. Casabianca

Department of Chemistry, Clemson University

Examining Binding Between Small Molecules and Polystyrene Nanoparticles: Results from Saturation-Transfer Difference NMR

As engineered nanoparticles become increasingly used both in biomedicine and everyday applications, they find their way into the body and into the environment. It is therefore important to have techniques by which to examine the structure and dynamics of molecules, such as pharmaceuticals, pollutants, and biological macromolecules, interacting with the nanoparticle surface.

We have used Saturation-Transfer Difference (STD)-NMR to explore the interaction between small molecules and the surface of polystyrene nanoparticles.¹ STD-NMR was originally developed to identify small-molecule ligands that bind to a protein receptor. Since this technique does not require the receptor to be seen by solution-state NMR, there is no upper limit to the size of the receptor that can be studied. This makes the STD-NMR technique an ideal one to study small molecules adsorbed on the surface of nanoparticles.

One advantage of the STD-NMR technique is that it can be used not only to screen potential ligands for binding to a specific receptor, but also to gain information about which part of the ligand is responsible for binding. Thus, we have used STD-NMR to explore the driving forces that are responsible for different ligands binding to polystyrene nanoparticle surfaces. By examining binding between amino acids and polystyrene nanoparticles,² we postulated three factors responsible for binding: electrostatic interactions, hydrophobic effects, and pi-pi interactions for aromatic amino acids. These interactions were further studied through experiments with zwitterionic nanoparticles.³Electrostatic effects were probed by performing STD-NMR experiments different pH values and salt concentrations. The influence of dispersion interactions was explored by establishing a structure-activity relationship for binding using a series of unnatural amino acids with different lengths of hydrophobic side chains. These results will be useful for predicting binding sites and driving forces for binding between peptides and nanoparticles in future studies.

References

[1] Y. Zhang, H. Xu, A. M. Parsons, L. B. Casabianca, “Examining Binding to Nanoparticle Surfaces Using Saturation Transfer Difference (STD)-NMR Spectroscopy.” J. Phys. Chem. C 2017, 121, 24678-24686.

[2] Y. Zhang and L. B. Casabianca, “Probing Amino Acid Interaction with a Polystyrene Nanoparticle Surface Using Saturation-Transfer Difference (STD)-NMR.” J. Phys. Chem. Lett. 2018, 9, 6921-9625.

[3] H. Xu and L. B. Casabianca, “Probing Driving Forces for Binding Between Nanoparticles and Amino Acids by Saturation-Transfer Difference NMR,” Sci. Rep., accepted.

Dan Conroy

Department of Chemistry and Biochemistry, The Ohio State University, Columbus OH

Observing Hoogsteen Base-Pairs in DNA using Dynamic Nuclear Polarization Solid-State NMR

Genomes of eukaryotes are organized into a complex, three-dimensional structure that plays crucial roles in development, physiology, and disease. DNA wraps around histones to form nucleosome particles (NCPs), which associate to form higher-order chromatin fibers and chromosomes. A widely accepted feature of double-stranded DNA is that the DNA helix is composed solely of G-C and A-T Watson-Crick (WC) base pairs (bps). However, alternative types of G-C and A-T bps, known as Hoogsteen (HG), have been observed in duplex DNA under torsional strain and at protein-DNA interfaces, but it is currently unknown whether or not HG bps exist under cellular conditions in chromatin to impart unique functionality to the DNA.

Dynamic nuclear polarization (DNP) is a powerful method to enhance the sensitivity of solid-state NMR spectroscopy. DNP-SSNMR offers a unique ability to study the structures of dsDNA bps in NCPs at an atomic resolution. We were able to observe DNP signal enhancements of 100-150 in DNA samples, doped with the AMUPol polarizing agent. 2D TEDOR experiments were used to identify the spectroscopic signatures of WC and HG bps, including, assigning the unique ¹³C and ¹⁵N chemical shifts of each form, and identifying conformation-specific, through-space cross-peaks. These strategies were then extended to study a Widom 601 sequence of DNA bound to a NCP and while no HG bps were initially observed, this illustrates the ability of DNP-SSNMR to study these DNA-protein complexes and opens the door to further in vitro studies to investigate the presence of HG bps in NCPs and nucleosome arrays.

David Weliky

Department of Chemistry, Michigan State University

²H NMR Evidence for Lipid Acyl Chain Disordering with Viral Fusion Peptides

Viruses such as HIV, influenza, and SARS CoV-2 are enveloped by a membrane that is obtained during budding from an infected host cell. Infection of a new cell requires fusion (joining) of the membranes of the virus and cell and is mediated by a protein in the virus membrane. Fusion proteins from different viruses don’t have sequence homology. Gp41 is the HIV fusion protein and hemagglutinin subunit 2 (HA2) is the influenza virus fusion protein. Each of these proteins has a ~25-residue N-terminal segment that is named the fusion peptide (fp) and at least for HA2, the fp is deeply-inserted in the final fused membrane. It is therefore proposed that the fp binds to the target membrane early in the fusion pathway. The gp41 and HA2 fp’s likely have additional roles in fusion beyond anchoring the target membrane, based on point mutations that greatly impair fusion without having a large effect on membrane binding. One possible fp function is modifying the lipid acyl chain conformations in a manner that increases the rate of forming a new bilayer with acyl chains from the outer leaflets of the virus and target membranes. ²H NMR spectra were acquired for samples with membrane with perdeuterated acyl chains and with GPfp, HAfp at pH5, or HAfp at pH 7. Spectra were also obtained for membrane without peptide. GPfp is oligomeric with intermolecular antiparallel b sheet structure and HAfp is monomeric with helical hairpin structure, and is more fusion-active at pH 5 vs. 7. Spectra were obtained at both higher temperatures for which the acyl chains were in the liquid-crystalline phase and at lower temperatures for which the chains were in the gel phase. At a particular temperature, spectra of membrane with fusion-active GPfp or HAfp, pH 5 were narrower than spectra of membrane without peptide or with less fusion-active HAfp, pH 7. Narrower spectra evidence larger amplitudes of fast motions of the acyl chains. For membrane in the liquid-crystalline phase, there was resolution of peaks for many -CD₂ sites, and the peak splittings exhibited larger fractional changes for sites closer to the tail vs. the headgroup. This is consistent with insertion of the fp’s into the hydrocarbon core of the membrane. The reductions in splittings were larger for GPfp vs. HAfp, pH 5, which supports greater disordering for b sheet oligomers vs. helical hairpin monomers. Acyl chain disordering by fusion-active fp’s may reduce barriers between the different topological states of the membrane during fusion.

Session 4, Saturday Afternoon

Sharon L. Campbell

Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill

Characterization of a novel pH sensing function of human Gα proteins

Ajit Prakash¹, Guowei Yin¹, Natalie Hewitt Valentin^1,2, Henrik G. Dohlman^1,2, Sharon L. Campbell^1,3

¹Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, ²Department of Pharmacology, University of North Carolina at Chapel Hill, ³UNC Lineberger Comprehensive Cancer Center

G-protein-coupled receptors (GPCRs) are the largest group of membrane receptors that are fundamental for most physiological processes spanning vision, smell, and taste to neurological, cardiovascular and endocrine functions. The GPCR binds to heterotrimeric G proteins (Gα, Gβ and Gγ) to activate downstream signaling pathways. Recently, Gα proteins have been shown to function as intracellular pH sensors (Isom et al, Mol. Cell., 2013). However, the molecular basis for Gαi mediated pH sensing has not been fully characterized and the specific mechanism by which pH modulates G-protein signaling is poorly understood. Using various biophysical methods including circular dichroism (CD), intrinsic tryptophan fluorescence and NMR, we have shown that changes in pH over the physiological pH range alter the structure and stability of Gαi bound to GDP. To further elucidate the mechanism of pH sensing, we conducted NMR and mutagenesis studies and discovered a pH sensing network in Gαi that is critical for a pH-mediated disordered to ordered transition. As changes in pH over the physiological pH range alter the structure, stability and dynamics of Gαi-GDP, we have investigated whether pH modulates the interaction of Gαi-GDP with its specific binding partners (e.g. Gβγ and GPCR).  Our preliminary data supports this hypothesis. We are now investigating the effect of pH on Gαi and Gβγ mediated signaling pathways. Finally, we find that pH sensing constitutes a conserved mechanism of regulation among different isoforms of Gα.  As pH changes play a key role in regulating both physiological and pathophysiological cellular processes, understanding Gα-mediated pH regulation in human cells will aid in elucidating this novel mechanism of pH regulation and may potentially direct new therapeutic approaches for pH associated diseases.

Rieko Ishima

Department of Structural Biology University of Pittsburgh School of Medicine, Pittsburgh, PA

Conformational change in the HIV-1 reverse transcriptase maturation

HIV-1 reverse transcriptase (RT) is translated as part of the Gag-Pol polyprotein that is cleaved by HIV-1 protease (PR), and finally forms the mature heterodimer, composed of a 66 kDa subunit (p66) and a p66-derived 51 kDa subunit (p51). Formation of homodimer, p66/p66, is critical for maturation of HIV-1 RT. Recently, we have shown that enhancement of RT maturation by PR in the presence of tRNA^Lys3 [1]. We have also shown how conformational asymmetry on the p66/p66 homodimer in HIV-1 RT is introduced upon binding of tRNA^Lys3 that is known as a primer for reverse transcription initiation [2]. We will discuss consistency of biochemical assay and NMR observation, and that among the NMR data recorded in various conditions, to further understand the mechanism. The work is supported by NIH P50AI150481.

[1] Ilina, et al. (2018) J Mol Biol. 430, 1891-1900.

[2] Slack, et al. (2019) Structure, 27, 1581-93.

Steven R. Van Doren

Biochemistry Dept. and Inst. for Data Science and Informatics, University of Missouri, Columbia

NMR Journey from Structures to Inhibitors of Transient MMP-7 Enzyme Interactions with Fluid Partners from Cell Surfaces

Anionic carbohydrates known as glycosaminoglycans (GAGs), as well as anionic bilayers, recruit the proteolytic enzyme MMP-7 to cell surfaces. This is strategic for activating anti-bacterial defenses in the intestines, healing of lungs, and tumor invasion. MMP-7 interactions with GAGs and bilayers are too fluid for crystallography and cryo-EM, but were captured using paramagnetic NMR relaxation enhancements (PREs). The proximity of proMMP-7 to spin-labeled lipids evident from PREs enabled docking of the NMR structure of the zymogen to disk-like bilayered micelles. This revealed switching from superficial association of proMMP-7 with zwitterionic bilayers to partial insertion into anionic bilayers. Spin-labeling of a heparin model of GAGs facilitated its docking to a basic swath that also binds anionic bilayers. A pocket within the MMP-7 interface for GAGs and anionic membranes was targeted by in silico screening. Ligand-detected NMR found more than 20 of the compounds to bind proMMP-7. Application of TREND NMR Pro software to TROSY spectra ranked the ligands and expedited NMR measurement of affinities. Though 10% of the true binders inhibit activation of proMMP-7 to MMP-7, most of the compounds that bind the same remote site are activating. Allostery connects the active site of MMP-7 with binding sites for bilayers, GAGs, and the new remote-binding compounds.

Supported by NIH and Hirshberg Foundation for Pancreatic Cancer Research.

Muriel C. Maurer

Chemistry Department, University of Louisville, Louisville, KY

Deciphering Conformational Changes Associated with the Maturation of Exosites Within Blood Coagulant Thrombin

Thrombin, derived from zymogen prothrombin, is a serine protease that participates in procoagulation, anticoagulation, and platelet activation. Functions of thrombin are controlled via anion-binding exosites I and II (ABE I and ABE II) that undergo maturation during activation. Such exosites are used to attract regulatory biomolecules.  Full knowledge of changes that occur at the individual residue level as pro-ABE I is converted to ABE I is lacking.  To address this issue, ¹H broadening studies and ¹H,¹⁵N-HSQC NMR titrations were used to monitor development of ABE I using peptides based on protease-activated receptors PAR3 and PAR1. Unique residues within the PAR1 (49-62) and PAR3 (44-56) peptides displayed increased affinity upon conversion of pro-ABE I to the mature ABE I.  Mature ABEs can utilize exosite-based communication to fulfill thrombin functions. Long-range communication was successfully examined by saturating ABE II with phosphorylated GpIbα (269-282, 3Yp) and monitoring the binding of PAR1 and PAR3 peptides to ABE I. Individual PAR residues exhibited increased affinities in this dual-ligand environment supporting the presence of inter-exosite allostery. Exosite maturation and beneficial long-range allostery are proposed to help stabilize an ABE I conformation that can effectively bind PAR ligands.

Session 5, Saturday Afternoon

Joshua J. Ziarek

Indiana University

Directed evolution as a tool to explore G protein-coupled receptor conformational dynamics

G protein-coupled receptors (GPCRs) are archetypal allosteric membrane signaling proteins involved in a wide variety of processes from vision, smell and taste to cell migration, pain management and drug addiction. Only 16% of the GPCR superfamily are clinically targeted – and yet they comprise nearly 35% of all marketed drugs. Despite their obvious therapeutic potential, the molecular details of signal transduction remain largely unknown. Biological assays have long suggested a complex activation landscape – making them intriguing targets for solution-state NMR measurements. Unfortunately, their high molecular weight, membrane environment, inherent instability and requirement for eukaryotic expression systems has limited studies primarily to mapping chemical shift perturbations of a subset of NMR probes (e.g. ¹³C-methionine, exogenous¹⁹F or selective ¹⁵N amino acids). Directed evolution of neurotensin receptor 1 (NTS1) to produce a thermostabilized variant (termed enNTS1) that expresses functionally in E. coli is a major technical advancement. NTS1 is one of only three receptors with structures in complex with both heterotrimeric G protein and arrestin downstream effectors. Directed evolution had little effect on resonance frequencies which enables the transfer of assignments to wildtype receptors via visual inspection. Prokaryotic expression is comparatively cheap, and the highly deuterated background affords rapid data acquisition using ¹⁵N- and methyl-TROSY based methods. We are applying ¹⁹F-, ¹³C-methyl, and ¹⁵N-uniform labeling strategies to the evolved receptor with the long-term goal of dissecting how ligand binding and effector association modulate receptor conformation and allosteric signaling pathways.

Kathleen B. Hall

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, 63110, USA

Backbone Dynamics of an RNA Recognition Motif Facilitate Protein Binding and RNA Specificity

Kathleen B Hall ¹ , Gregory T DeKoster ¹ , Gert Weber ^{2 ,} Nicole Holton ² , Markus C Wahl ^{2, 3}

¹ Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, 63110, USA. ² Laboratory of Structural Biochemistry, Freie Universität Berlin, Takustraße 6, D-14195, Berlin, Germany. ³ Helmholtz-Zentrum Berlin für Materialien und Energie, Macromolecular Crystallography, Albert-Einstein-Straße 15, D-12489, Berlin, Germany.

Among protein domains that bind RNA, the RNA Recognition Motif (RRM) is the most common. Its characteristic α/β structure is used to bind single-stranded RNAs that are typically draped over its β-sheet. The canonical example of RRMs that bind RNA with high affinity and specificity is the U1A/U2B”/SNF family that is found in the U1 and U2 snRNPs. As part of these Ribonucleoprotein particles (RNP), these RRMs bind two different RNA hairpin loops, and in addition, U2B” and SNF RRMs bind another protein. Here we investigate the molecular mechanisms that allow SNF RRM to bind both RNAs and protein, and to use that differential binding to allosterically control relative affinity for each molecule. Using x-ray crystallography, we map the atomic interactions between SNF’s RRM and RNAs and the LRR domain of the U2A’ protein. Using NMR, we measure how the backbone dynamics of the RRM respond to binding of RNA and LRR domain. The combination of structure and dynamics reveals how binding alters the pattern of motions throughout the RRM, allowing it to form its mutually exclusive complexes of SNF/RNA1 and SNF/LRR/RNA2. SNF RRM serves as a model for how RNA binding by other RRMs could be modulated by protein:protein interactions, a mechanism that is likely to be especially relevant in the context of RNPs that are now being found associated with mRNAs and lncRNAs in vivo.

Michael A. Kennedy

Department of Chemistry and Biochemistry, Miami University, Oxford, OH, United States of America

NMR investigation of the molecular basis for P[II] human rotavirus VP8* domain recognition of human histo-blood group antigen (HBGA) host cell receptors

 Shenyuan Xu¹, Luay U. Ahmed¹, Michael Robert Stuckert¹, Kristen Rose McGinnis¹, Yang Liu^2,4, Ming Tan^2,3, Pengwei Huang², Weiming Zhong², Dandan Zhao², Xi Jiang^2,3*, Michael A. Kennedy^1*

¹ Department of Chemistry and Biochemistry, Miami University, Oxford, OH, USA. ² Division of Infectious Diseases, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA. ³ University of Cincinnati College of Medicine, Cincinnati, OH, USA. ⁴ Tianjin Key Laboratory of Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China

Rotavirus (RV) infections are responsible for ~130 million episodes of gastroenteritis each year in children age <5 years and cause ~200,000 deaths annually with an estimated cost of ~$1 billion each year in the United States alone. The two existing RV vaccines, Rotarix and RotaTeq, which are based on live-attenuated RVs, are largely ineffective for children in developing countries, expensive, known to cause intussusception, and have a known risk of becoming revertant. In this project, we are generating knowledge required to develop a next-generation synthetic nanoparticle-based RV vaccine with superior efficacy, safety, and cost. Our recent studies establish that P[4], P[6], P[8] and P[19] P[II] genogroup RVs responsible for >90% of human infections have distinct receptor binding specificities to human histo-blood group antigens (HBGAs), which are synthesized in a developmentally-regulated, step-wise manner during early lives of children, and that are evolutionarily conserved in some animals. This hypothesis potentially explains why many RVs exclusively infect only humans or animals while others infect both humans and animals, and why some RVs infect neonates and young infants while others only infect older children. Initial cell attachment of rotavirus (RV) to specific cell surface glycan receptors, which is the essential first step in RV infection, is mediated by the VP8* domain of the spike protein VP4. RV strains in the P[4] and P[8] genotypes of the P[II] genogroup share common recognition of the Lewis b (Le^b ) and H type 1 antigens, however, the molecular basis of receptor recognition by the major human P[8] RVs remains unknown due to lack of experimental structural information. In this study, we have used nuclear magnetic resonance (NMR) spectroscopy-based titration experiments and NMR-derived high ambiguity driven docking (HADDOCK) modeling to elucidate the molecular basis for P[8] VP8* recognition of the Le^b (LNDFH I) and type 1 HBGAs. We also used X-ray crystallography to determine the molecular details underlying P[6] recognition of H type 1 HBGAs. Unlike P[6]/P[19] VP8*s that recognize H type 1 HBGAs in a binding surface composed of an α-helix and a β-sheet, referred as the “βα binding site”, the P[8] and P[4] VP8*s bind Le^b HBGAs in a previously undescribed pocket formed by the edges of two β-sheets, referred to as the “ββ binding site”.

Shenyuan Xu ¹, Luay U Ahmed ¹, Michael Robert Stuckert ¹, Kristen Rose McGinnis ¹, Yang Liu ^2 3, Ming Tan ^2 4, Pengwei Huang ², Weiming Zhong ², Dandan Zhao ², Xi Jiang ^2 4, Michael A Kennedy ¹ Molecular Basis of P[II] Major Human Rotavirus VP8* Domain Recognition of Histo-Blood Group Antigens. PLoS Pathog. 2020 Mar 24;16(3): e1008386. doi: 10.1371/journal.ppat.1008386.