Strong LaboratoryProgram in Structural Molecular Biology
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C-type Lectin-like Immunoreceptor/MHC Class I-like Ligand Interactions
Siderocalins: siderophore-specific, anti-bacterial responses through iron sequestration
Development of novel AIDS vaccine immunogens
C-type Lectin-like Immunoreceptor/MHC Class I-like Ligand Interactions
Collaborators: Thomas Spies (Division of Clinical Research,
FHCRC), Effie Petersdorf (Division of Clinical Research,
FHCRC), Dan Geraghty (Division of Clinical Research,
FHCRC), David Baker (Department of Biochemistry,
University of Washington)
MHC class I proteins present peptide fragments of endogenous proteins on the cell surface, allowing T cells to monitor the proteome of a given cell for the expression of aberrant proteins associated with infection or tumorigenesis:
Ribbon and space-filling diagrams of a typical MHC class I protein.
Natural killer (NK) cells function through a diverse array of cell-surface receptors that can be divided
into two classes either on the basis of structural homology (immunoglobulin- or C-type lectin-like) or
by function (inhibitory or stimulatory). NK receptors specific for classical and non-classical MHC class
I proteins play an important role in immunosurveillance against cells that have dysregulated MHC class I
expression as a result of infection or transformation. An example of such an interaction is the
recognition of the non-classical MHC class I protein HLA-E by heterodimeric NKG2x/CD94 NK receptors
(where "x" is A, B, C, E or H). Another related immunoreceptor, NKG2D, is a homodimeric, activating NK
cell receptor first identified on NK cells but subsequently found on macrophages and a variety of T cell
types. Human NKG2D ligands include the CMV UL16 binding proteins (ULBPs) and the polymorphic, cell
stress-inducible proteins MIC-A and MIC-B. NKG2D-MIC recognition events provide co-stimulatory signals
to T cells and have been implicated in anti-viral and anti-tumor immune responses by both T and NK
cells.
We are studying the interactions between C-type lectin-like NK cell receptors and their ligands
by 1) crystallographic analyses of the molecules in isolation and as complexes; 2) by examining the
interaction of soluble forms of these proteins through biochemical and mutagenesis studies; and 3)
establishing cell-based activation assays to determine the effect microscopic interaction parameters
(affinities, kinetics, thermodynamics) have on relative signal output. The goal is to elucidate the
signal integration mechanism/s employed by NK cells to control activation of effector functions.
For our biochemical and crystallographic studies, we generally express truncated, cell-surface glycoproteins recombinantly as inclusion bodies in bacteria, denature and then re-fold in vitro. The basic StrongLab re-folding protocol: PDF.
CenterNews article on the NKG2D/MIC-A complex structure
Views of various immunoreceptor/ligand complexes
See - StrongLab Structures & Publications - for more detailed information
Siderocalins: siderophore-specific, anti-bacterial responses through iron sequestration
Collaborators: Niels Borregaard (The Granulocyte Research Laboratory, Rigshospitalet), Kenneth N. Raymond (Department of Chemistry,
University of California, Berkeley), Isabelle Schalk (Ecole Superieure de Biotechnologie de Strasbourg),
Kelly D. Smith (Institute for Systems Biology), Alan Aderem (Institute for Systems
Biology), Jonathan Barasch
(Columbia University)
Lipocalins are a diverse family of small, secreted proteins that generally bind small, hydrophobic ligands and specific cell-surface receptors. Despite limited sequence similarity across the family, the lipocalin fold is remarkably well conserved. The core structure consists of an eight-stranded anti-parallel beta-barrel which defines a calyx, or cup-shaped structure, that defines the ligand binding site. Lipocalins, along with the avidins and the fatty-acid binding proteins, comprise the calycin superfamily of proteins:
For more information about lipocalins, link to the Lipocalins Website
Siderocalin (aka NGAL, 24p3 or lipocalin 2) is a lipocalin
that is secreted from neutrophil granules or epithelial cells in response to inflammation,
bacterial infection or transformation. We have shown that siderocalin tightly binds the catecholate-type
ferric siderophores of enteric bacteria and the mixed-type carboxymycobactin siderophores of
mycobacteria. Siderophores are powerful iron chelators that allow microorganisms to acquire this scarce
resource, either in the environment or in the body during infection. Our structural and biophysical
studies of these interactions reveal that siderocalin employs a unique type of recognition degeneracy to
bind these disparate compounds tightly. We have also shown, through in vitro bacterial growth assays
and collaborative studies of siderocalin KO mice, that siderocalin mediates key anti-bacterial innate
immune responses, functioning to deprive invading pathogenic bacteria of essential iron.
Our current studies focus on 1) dissecting the details of degeneracy, through mutagenesis, synthesis of
model siderophores, binding/kinetic studies and computational modeling of the interactions; 2) analyzing
siderocalin's pleiotropic functions in endogenous iron transport and receptor-mediated cellular effects;
and 3) searching for additional, siderophore-binding lipocalins that may complement siderocalin's
anti-bacterial activity.
CenterNews article on siderocalin's role in immunity & iron metabolism
CenterNews article on the siderocalin knock-out mouse
Some bacterial siderophores.
See - StrongLab Structures & Publications - for more detailed information
Development of novel AIDS vaccine immunogens
Collaborators: David Baker (Department of Biochemistry,
University of Washington), Pamela Bjorkman (Caltech), Bill Schief (Department of Biochemistry, University of Washington),
Leo Stamatatos (Seattle Biomedical Research Institute)
As part of the Bill & Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery (CAVD), we are participating in a consortium to computationally design effective AIDS immunogens as the basis of broadly protective humoral vaccines that could be used prophylactically. Our role is to biophysically evaluate designed immunogens, and elicited serum responses, in an iterative, integrative effort to achieve a useful vaccine candidate, as well as analyzing antibody/HIV interactions in order to define biophysical correlates of - or thresholds for - neutralization.
CenterNews article on the Gates Foundation CAVD awards to the Center
Nature News article on Gates Foundation funding of AIDS vaccine development
Seattle Post-Intelligencer news article on Gates Foundation grants for AIDS vaccine development
See - StrongLab Structures & Publications - for more detailed information
