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Principal Investigator: Benjamin James Wylie
Organization: TEXAS TECH UNIVERSITY
Fiscal Year: 2024
Award: $428,400
Funding agency: National Institute of General Medical Sciences
Inward-rectifier K+ (Kir) channels and G protein-coupled receptors (GPCRs) are membrane proteins that are
regulated by cholesterol and anionic lipids found in their native membranes. We will use solid-state NMR
(SSNMR) to study proteins with functional lipids in bilayer environments ranging from proteoliposomes to
biological membranes. These measurements will compliment functional assays, fluorescence techniques, and
molecular dynamics (MD) simulations under identical conditions. Kir channels are involved in long-QT syndrome,
hypoglycemia, Bartter’s syndrome, epilepsy, substance abuse, and periodic paralysis. Kir Channels are ligand
gated, but details of the structure and dynamics of gated channels are largely unknown. The Kir2 channel family
is gated by the anionic lipid phosphatidylinositol 4,5-bisphosphate (PIP2) but inactivated by cholesterol which
competes with PIP2 to access the protein. G protein-activated Kir channels (GIRK, Kir3) are gated by the coaction
of PIP2 and Gbγ protein heterodimers. In the Kir3 family, cholesterol increases rather than suppresses activity.
Here we will explore the differing roles of functional lipids and quantify the structure and dynamics of the observed
active and inactivated states. We will continue our studies of the Kir channel, KirBac1.1. We assigned 90% of
the 15N and 13C chemical shifts in this protein (over 1600 unique heavy atoms) and used these assignments to
identify allostery, the activation mechanism, the inactivated structure bound to a cholesterol dimer, refined the
structure of the closed state, and solved the structure of the open state of the channel. Now we will measure the
channel dynamics and identify the multiple gated states of the channel reflected in our data. We will study
structural changes in the channel under voltage and identify discrete channel states and lipid contacts using
freeze-trapped Dynamic Nuclear Polarization. In tandem, we will also study the Kir3.1-KirBac1.3 channel
chimera. Preliminary data identifies PIP2 binding residues and membrane-water interfacial residues key for
channel function. The eventual goal will be the mammalian Kir3.2 (GIRK2) channel and its full complement of
functional activators. In a second project we will study the CC motif chemokine receptor CCR3 with the CCL11
chemokine in lipid bilayers. No drug trial targeting CCR3 has succeeded, which is unfortunate as it is involved in
cancer metastasis, HIV entry, and the COVID19 cytokine storm. To date, we identified both CCL11 docking, and
signal transduction are dose dependent upon bilayer cholesterol. Preliminary SSNMR studies found cholesterol
conformationally selects for optimal ligand binding configurations of the receptor. We plan to fully assign the 15N
and 13C chemical shifts of CCR3 in cholesterol and anionic lipid enriched membranes. The structures of this
protein with CCLL11 in different functional states will be solved, and regional dynamics measured following a
similar workflow established for KirBac1.1. NMR will also be used to solve the structures of CCL11 in solution
and in complex with CCR3. We will pursue cholesterol oligomerization, CCR3 dimerization, and the relationship
between these events. Throughout we will examine lipid oligomerization, dynamics, and protein affinity.
Terms: <AD dementia><AIDS Virus><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome Virus><Affinity><Alzheimer Type Dementia><Alzheimer disease dementia><Alzheimer sclerosis><Alzheimer syndrome><Alzheimer's><Alzheimer's Disease><Alzheimers Dementia><Assay><Barter's Disease><Bartter Disease><Bartter Syndrome><Bartter syndrome (BS)><Binding><Bioassay><Biochemical><Biological><Biological Assay><CC CKR3><CC chemokine receptor 3><CCL11><CCL11 gene><CCR3 receptor><COVID-19 associated cytokine storm><COVID-19 cytokine storm><COVID-19 induced cytokine storm><COVID-19 related cytokine storm><Cell Communication and Signaling><Cell Membrane Lipids><Cell Signaling><Chemical Dependence><Chemicals><Chemokine Receptor Gene><Chemotactic Cytokines><Chimera><Chimera organism><Cholesterol><Complement><Complement Proteins><Complex><Data><Development><Dimerization><Disease><Disorder><Docking><Dose><Drug Addiction><Drug Dependence><Drug Dependency><Dysfunction><Environment><Epilepsy><Epileptic Seizures><Epileptics><Event><Family><Fluorescence><Freezing><Functional disorder><G Protein-Complex Receptor><G Protein-Coupled Receptor Genes><G-Protein-Coupled Receptors><G-Proteins><GIRK2><GIRK2 subunit, G protein-coupled inwardly-rectifying potassium channel><GPCR><GTP-Binding Proteins><GTP-Regulatory Proteins><Goals><Guanine Nucleotide Coupling Protein><Guanine Nucleotide Regulatory Proteins><HIV><Homologous Chemotactic Cytokines><Human><Human Immunodeficiency Viruses><Hydrogen Oxide><Hypoglycemia><Inositide Phospholipids><Inositol Phosphoglycerides><Inositol Phospholipids><Intercrines><Intracellular Communication and Signaling><K channel><LAV-HTLV-III><Ligand Binding><Lipid Bilayers><Lipids><Long QT Syndrome><Lymphadenopathy-Associated Virus><MGC22554><Measurement><Measures><Membrane><Membrane Lipids><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Metastasis><Metastasize><Metastatic Lesion><Metastatic Mass><Metastatic Neoplasm><Metastatic Tumor><Modern Man><Molecular><Molecular Configuration><Molecular Conformation><Molecular Dynamics Simulation><Molecular Interaction><Molecular Stereochemistry><Neoplasm Metastasis><Nuclear><PIP2><Paralysis Agitans><Parkinson><Parkinson Disease><Phosphatidyl Inositol><Phosphatidylinositol 4,5-Biphosphate><Phosphatidylinositol 4,5-Diphosphate><Phosphatidylinositol-4,5-Bisphosphate><Phosphatidylinositols><Phosphoinositides><Physiopathology><Potassium Channel><Potassium Ion Channels><Primary Parkinsonism><Primary Senile Degenerative Dementia><Protein Dimerization><Proteins><PtIns 4,5-P2><PtdIns><PtdInsP2><Receptor Protein><Role><SARS-CoV-2 associated cytokine storm><SARS-CoV-2 cytokine storm><SARS-CoV-2 induced cytokine storm><SARS-CoV-2 related cytokine storm><SCYA11><SIS cytokines><SSNMR><Secondary Neoplasm><Secondary Tumor><Seizure Disorder><Severities><Signal Transduction><Signal Transduction Systems><Signaling><Structure><Substance abuse problem><Surface Proteins><System><Techniques><Virus-HIV><Water><abuse of substances><aldosteronism-normal blood pressure syndrome><autoinflammatory><biologic><biological signal transduction><cancer metastasis><chemoattractant cytokine><chemokine><chemokine receptor><chimeras><complementation><conformation><conformational><conformational state><conformationally><conformations><coronavirus disease 2019 associated cytokine storm><coronavirus disease 2019 cytokine storm><coronavirus disease 2019 induced cytokine storm><coronavirus disease 2019 related cytokine storm><developmental><dimer><epilepsia><epileptogenic><hypoglycemic><hypoglycemic episodes><insight><interfacial><juxtaglomerular hyperplasia syndrome><ligand gated channel><lipid bilayer membrane><membrane structure><molecular dynamics><pathophysiology><periodic paralysis><primary degenerative dementia><protein structure><protein structures><proteins structure><proteoliposomes><receptor><senile dementia of the Alzheimer type><severe acute respiratory syndrome coronavirus 2 associated cytokine storm><severe acute respiratory syndrome coronavirus 2 cytokine storm><severe acute respiratory syndrome coronavirus 2 induced cytokine storm><severe acute respiratory syndrome coronavirus 2 related cytokine storm><social role><solid state NMR><solid state nuclear magnetic resonance><substance abuse><targeted drug trials><targeted pharmaceutical trials><targeted therapy trials><targeted treatment trials><tumor cell metastasis><voltage>