Stefan Stoll, Ph.D. - US grants

DESCRIPTION (provided by applicant): Substantial evidence implicates EGF-like growth factors (GFs) in promoting the survival, proliferation, migration and invasiveness of normal and malignant epithelial cells. Several different signaling pathways have been shown to promote shedding of membrane-bound EGF-like GFs. Our published data demonstrate that metalloproteinases (MPs) are a major stimulus for ErbB signaling, ERK activation and subsequent gene expression in human skin organ culture. Our preliminary data indicate that MPs are required for autocrine ERK activation in normal human keratinocytes (NHK). They also demonstrate that MP inhibitors block proliferation and wound-induced migration of NHK, and that different ligands appear to be involved in LPA stimulation, autocrine proliferation, and wound-induced migration of NHK. Together, these data indicate that autocrine keratinocyte (KC) signaling requires context-dependent cleavage of membrane-bound ErbB ligands by MPs. The MP(s) involved in transactivating ErbB receptors in skin and KCs have not yet been elucidated. Several members of the ADAM family are strongly implicated in ectodomain processing of various membrane-bound proteins. ADAM10 was recently shown to mediate the basal as well as G protein-coupled receptors (GPCR)-stimulated activation of EGF receptors in mammary epithelial cells. Our preliminary data indicate that ADAM10 is abundantly expressed in NHK, and that expression of the active form of ADAM10 correlates with EGF-independent migration and ERK activation. Based on these observations, we hypothesize that KCs utilize ADAM10 to catalyze shedding of one or more transmembrane EGF-like GF precursors in a context-dependent manner, leading to stimulus-specific KC responses. To test this hypothesis, we propose the following specific aims: Specific Aim 1: To (a) determine which ErbB receptor ligands are shed from KC via MP-dependent proteolysis in three cellular contexts (LPA stimulation, autocrine proliferation, and wound-induced migration), and (b) to elucidate the context-dependent functions of the various autocrine ErbB ligands identified in Aim la. Specific Aim 2 determines whether and in which contexts ADAM10 is involved in the shedding of ErbB ligands in human KCs.

DESCRIPTION (provided by applicant): Hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels were first discovered in photoreceptors where they shape the light response. They exhibit several properties that make them specialized for retinal signaling: 1) they are activated by membrane hyperpolarization instead of depolarization, 2) they are regulated by the direct binding of cyclic nucleotides to an intracellular domain, and 3) they are expressed in the distal dendrites of neurons. Recently an accessory subunit of HCN channels in photoreceptors and other neurons was discovered called TRIP8b that has a profound effect on each of these important channel properties. Our long term goal is to understand the molecular mechanisms for these properties. In previous funding periods we have made great progress toward achieving this goal. We have solved the X-ray crystal structure of the cyclic nucleotide-binding domain of HCN2 and the structure of TRIP8b bound to HCN2. We have also invented three ground-breaking new fluorescence methods that allow us to record molecular rearrangements in intact channels simultaneous with electrophysiological recording. In this funding period, we propose to combine these methods with double electron-electron resonance (DEER), a powerful magnetic resonance-based method, and molecular dynamics simulations, to measure and model the structure and dynamics of the HCN channel and its interaction with TRIP8b. These experiments will lead to the first dynamic picture for how HCN channels regulate the excitability of photoreceptors and other neurons.

In this project funded by the Chemical Measurement and Imaging program of the Chemistry Division, Professor Stefan Stoll of the University of Washington is developing high-sensitivity instrumentation and methodology for the determination of the nanoscale structure of flexible proteins. The flexibility of proteins is a prerequisite for their activity, but is difficult to quantify experimentally. The methods developed in this project provide tools to accurately probe fundamental chemical processes underlying life at the molecular level. The broader impacts include potential benefits to human health, as the molecular-level knowledge attainable through the methods developed in this project furthers our ability to rationally design new drugs and therapies.

This project focuses on instrumentation development of high-sensitivity pulse electron paramagnetic resonance (EPR) spectroscopies for biostructural applications, specifically double electron-electron resonance (DEER) spectroscopy. DEER is a pulse EPR technique that can quantitatively determine protein conformational distributions and flexibility by measuring nanometer-scale distances between spin labels attached to proteins. The research approach consists of three thrusts: (1) Develop a pulse EPR spectrometer with improved sensitivity for DEER and related methods to be able to accurately measure conformational landscapes in proteins and complexes even at very low concentrations. (2) Quantify protein conformational change in a small soluble ligand-binding protein to benchmark the methodology and to gain insight into conformational dynamics and equilibria involved in ligand binding. (3) Elucidate the conformational landscape in fragments of a cardiac ion channel, in order to test and validate the methodology on a more complex membrane protein system.

Project Summary/Abstract We propose to upgrade the existing DEER/EPR spectrometer at the University of Washington (UW) to achieve higher sensitivity and higher sample throughput. DEER is a structural technique for the quantification of protein conformational landscapes and protein motions on the nanometer scale. Protein motions underlie the molecular processes at the basis of human life and disease. Therefore, DEER is a crucial technique that provides insights that contribute to the knowledge base necessary for drug development. In combination with X-ray crystallography, NMR and cryo- electron microscopy, DEER is part of a complementary set of experimental biostructural tools. It is especially important for the study of membrane proteins. The number of University of Washington (UW) researchers active in the area of biostructural and molecular biomedical sciences has increased substantially in recent years. There is increased demand for DEER measurement time from a variety of NIH-funded researchers at UW. The existing DEER instrument is old and not able to meet the increased demand due to its inherently low sensitivity. The upgrade requested in this project will (a) dramatically enhance sample throughput to provide more DEER data to more investigators, (b) provide increased resolving power with respect to conformational populations and conformational changes in proteins, (c) provide significantly higher sensitivity that will allow the investigation of proteins and protein complexes at lower concentrations, including full-length membrane proteins. This will expand the scope of systems that can be studied. Research projects that will benefit from the new upgraded DEER capabilities include the study of the molecular mechanisms of allosteric regulation in a broad range of ion channels (HCN, KCNH, TRPV1), the development of new molecular probes for protein kinases from the SRC family, the study of ubiquitin ligases from pathogenic bacteria, and the evaluation of de novo designs of proteins for health applications. Overall, the upgrade will significantly expand the scope of biostructural research at UW, which is conducted in a collaborative multidisciplinary environment.

PROJECT SUMMARY/ABSTRACT This project develops new computational analysis and modeling methods for DEER (double electron- electron resonance) spectroscopy. DEER is a biostructural technique for the quantification of protein conformational landscapes and protein motions on the nanometer scale. Protein motions are crucial for many key molecular processes at the basis of human life and disease. Therefore, DEER provides important insights that contribute to the knowledge base necessary for drug development. In combination with X-ray crystallography, NMR and cryo-EM, DEER is part of a complementary set of integrative experimental biostructural tools. It is especially important for the study of membrane proteins. A major barrier in the field is the lack of integrated analysis tools for biomedical researchers. This project addresses this issue by (a) developing methods and tools based on Bayesian statistics for the rigorous and reproducible analysis of experimental DEER data, providing comprehensive methods for uncertainty quantification (error bars) in DEER data, and (b) creating advanced computational approaches that utilize DEER data in the refinement of protein structures based on atomic-resolution ensemble models. Overall, the goal of the project is to significantly accelerate the workflow of DEER data analysis, interpretation, and modeling and to increase its rigor, reproducibility and accessibility. This will enable the study of the structure and dynamics of larger and more complex proteins and protein assemblies, which are increasingly important in biomedical research.

Cyclic nucleotide-regulated ion channels are exquisite molecular machines that underlie important physiological functions. Cyclic nucleotide-gated (CNG) channels generate the primary electrical response to light in photoreceptors and to odorant in olfactory receptors. The related hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the pacemaker activity of the heart and many neurons in the brain. These cation selective channels are opened by the direct binding of cyclic nucleotides (cAMP and cGMP) to an intracellular domain of the channel. Our goal is to reveal the molecular mechanism for this allostery in CNG channels. Our approach will be to study bacterial CNG channels as a model system for the eukaryotic channels because of the huge advantages they provide for our biochemical methods . We will leverage the power of four different methodologies to determine the structure, conformational heterogeneity, and dynamics of these channels: 1) cryoelectron microscopy (cryo-EM), 2) double electron-electron resonance (DEER), 3) microfluidic rapid freeze quench (µRFQ) in combination with DEER, and 4) Rosetta-based molecular modeling. The proposal includes four investigators who are pioneers in each of these methods. The use of all four methods on the same ion channel under the same conditions is synergistic and ultimately will lead to a comprehensive structural and energetic model for the allostery of this channel. Ultimately a molecular understanding of these channels would inform not only the physiology and pathophysiology of the heart and brain, but also the general mechanisms for allosteric control of many enzymes.

PROJECT SUMMARY/ABSTRACT This project develops new computational analysis and modeling methods for DEER (double electron-electron resonance) spectroscopy. DEER is a biostructural technique for the quantification of protein conformational landscapes and protein motions on the nanometer scale. Protein motions are crucial for many key molecular processes at the basis of human life and disease. Therefore, DEER provides important insights that contribute to the knowledge base necessary for drug development. In combination with X-ray crystallography, NMR and cryo-EM, DEER is part of a complementary set of integrative experimental biostructural tools. It is especially important for the study of membrane proteins. A major barrier in the field is the lack of integrated analysis tools for biomedical researchers. This project addresses this issue by (a) developing tools based on Bayesian statistics for the rigorous and reproducible analysis of experimental DEER data, providing comprehensive methods for uncertainty quantification (error bars) in DEER data, and (b) creating advanced computational tools to utilize DEER data in the refinement of protein structures based on atomic-resolution ensemble models. Overall, the goal of the project is to significantly accelerate the workflow of DEER data analysis, interpretation and modeling and increase its rigor, reproducibility and accessibility. This will enable the study of the structure and dynamics of larger and more complex proteins and protein assemblies, which are increasingly important in biomedical research.