G protein-coupled receptors (GPCRs) are largest family of membrane receptor protein and the target of most pharmaceuticals made today. The first step in the activation mechanism of most GPCRs is the binding of a signaling ligand. Ligand binding to the extracellular loops or within the transmembrane helical bundle of these receptors leads to an allosteric conformational change that promotes G protein activation. The precise location of the activating ligand and the conformational changes triggered by ligand binding are unknown for any GPCR.
Our research on signal transduction mechanisms mediated by GPCRs mainly involves the visual pigment rhodopsin. Rhodopsin is the receptor in vertebrate rod cells responsible for vision in dim light. However, we have recently begun structural studies on CCR5, a chemokine receptor in T-cells, and the b 2-adrenoreceptor. The b 2-adrenoreceptor mediates physiological responses to adrenaline and noradrenaline, and plays a critical role in the regulation of the cardiovascular system.
The structure of rhodopsin consists of a bundle of seven transmembrane helices that surround the photoreactive chromophore, 11-cis retinal. Absorption of a single photon of light is sufficient to isomerize the retinal from cis to trans, and activate the protein. Using solid-state NMR spectroscopy, we can define the position of the retinal in the active and inactive states of rhodopsin, and the structural changes within the retinal binding site that lead to receptor activation. The location of the retinal in activated rhodopsin and its interaction with sequence motifs that are highly conserved across the pharmaceutically important class A GPCR family has provided the basis for a general mechanism of GPCR activation.
Amyloid assemblies are found in many neurodegenerative pathologies including Alzheimer’s, Huntington’s, and prion diseases. Although much is known about the physiological consequences of these assemblies, very little is known about the high resolution structures of these b -sheet rich fibrils and their oligomeric precursors. A combination of several biophysical methodologies including solid-state NMR, infrared spectroscopy, fluorescence assays, electron microscopy, and atomic force microscopy are being used to obtain high resolution structural information of these multimeric complexes. The data obtained is being used to design novel peptide and small molecule inhibitors to disrupt the formation of these assemblies and reduce their toxicity to cultured neurons.
Cytokine receptors are a family of transmembrane proteins that include the erythropoietin receptor and the thrombopoietin receptor, among others. These proteins bind ligand (hormones) through an extracellular domain, transmitting a signal through the membrane that results in intracellular effects that include inhibition of apoptosis and usually mitogenesis. Though it is known that these effects are mediated by receptor dimerization and the Jak/STAT pathway, the precise mechanism by which these tasks are accomplished remains elusive.
Erythropoietin (Epo) stimulates erythropoiesis in hematopoietic stem cells. It does this by binding to the extracellular domain of the erythropoietin receptor, transmitting a signal through the transmembrane domain to the intracellular domain, resulting in activation of the Jak/STAT pathway and production of red blood cells. It has been shown that the Epo receptor exists as preformed dimers, though the point of monomer association is a hotly contested issue. We believe that dimerization is mediated through the TM domain that locks the receptor in an “off” position in the absence of ligand. Epo binding results in a change in TM domain conformation that somehow allows Jak2 to phosphorylate the cytoplasmic tyrosine residues on the receptor and other Jak2 molecules. This results in stimulation of mitogenesis and production of red blood cells.
While many groups have studied functional receptor mutants and a crystal structure of the extracellular domain exists, the complete structure remains to be solved. Using recombinantly expressed protein constructs and solution NMR spectroscopy, we aim to solve the complete structure of the Epo receptor and propose a model of receptor activation that may extend to other members of the hematopoietin receptor family. Insights into the structure-function of the Epo receptor may provide a general model for the activation of other cytokine receptors and could facilitate the development of pharmacotherapies for the treatment of erythropoietic diseases.
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 or PIP2) has emerged as a key regulator of signal transduction in cell membranes. Hydrolysis of PI(4,5)P 2 by phospholipase C generates two important second messengers, inositol triphosphate (IP 3 ) and diacylglycerol (DAG). Phosphorylation of PI(4,5)P 2 by PI-3 kinase generates another second messenger, PI(3,4,5)P 3 . In addition, PI(4,5)P 3 itself plays a crucial role in many different cellular processes including membrane trafficking and transport, exocytosis and endocytosis, and cytoskeletal attachment.
We are interested in how the concentration of PIP2 is regulated in cell membranes so that it can carry out its many functions in a coordinated fashion. One mechanism of regulation is by MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate protein), a membrane-associated protein that participates in many cell signaling pathways. MARCKS is able to sequester PIP2 in lateral membrane domains when its highly positively charged effector domain (151-175) is not phosphorylated. Phosphorylation by PKC releases the effector domain from the membrane surface , which in turn frees locally sequestered PIP2. We are in the process of determining the structure of the membrane bound effector domain of MARCKS and how it interacts with PI(4,5)P 2 .
We are also interested in the mechanism of gating and selectivity in ion channels. Research in our lab is focused on the structure and function of phospholamban, a 52-residue ion channel protein found in cardiac muscle cells that regulates calcium levels across the sarcoplasmic reticulum membrane.
Phospholamban is essential in b -adrenergic response in the heart. During muscle contraction, phospholamban binds to the Ca 2+ pump and prevents Ca 2+ from being pumped back into the SR. During muscle relaxation, phospholamban is phosphorylated by Protein Kinase A at Ser16 and Thr17 which removes the inhibition and restores low calcium levels in the cytoplasm. Though the 52-residue peptide is most inhibitory as a monomer, phospholamban also associates into pentamers that have been shown to be selective for Ca 2+ ions.
The structure of phospholamban is central to its function. We are using various biochemical and biophysical methods to investigate the structure and function of this peptide and to understand its mechanism. We are able to determine its global secondary structure by CD and FTIR spectroscopy, and full three dimensional structure by solid state NMR.
Membrane proteins commonly fold into bundles of helices, and helix interactions are important for their folding, stability and function. However, the nature and distribution of the amino acids in membrane proteins is very different than in soluble proteins. The difference in the composition of the surface-exposed residues is well known and simply reflects the environment of the protein, i.e. in soluble proteins polar and charged residues are on the water-accessible surface, whereas in membrane proteins hydrophobic residues cover the lipid-exposed surface. Much less is known about the nature and distribution of amino acids in the interiors of membrane and soluble proteins.
Combining structural studies of membrane proteins with bioinformatics approaches, we have shown that both helical membrane and soluble proteins make use of a general motif for helix interactions which relies mainly on four residues (Leu, Ala, Ile, Val) to mediate helix interactions in a fashion characteristic of left-handed helical coiled-coils. However, a second ‘motif’ for mediating helix interactions is revealed by the high occurrence and high average packing values of small and polar amino acids (Ala, Gly, Ser, Thr) in the helix interfaces of membrane proteins. There is a strong linear correlation between the occurrence of residues in helix-helix interfaces and their packing values. Based on this correlation, we introduced the concept of a helix packing moment to predict the orientation of helices in helical membrane proteins and membrane protein complexes. The helix packing moment is a complementary tool to the helical hydrophobic moment in the analysis of transmembrane sequences. Helix packing moments also help to identify the packing interfaces in membrane proteins with multiple transmembrane helices, where a single helix can have multiple contact surfaces.
Analyses on class A G protein-coupled receptors (GPCRs) with 7 transmembrane helices show that helix packing moments are conserved across the class A family of GPCRs and correspond to key structural contacts in rhodopsin. These contacts are distinct from the highly conserved signature motifs of GPCRs and have not previously been recognized. The specific amino acid types involved in these contacts, however, are not necessarily conserved between subfamilies of GPCRs, indicating that the same protein architecture can be supported by a diverse set of interactions. In GPCRs, as well as membrane channels and transporters, amino acids with small side chains ( Gly, Ala, Ser, Cys) allow tight helix packing by mediating strong van der Waals interactions between helices. Closely packed helices, in turn, facilitate interhelical hydrogen bonding of both weakly polar (Ser, Thr, Cys) and strongly polar (Asn, Gln, Glu, Asp, His, Arg, Lys) amino acids.
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