Steven O. Smith, Ph.D.
Director of Structural Biology
Department of Biochemistry and Cell Biology
Center for Structural Biology
138 Centers for Molecular Medicine
Stony Brook, NY 11794-5215
Office telephone: 631-632-1210
Lab telephone: 631-632-1211 or 1212
G Protein-Coupled Receptors
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 Fibril Structure, Formation and Inhibition
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.
Basic – Aromatic Clusters in Peripheral Membrane Proteins
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
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 Protein Folding and Structure
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
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)
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