Oral Presentation Abstracts
Day 1 | May 12th
Scientific Session 1: Frontiers in Bioinformatics
Dr. Genki Terashi; Purdue University, Biological Sciences | 9:00 am - 9:40 am
VESPER: Global and Local Cryo-EM Map Alignment and Database Search Using Local Density Vectors
An increasing number of density maps of biological macromolecules have been determined by cryo-electron microscopy (cryo-EM). Although individual maps provide valuable structure information of macromolecules, the value of the maps in the database would be significantly raised if a computational method is available that performs accurate global and local map alignment and database search. Previously, we have developed EM-SURFER (http://kiharalab.org/em-surfer/), a web-based tool for real-time global matching and database search for EM maps. Using 3DZernike Descriptors, an EM-SURFER search against the entire EMDB can be finished in a few seconds. On the other hand, EM-SURFER does not provide a map alignment because it uses rotation-invariant descriptors and also it only performs global matching. Here, we developed a new method VESPER (VEctor-based local SPace ElectRon density map alignment), which performs accurate global and local alignment and comparison of EM maps. The advantage of the VESPER algorithm is it matches two maps by considering local gradients represented by unit vectors, which can effectively capture underlined macromolecular structures in the maps. Thus, the directions of the vectors capture local structures embedded in the map, which turned out to be effective in obtaining accurate global and local map alignment. An alignment of maps is evaluated by a score defined as the sum of dot products of matched vectors from two maps and the best alignment with the maximum score is sought using fast Fourier transform (FFT). Compared on benchmark datasets, VESPER showed a higher accuracy in map retrieval as well as global and local map matching than other existing methods. VESPER also showed higher accuracies in both global and local EM map alignment. Our VESPER algorithm was published this year in Nature Communications.
Xiao Wang; Purdue University, Computer Science | 9:40 am — 10:00 am
Emap2sec+: Detecting Protein and DNA/RNA Structures in Cryo-EM Maps of Intermediate Resolution Using Deep Learning
An increasing number of density maps of macromolecular structures, including proteins and protein and DNA/RNA complexes, have been determined by cryo-electron microscopy (cryo-EM). Although maps at a near-atomic resolution are routinely reported, there are still substantial fractions of maps determined at intermediate (~ 4Å) or lower resolutions, where extracting structure information is difficult. Considering limited approaches developed for maps with protein-nucleic acid complexes under intermediate resolution, we report a new computational method, Emap2sec+, which identifies DNA or RNA as well as the secondary structures of proteins in cryo-EM maps of 5 to 10 Å resolution. Emap2sec+ employs the 3Ddeep residual convolutional neural network (3D-ResNet) as its core of the architecture. Emap2sec+ assigns structural labels with associated probabilities at each voxel in a cryo-EM map, which can help structure modeling in an EM map. Emap2sec+ adopts a two-phase stacked neural network architecture, where predictions in the first phase are further smoothed in the subsequent second phase by incorporating the context of neighboring voxels. Emap2sec+ showed stable and high assignment accuracy for nucleotides in low resolution maps and improved performance for protein secondary structure assignments than its earlier version Emap2sec when tested on simulated and experimental maps. Detection accuracy is remarkably stable for nucleotides even in the maps of a low resolution. More description is available at https://www.biorxiv.org/content/10.1101/2020.08.22.262675v1. The code is available at https://github.com/kiharalab/Emap2secPlustogether with other cryo-EM software https://kiharalab.org/emsuites.
Barrett Davis; Purdue University, Biomedical Engineering | 10:00 am — 10:20 am
Computational Model of PDZ-Binding Competition and AMPA Receptor Insertion in the Postsynaptic Density (PSD)
Neurodevelopmental disorders (NDD), including Autism Spectrum Disorders;(ASD) are estimated to affect between 2-5% of all children, with an associated US economic burden of over $100B. Though NDDs often stem from de novo mutations, mounting evidence has identified some common neuronal pathophysiology in trans-and postsynaptic scaffolding proteins including neuroligin (NLGN), leucine-rich repeat transmembrane protein (LRRTM) and post-synaptic density protein 95 (PSD-95).This “common pool” interacts through shared PDZ-binding motifs and is essential to the fine-tuning of long-term synaptic plasticity. At the molecular level, this tuning process is modulated by both glutamatergic receptor availability (i.e., AMPAR) and molecular competition for PDZ-binding sites which then anchor these receptors into the post synaptic density (PSD). While knockouts of PDZ-binding partners like NLGN and LRRTM have been shown to strongly bias synapses towards long-term potentiation (LTP), at present we lack adequate mechanistic models to describe the transient nature of receptor availability and scaffolding protein competition during calcium stimulation. Therefore, building from previous work in our lab, here we present a compartmentalized, rule-based model for activity-dependent insertion of AMPARs into the post-synaptic density. We further extend this work by simulating in vitro models of disease and characterizing the effects of simulated genetic and pharmaceutical therapies.
Daipayan Sarkar; Purdue University, Biological Sciences | 10:20 am — 10:40 am
Mem-LZeRD: Modeling transmembrane protein interactions using topological and biophysical principles
Modeling interactions of membrane proteins is an important subcategory of protein docking as a relatively lower fraction of structures are available in Protein Data Bank (PDB) despite their significant biological importance, especially since membrane proteins are important targets for drug discovery. Docking of membrane proteins typically starts by placing two membrane proteins in the correct topology relative to the membrane. Then docking conformations are sampled under the constraints that interacting residues from two protein chains are at a similar height relative to the membrane and maintain a tilt angle for a given conformation. Prior to docking, the membrane proteins are initially oriented using the Orientations of Proteins in Membranes (OPM) database and the PPM server, which determines the spatial position of a protein in the membrane by optimizing the transfer free energy from solvent to the homogeneous lipid bilayer. In this work, we perform bound and unbound docking experiments to test and validate our docking method on alpha-helical and beta-barrel transmembrane protein targets. After docking individual protein chains in the transmembrane region, we develop a scoring function using a logistic regression model that combines several energy-based scores. Our results indicate that we obtain good overall performance, both for bound and unbound docking, using our membrane docking method. This docking program is part of the LZeRD protein docking software suite (https://lzerd.kiharalab.org, https://kiharalab.org/proteindocking/).
Keynote Lecture
Dr. Doug Lauffenburger; MIT, Department of Biological Engineering | 11:00 am — 12:00 pm
Hijacked by Covid: Antibody-Centric Immune Systems Analysis, Intensely Applied
Systems Immunology is a field of scientific research aimed at understanding how host immune responses to insult, injury, and infection operate, from an encompassing perspective that naturally benefits from intimate combination of experimental and computational approaches addressing integration of multiple diverse facets of biological mechanistic processes. Our laboratory has been engaged in this field for approximately a decade, mainly working in close collaboration with the Ragon Institute of MGH / MIT / Harvard, to develop insights concerning how the antibody-mediated arm of the immune response functions in choreographing much of the larger system performance. Originally focused on complex pathogens such as HIV and others, the year 2020 brought the urgent new application in Covid for our systems immunology efforts, altering not only some of the substance but also aspects of style of academic research. This talk will offer a travelogue from the past year-plus on our contributions.
Scientific Session 2: Enzymology Across Life
Dr. Angeline Lyon; Purdue University, Chemistry | 1:00 pm — 1:40 pm
A Structure-Based Approach to Understanding Phospholipase C Function
Phospholipase Cβ and ε enzymes are essential for normal cardiovascular function, as they hydrolyze phosphatidylinositol lipids at cellular membranes to generate second messengers that increase intracellular calcium and activate downstream kinases. These proteins also have low basal activity but are stimulated following the activation of G protein coupled receptors and receptor tyrosine kinases through direct interactions with G proteins. Using PLCβas a model system, we have begun characterizing how the conformational dynamics of the protein contributes to its regulation and membrane association. We are also using structural biology and biochemical assays to begin characterizing the structure and regulation of PLCε, alone and upon binding the Rap1AGTPase. Rap1A is the best characterized activator PLCε in the heart, and chronic activation results in cardiovascular disease. We have shown that PLCε is conformationally heterogeneous in solution, with its PH and RA domains contributing to maximum basal activity and stability. The C-terminal RA domain has been previously identified as the Rap1A binding site. We propose the conformational flexibility of this RA domain is essential for Rap1A-dependent activation, and that the mechanism has an allosteric component. Taken together, these studies provide the insights into the molecular basis underlying basal and activated phosphatidylinositol hydrolysis by PLCε.
Chelsea St. Germain; Purdue University, Chemistry | 1:40 pm — 2:00 pm
Characterization of the binding site of the zinc metalloprotease, Ste24
The human zinc metalloprotease ZMPSTE24 is a seven transmembrane domain protein that resides in the ER and inner nuclear membranes of most mammalian cells. Defects in the enzymatic function of ZMPSTE24 have been shown to cause premature aging disorders. The crystal structure of both the mammalian and yeast homologs have been solved revealing a novel surprising structure. Both contain seven transmembrane helices all surrounding a water-filled chamber which is capped on both sides, with the catalytic HExxH zinc-binding motif lying inside the chamber. In addition to cleaving the bonafide substrates (prelamin A in mammals and a-factor in yeast).Mammalian ZMPSTE24 is believed to play a protective role in both Type 2 diabetes and several viral infections by declogging the cellular translocon. ZMPSTE24 is also significantly inhibited by several HIV aspartyl protease inhibitors used as drug treatments for HIV patients and could be the cause of several of the side-effects of these drugs. As of now, no precise location or recognition pattern for substrate binding has been identified. Thus, the goal of this project is to localize important regions that comprise the binding site to define possible requirements for substrate binding in the yeast model protein Ste24 which functionally complements the activity of ZMPSTE24.My project aims to explore the binding site of Ste24 in three ways. First, the development of a biophysical assay for the direct analysis of Ste24 mutations on the binding affinity (Kd) to a-factor. Secondly, a targeted proteomics experiment is being utilized to precisely locate the area of interaction of the prenyl group of the substrate during Ste24 activity. Thirdly, analyses of conserved aspartate residues are being utilized to identify how HIV drugs are interacting with Ste24 to inhibit function, allowing for the future development of more targeted HIV drugs with fewer off-target side-effects. Thus far, these assays have shown promising results in evaluating the Kd of Ste24 mutants, the location of the prenyl group during Ste24 activity, and the role of the conserved aspartate residues.
Karthik Srinivasan; Purdue University, Biological Sciences | 2:00 pm — 2:20 pm
Characterizing Fabs for the isolation of the TOC Complex
Protein translocation across the chloroplast outer membrane is essential for photosynthesis in all green plants. This is because most chloroplast proteins (over 90%) are encoded in the nucleus, translated in the cytoplasm, and must be imported into the chloroplasts to perform their functions. The translocon on the outer chloroplast membrane (TOC) complex orchestrates this vital translocation process and consists of three components: Toc75, Toc33/34 and Toc159 with unknown stoichiometries. Our lab seeks to elucidate the structural architecture of the TOC complex to gain mechanistic insights into protein translocation in chloroplasts. Toc75 is a β-barrel membrane protein that forms the channel of the TOC translocon and our lab has previously reported a crystal structure of the N-terminal polypeptide transport-associated (POTRA) domains. In this work, we demonstrate the generation of antigen-binding fragments (Fabs) that specifically recognize the POTRA domains. Further, we characterize this interaction using size exclusion chromatography coupled with small angle X-ray scattering, isothermal titration calorimetry, and X-ray crystallography. Future directions include using these Fab fragments to pull down the TOC complex from natural sources which may then be used for structural elucidation using cryo electron microscopy.
Tupiwa Chiura; Saint Louis University, Chemistry | 2:20 pm — 2:40 pm
Resonance Raman unveils a tilted configuration for the cyano adducts of human heme oxygenase
Free iron protoporphyrin IX (heme) is extremely toxic to living cells, therefore, its effective catabolism is essential in human physiology. Human heme oxygenase (hHO-1) is an enzyme that degrades heme through a multi-step reaction into biliverdin, with the release of CO and ferrous iron. The mechanism of the heme degradation is complex and a subject of multiple studies. This reaction is stereospecific to the heme α -meso carbon, the specificity being facilitated by a hydrogen bond network cluster that comprises the aspartate 140 residue and two water molecules in the distal heme site. To probe this hydrogen bond network, we investigated the cyanide adducts of hHO-1 using resonance Raman spectroscopy (rR). Cyanide is highly anionic and sensitive to subtle changes in active site polarity and steric factors; therefore, it is an efficient probe of the active site perturbations. The rR studies of CN-adducts are reported and the assignment of modes associated with Fe-C-N fragment was supported using isotopic substitution and deconvolution procedures. It was found that the CN-adducts of wild-type hHO-1 and D140 mutants have tilted conformation and the tilting angle of the Fe-C-N linkage is much stronger than in other heme proteins. A larger than typical tilting angle can be reasonably associated with the self-oxidation reaction catalyzed by these enzymes.
Scientific Session 3: Investigating Human Disease with Novel Techniques
Dr. Shalini Low-Nam, Purdue University, Chemistry and Physics & Astronomy | 3:00 pm — 3:40 pm
Physical mechanisms of T cell signaling and activation at the single molecule, single cell level
T cells exquisitely discriminate between antigenic and self-peptide ligands presented on major histocompatibility complexes (pMHCs) despite subtle differences in affinities. This feature of adaptive immunity is required for robust, specific, and highly selective T cell activation and to avoid autoimmunity in the background of more abundant self pMHCs. We recently introduced a reconstitution-based molecular impulse-response assay that showed how T cell receptors(TCR) execute this single molecule precision in antigenic pMHC detection using a spatiotemporally-correlated mechanism which translates a series of input ligand:TCR binding events into activation responses. Cell-based immunotherapies reprogram T cells to eliminate cancer cells through the engineering of tumor-associated antigen-specific (TAA) chimeric antigen receptors (CARs). Many TAAs are epitopes of highly overexpressed cell surface proteins but are rarely tumor-exclusive. CARs have been highly effective in eliminating cancers of blood cells but continue to be minimally effective against solid tumors. The hyperactivation of CAR T cells, as well as off-target toxicity, have plagued the translation of mobilizing T cells as a therapeutic. We propose that a mechanistic, single molecule understanding of the CAR T cell input:response function is needed to optimally harness anti-tumor responses and minimize side effects. I will discuss our efforts to measure molecular activation thresholds in primary, anti-tumor CAR T cells.
Fan Xu; Purdue University, Biomedical Engineering | 3:40 pm — 4:00 pm
Three-dimensional super-resolution imaging in whole-cell and tissue specimens
Super-resolution fluorescence microscopy is a powerful tool in visualizing organelle structures, interactions, and protein functions in biological research. However, inhomogeneous refractive indices inside cells and tissues distort the fluorescent signal emitted from single-molecule probes, which rapidly degrades resolution with increasing depth. We developed an in situ PSF retrieval (INSPR) method that enables the construction of an insitu3D response of single emitters directly from single-molecule blinking datasets and therefore allows for pin-pointing their locations with Cramér-Rao lower bound achieving precision and fidelity in whole cells and tissues. This advancement expands the routine applicability of super-resolution microscopy from selected cellular targets near coverslips to intra-and extracellular targets deep inside tissues. The newfound capacity of visualization could allow for better understanding for neurodegenerative diseases such as Alzheimer’s, and many other diseases affecting the brain and various parts inside the body.
Muriel Eaton; Purdue University, MCMP | 4:00 pm — 4:20 pm
Substantial reduction of Scn2aexpressionrendersbehavioral abnormalities in mice indicative of autism spectrum disorder
Recent exome sequencing studies have discovered a strong correlation between the voltage-gated sodium ion channel Nav1.2 (encoded by gene Scn2a) and autism spectrum disorder (ASD), as well as other neurodevelopmental disorders. Nav1.2, together with Nav1.1 and Nav1.6, are major sodium channels expressed in the central nervous system playing major roles in contributing to neuronal excitability. Homozygous knockout of Scn2a-/- is perinatal lethal. Heterozygous Scn2a+/- mice display some mild abnormalities but do not seem to recapitulate severe disease phenotypes. To further explore the relationship between the dysfunction of Scn2a, ASD, and its comorbidities, our lab acquired a mouse model in which the expression of Scn2a is substantially reduced. We are currently conducting a behavioral battery to assess the phenotype of these mice including sociability, anxiety, and innate behavior. We plan to test both males and females to determine if there are sex differences. The data we obtain will justify that these mice can be used as a model to study severe ASD associated with Scn2a loss-of-function and its related comorbidities.
Marco Hadisurya; Purdue University, Biochemistry | 4:20 pm — 4:40 pm
Quantitative proteomics and phosphoproteomics of urinary extracellular vesicles define diagnostic and prognostic biosignatures for Parkinson's Disease
Compared to cancer, relatively lower genetic contribution to Parkinson's disease (PD)propels the search for protein biomarkers for early-detection of the disease. Utilizing 82 biological urine samples from the underlying populations-21 healthy individuals (control), 13 healthy individuals with G2019S mutation in the LRRK2gene (non-manifesting carrier/NMC), 28 PD individuals without G2019S mutation (idiopathic PD/iPD), and 20 PD individuals with G2019S mutation (LRRK2 PD), here we introduce a novel strategy to determine potential diagnostic and prognostic biomarkers for PD from urinary extracellular vesicles (EVs). After efficient isolation of urinary EVs through chemical affinity followed by mass spectrometric analyses of EV peptides and enriched phosphopeptides, we identified and quantified 4,480 unique proteins and 2,682 unique phosphoproteins. We established4panelsof proteins and phosphoproteins as novel candidates for disease, genotype, risk, and progression biomarkers, which were further substantiated using ROC, machine learning, and in-depth network analysis. These findings demonstrate a general strategy of utilizing biofluid EV proteome/phosphoproteome as an outstanding and non-invasive source for a wide range of disease exploration.
Day 2 | May 13th
Scientific Session 4: Fighting Bacterial Pathogens through Biophysics
Dr. Nicholas Noinaj; Purdue University, Biological Sciences | 9:00 am — 9:40 am
The β-barrel Assembly Machinery in Motion
β-barrel outer membrane proteins (OMPs) are found within the outer membranes (OM) of Gram-negative bacteria and are essential for nutrient import, signaling, and adhesion. While the exact mechanism for the biogenesis of these OMPs is unknown, it is known that a 200 kDa five component complex called the β -barrel assembly machinery (BAM) complex is responsible for this task. We have previously used X-ray crystallography and MD simulations to establish that BamA, the central and essential component, may serve as a catalyst on the membrane to reduce the energy required for insertion of new OMPs. Further, we have shown that lateral opening of the barrel domain of BamA is required for function, suggesting a route for the insertion of new OMPs directly into the membrane. Despite these studies, it is known that the BAM complex functions most efficiently when fully assembled. To gain insight into this intriguing mechanism, recently, we reported the structure of the BAM complex from E. coli, revealing unprecedented conformational changes in the barrel domain of BamA, which may be regulated by the accessory proteins BamB, C, D, and E. The periplasmic domain of BamA was found in a closed state that prevents access to the barrel lumen from the periplasm, indicating substrate OMPs likely do not enter the barrel during biogenesis, but rather may be inserted directly at the lateral gate. In this talk, I will review the previous studies with BamA, present the new recent structural studies of the fully assembled BAM complex, and put forth two plausible mechanisms for how the BAM complex may function in the biogenesis of new OMPs.
Shalini Iyer; Purdue University, Chemistry | 9:40 am — 10:00 am
Modulation of host signaling by two syntenic genes of Legionella pneumophila
Legionella pneumophilais a facultative intracellular pathogen that uses the Dot/Icm T4SS to translocate a vast number of Icm/Dot-translocated substrates (IDTS) (commonly known as effectors) into its host in order to establish a safe, replicative intracellular environment for its survival. The phagocytosed bacteria reside in a vacuolar structure known as the Legionella-containing vacuole (LCV) within the host cells, sweet-talk the host factors in a multitude of different ways and quickly subvert a variety of signaling events. These effectors also contribute to bacterial virulence by positively or negatively regulating the activity of one another. A recently discovered pair of such proteins with opposing activities is MavC and MvcA, two co-directionally encoded effectors found on the same genetic locus in Legionella pneumophila. Despite weak sequence similarity both MavC and MvcA have extremely homologous structures and interact with Ube2N,an important E2-conjugating enzyme central to the strategy employed by the pathogen to suppress host immune response and ensure its longevity within the host. I will be presenting the crystal structures of MavC in complex with its substrate as well as the crosslinked product. These structures provide insights into the enzyme’s unique ubiquitinating activity via a transglutaminase mechanism, which is very different from the canonicalE1/E2/E3 pathway. I will also be presenting the underlying protein dynamics involved and the crucial role played by the structurally unique insertion domain towards the exquisite specificity of MavC towards Ube2Nover other structurally similar E2 proteins.
Ravi Yadav; Purdue University, Biological Sciences | 10:00 am — 10:20 am
Structural insight into dual function of Neisserial lactoferrin binding protein B
Lactoferrin binding protein B (LbpB) is a surface exposed lipoprotein that plays dual rolesin iron import from host lactoferrin protein, and protection against antimicrobial cationic peptide lactoferricin. LbpB is an attractive target for antimicrobial drugs and vaccine design. However, molecular mechanisms of LbpB’s functions are unclear. In the current study, we determined the structures of Neisserial LbpBs in complex with human lactoferrin using X-ray crystallography and cryo-electron microscopy. The structures depict the bi-lobe architecture of LbpB and revel the binding interface with the lactoferrin. Structural and functional analysis of the LbpB proteins indicate that iron-loaded lactoferrin selectively binds to N-lobe of LbpB. In contrast, we show that lactoferricin binding site is in the C-lobe of LbpB. Furthermore, our data suggests that lactoferrin and lactoferricin binding to LbpB are independent. Together, these results provide insights into lactoferrin and lactoferricin recognition, and create a framework for understanding the dual function of LbpB protein.
Vatsal Purohit; Purdue University, Biological Sciences | 10:20 am — 10:40 am
Ionic Inhibition utilized to develop a pH-dependent reaction triggering method in PmHMGR crystals
HMG-CoA reductase (Pseudomonas mevalonii, PmHMGR) utilizes mevalonate and cofactors coenzyme-A (CoA) and NAD in a complex mechanism involving two hydride transfers with cofactor exchange, accompanied by large conformational changes by a 50-residue subdomain, to generate HMG-CoA. Details about this mechanism such as the conformational changes that allow intermediate formation, cofactor exchange and product release remain unknown. The formation of the proposed intermediates has also not been observed in structural studies with natural substrates. Having been shown to be an essential enzyme for the survival of gram-positive antibiotic resistant pathogenic bacteria, studying its mechanism in detail will be beneficial in developing novel antibacterials. The enzyme has been shown to be catalytically active inside the crystal with dithio-HMG-CoA and NADH but curiously is found to be inactive in the reverse direction in the structure bound to mevalonate, CoA and NAD. To understand the factors limiting activity in the HMGR crystal with mevalonate, CoA and NAD, we studied the effect of the major ionic components in the crystallization environment on enzymatic activity. By measuring its turnover, we observed a strong inhibition in PmHMGR activity in the crystallization buffer in comparison to optimized activity assay conditions. The inhibition was also found to be dependent on ionic concentration of the crystallization precipitant ammonium sulfate and independent of its ionic composition. Using crystallographic studies, we have determined that the crystal environment inhibits the mevalonate oxidation step and if overcome can allow interaction between the proposed reaction intermediates. Using pH-rate profile measurements we observed an increase in enzyme turnover with pH. We have demonstrated using UV-Vis spectroscopic measurements in PmHMGR crystals that we can initiate mevalonate oxidation in a modified crystal environment using pH-changes. The utilization of ionic inhibition in a crystalline environment in combination with the change in turnover with pH, can hence be utilized as a novel approach for triggering the PmHMGR reaction in crystals and capturing information about unknown intermediate states along the reaction pathway.
Scientific Session 4: Design and Characterization of Peptide Analogs
Dr. Daniel Flaherty; Purdue University, MCMP | 1:00 pm — 1:40 pm
Optimization and structural studies of inhibitors for bacterial carbonic anhydrases
Currently the pace of new antibiotic development is lagging behind that of which bacteria are developing resistance to approved classes of therapeutics. To add to the problem it has not been since the early 2000’s that a novel antibiotic scaffold was approved by the FDA. All new antibiotics brought to market are derivations of existing scaffolds that hit established targets for which resistance is already documented. Thus, there is an urgent need to identify: 1) novel antibacterial targets and 2) novel molecular scaffolds. To this end our lab has shown that human carbonic anhydrase inhibitors are detrimental to the growth of two high-priority drug-resistant pathogens: vancomycin resistant enterococcus (VRE) and N. gonorrhoeae. We have since synthesized and tested over 60 analogs and assessed them for antimicrobial efficacy, in vitro potency against the bacterial carbonic anhydrase, and shown that they are effective at treating infections in murine models. Finally, we have recently solved ligand bound structures of analogs to the N. gonorrhoeae carbonic anhydrase that suggest a novel binding mode not previously observed for carbonic anhydrase inhibitors. Taken together our studies have validated bacterial carbonic anhydrases as new antibiotic targets and developed new scaffolds to combat drug-resistant pathogens.
Nicolas Varas; Indiana University, Biochemistry & Molecular Biology | 1:40 pm — 2:00 pm
A new fibrillation-resistant glucagon analogue
One of the main side effects of Insulin treatment in diabetic patients is the high rate of hypoglycemic episodes. Most of these episodes are manageable by sugar ingestion, but their continuous occurrence increases the probability of ischemic events, arrhythmias, neurological damage, coma, and even death[1, 2]. Consequently, the risk of hypoglycemia remains the most significant obstacle that prevents insulin-dependent patients from attaining acceptable metabolic control. A major restraint for advancements comes from the lack of hyperglycemic agents to counteract the exogenous insulin action. Glucagon is the natural counter-regulatory hormone, which fulfills its role by binding in a α-helical structure to glucagon receptor and subsequently stimulating hepatic glycogenolysis and gluconeogenesis[3] . Previous clinical studies have shown a significant decrease in hypoglycemic events when using a dual-hormone, insulin/glucagon, artificial pancreas [4-6]. But, despite its great potential as a drug, native glucagon has low solubility at physiological pH, it is prone to chemical degradation under acidic or basic buffers and has an intrinsic propensity to rapidly form β-amyloid fibrils, which nullify its activity and can make it cytotoxic [7-10]. To address this problem our group is exploring different approaches to create novel, stable, and fibrillation-resistant glucagon analogs that could be suitable for dual-hormone pumps. One of those methods is to lock-in an α-helix turn through side-chain cyclization, particularly a lactam side-chain bond at positions i,i+4. We generated glucagon analogs by solid-phase peptide synthesis and induce side-chain cyclization using selective deprotection with subsequent amide bond formation. This strategy gave us a glucagon analogue with fibrillation-resistance for at least 11 days at 100μM in PBS buffer, under agitation and 37°C. This analog proved to be active in vitro and in vivo, even after a week of agitation at 37°C. To date, there is no glucagon analog or formulation commercially available for use in dual-hormonal pumps, and the need for an ultra-stable game-changing analog that moves beyond emergency hypoglycemia rescue is still present[6] . We hope this lactam restrained glucagon will be the base for a new therapeutic with the goal of improving the therapeutic management of Type 1 Diabetes Mellitus.
Austin Cool; The Ohio State University, Chemistry & Biochemistry | 2:00 pm — 2:20 pm
Computational Methods Elucidate Consequences of Mutations and Post-Translational Modifications on Troponin I Effective Concentration to Troponin C
Ca2+ binding to cardiac troponin C (cTnC) causes a conformational shift that exposes a hydrophobic patch (cTnCHP) for binding of the cTnI switch peptide (cTnISP), ultimately resulting in contraction of the heart. The inhibitory peptide (cTnIIP), attached at the N-terminal end of the cTnISP, serves as a tether for the cTnISP to the rest of the troponin complex. Due to this tethered nature, the cTnISP remains within proximity of the hydrophobic patch region, resulting in the cTnCHP experiencing an elevated “effective concentration” of the cTnISP. Mutations to the cTnIIP region have been hypothesized to cause disease by effecting the ability of the cTnISP to ‘find’ the hydrophobic patch, resulting in alterations to the heart’s ability to contract normally. We tested this hypothesis using molecular dynamics (MD) simulations of the troponin complex using a model that contained all three subunits of troponin: C, I, and T (cTnT). We developed methods that allowed us to quantitatively measure the effective concentration of the cTnISP from the simulations. A significant reduction in cTnISP effective concentration was observed when the cTnIIP was removed from the system, showcasing the importance of a tethered cTnISP. Through accelerated MD methods, we proposed the minimum effective concentration of a tethered cTnISP to be approximately 21mM. Modification of the cTnIIP via PKC mediated phosphorylation of T143 was shown to significantly increase the estimated effective concentration of cTnISP, help the cTnISP find the cTnCHP faster, and maintain the relative shape of the cTnIIP when compared to the native model. All of this data indicates that pT143 may be able to help promote binding of cTnISP to the cTnCHP. We then tested six mutations within the cTnIIP region that are known cTnC Ca2+ sensitizing mutations, and have been linked with cardiomyopathy. We did not observe a significant reduction in effective concentration upon introduction of these mutations, however we did observe increased variability in the flexibility and dynamics of the cTnIIP region when compared to native. Our observations led us to hypothesize that the mechanism by which these cardiomyopathic mutations effect Ca2+ sensitivity is by altering the off rate of cTnISP from the hydrophobic patch.
Christine Muli; Purdue University, MCMP | 2:20 pm — 2:40 pm
Binding Site Discovery of a Peptidomimetic Probe on Proteasome Ubiquitin Receptor, Rpn-13
Targeting the ubiquitin-proteasome system has greatly contributed to the treatment of hematological cancers over the past fifteen years. Despite the success of these proteasome-targeted drugs (such as bortezomib, Btz) that inhibit the proteasome’s catalytic function, eventual drug resistance and off-target toxicities demand for alternative therapies to treat patients that become resistant to current treatments. To overcome the challenges with current therapeutics, many groups have begun targeting the regulatory subunits of the proteasome.Rpn-13, a secondary ubiquitin receptor on the proteasome’s regulatory particle, has been one of the most promising targets as it is not an essential proteasome subunit in healthy cells but has been shown to be required for cell survival of hematological cancers. Our long-term goal is to develop Rpn-13 inhibitors with improved potency and physicochemical properties that bind to the same biologically-relevant surface asRpn-13’s only selective inhibitor,KDT-11. To accomplish this, we have conducted numerous biophysical studies with fluorescence polarization, circular dichroism, cross-linking, mass spectrometry, and protein NMR to identify KDT-11’s binding site on the non-catalytic ubiquitin receptor. This helical peptoid probe was discovered to bind within the Pru domain at a surface different from any of Rpn-13’s known protein-protein interactions, suggesting that binding at this site decreasesRpn-13’s ability to perform its proteasomal function. Determination of the peptoid probe’s binding site allows us and others for rational-based drug design for new scaffolds ofRpn-13 inhibitors. This not only contributes a newly discovered mechanism of inhibition for Rpn-13 but also provides a thorough guide for binding site identification of poorly soluble peptide/peptidomimetic probes on non-catalytic protein targets.
Scientific Session 5: Innovative Biophysical Methodologies
Dr. Fang Huang; Purdue University, Biomedical Engineering | 3:00 pm — 3:40 pm
Revealing Subcellular Structures with Live-cell and 3D Fluorescence Nanoscopy
We are in an exciting era of biomedical imaging where the inner-workings of cells and tissues can be explored by rapidly developing imaging methods. Labeling specificity and live cell compatibility make fluorescence microscopy an important tool in biomedical research. Its resolution, however, is limited by diffraction to ~250 nm, preventing us from resolving detailed structures within the cell. The recent advent of single molecule switching nanoscopy methods (SMSN, also known as PALM/STORM), overcomes this fundamental limit by stochastically switching single fluorophores on and off so that their emission events can be localized with high precision resulting in a reconstructed image with down to ~25 nm lateral resolution. However, its application has been largely limited to fixed and flat samples due to the poor temporal resolution, inferior resolution in z, and rapidly deteriorating resolution in thick samples. In this talk, I will present some of our most recent developments which synergistically combine newly available sensors/devices such as sCMOS cameras and deformable mirrors, analytical methods such as deep learning and novel instrumentation to allow SMSN imaging in live cells and tissue specimens. Dr. Huang will demonstrate the capabilities of these new imaging systems in revealing the fine details of subcellular structures from a diverse set of biological systems including viruses, bacteria, yeasts, mammalian cells and brain sections.
Andrew DeMarco; Purdue University, Biochemistry | 3:40 pm — 4:00 pm
Phosphatase and Kinase Substrate Specificity Profiling with Pool Synthetic Peptides and Mass Spectrometry
Reversible phosphorylation is a pervasive regulatory event in cellular physiology controlled by reciprocal actions of protein kinases and phosphatases. Determining the inherent substrate specificity of kinases and phosphatases is essential for understanding their cellular roles. Synthetic peptides have long served as substrate proxies for defining intrinsic kinase and phosphatase specificities. Here, we describe a high throughput protocol to simultaneously measure specificity constants (kcat/KM) of many synthetic peptide substrates in a single pool using label-free quantitative mass spectrometry. The generation of specificity constants from a single pooled reaction provides a rigorous and rapid comparison of substrate variants to help define an enzyme’s specificity. Equally applicable to kinases and phosphatases, as well as other enzyme classes, the protocol consists of 3 general steps: 1) reaction of enzyme with pooled peptide substrates, each ideally with a unique mass and at concentrations well below KM, 2) analysis of reaction products using liquid chromatography-coupled mass spectrometry (LC-MS), and 3) automated extraction and integration of elution peaks for each substrate/product pair. We incorporate an ionization correction strategy allowing direct calculation of reaction progress, and subsequently kcat/KM, from substrate and product peak areas in a single sample, obviating the need for stable isotope labeling. Peptide consumption is minimal, and high peptide purity and accurate concentration measurements are not required. Access to a high-resolution LC-MS system is the only non-standard equipment need. We present an analysis pipeline consisting entirely of established open-source software tools and demonstrate proof of principle with the highly selective cell cycle phosphatase Cdc14 from multiple fungal pathogen orthologs.
Ryan Benke; Purdue University, Biochemistry | 4:00 pm — 4:20 pm
Using Metabolomics and Association Genetics to Map Lesion Mimic Mutants
Plant mutants that form lesions and/or undergo spontaneous cell death in the absence of pathogens or stress have been isolated from a wide variety of genetic systems. These mutants form spontaneous lesions due to constitutively active disease signaling or defects in metabolism that result in cell death. Of the more than 50 maize lesion-forming mutants, the etiologies of only four of these are known. Using untargeted metabolite profiling we have explored the metabolic consequences of 24 maize lesion-forming mutants and compared them to mutants with constitutively activated hypersensitive-response (Rp1-D21) and defects in chlorophyll metabolism (e.g. Oy1-N1989). Clustering on metabolite abundances clearly indicates a metabolic syndrome similar to the HR-signaling mutant Rp1-D21 in a subset of these 24 mutants. The dominant mutant Les10 exhibits similar shifts in metabolite levels, including predicted defense metabolites (e.g. salicylic acid) and chlorophyll breakdown products, as observed in Rp1-D21.The Les10 gene was previously mapped to chromosome 2L using translocation stocks and mutant linkage. We used genome-wide association to explore whether natural variation encoded suppressor or enhancer alleles affecting Les10 phenotypic severity. An F1-hybrid population was generated by crossing an association panel by Les10/+pollen parents and phenotyped for lesion severity, chlorophyll content, and plant height. The polymorphisms with the strongest association with these traits mapped to the region encoding Les10, suggesting that a cis-QTL affecting the Les10 locus itself may be responsible for the largest genetic effect on the mutant phenotype in this population. The top SNP is near anankyrin-domain containing gene similar to Arabidopsis ACCELERATED CELL DEATH6. Adominant allele of acd6 in Arabidopsis undergoes spontaneous cell death, suggesting this maize gene is a good gene candidate for Les10.
Soutick Saha; Purdue University, Physics & Astronomy | 4:20 pm — 4:40 pm
Capacity to multitask limits cellular chemotaxis
Chemotaxis is the biased movement of cells in response to chemical gradients. Cellular signal processing capacity has been thought to be important to cells’ chemotaxis behavior. However, it still remains elusive how cells sense and decipher multiple chemical cues. In this study, we test a hypothesis that the chemotactic performance of cells is constrained by the capacity to “multitask.” Specifically, if the intracellular signal processing capacity to respond to multiple cues is saturated, the effect of chemoattractants can become antagonistic rather than synergistic or even independent. We experimentally investigate the migratory behavior of two types of cancer cells under single and combined cues of chemoattractants –transforming growth factor-beta and epidermal growth factor. For both cell types, the results show that the combination of the two attractants suppresses the chemotactic performance of cancer cells even though cells use independent receptors for sensing the two signals away from the receptor saturation regime. We show that both a mutual repression and a convergent pathway mechanism can explain antagonism behavior but only the convergent pathway mechanism correctly predicts that (i) chemotactic performance can alternatively be suppressed by elevated background signal on top of a gradient and (ii) the speed of migration remains unchanged when different kinds of signals are introduced. We confirm these predictions with further experiments on both cell lines. Our framework provides new insight into the intracellular mechanism of signal processing and allows one to predict cellular response under complex chemical cues.