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PDBsum entry 2ipk
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Immune system
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PDB id
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2ipk
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Contents |
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179 a.a.
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190 a.a.
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14 a.a.
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232 a.a.
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References listed in PDB file
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Key reference
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Title
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Fluorogenic probes for monitoring peptide binding to class ii mhc proteins in living cells.
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Authors
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P.Venkatraman,
T.T.Nguyen,
M.Sainlos,
O.Bilsel,
S.Chitta,
B.Imperiali,
L.J.Stern.
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Ref.
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Nat Chem Biol, 2007,
3,
222-228.
[DOI no: ]
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PubMed id
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Abstract
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A crucial step in the immune response is the binding of antigenic peptides to
major histocompatibility complex (MHC) proteins. Class II MHC proteins present
their bound peptides to CD4(+) T cells, thereby helping to activate both the
humoral and the cellular arms of the adaptive immune response. Peptide loading
onto class II MHC proteins is regulated temporally, spatially and
developmentally in antigen-presenting cells. To help visualize these processes,
we have developed a series of novel fluorogenic probes that incorporate the
environment-sensitive amino acid analogs
6-N,N-dimethylamino-2-3-naphthalimidoalanine and
4-N,N-dimethylaminophthalimidoalanine. Upon binding to class II MHC proteins
these fluorophores show large changes in emission spectra, quantum yield and
fluorescence lifetime. Peptides incorporating these fluorophores bind
specifically to class II MHC proteins on antigen-presenting cells and can be
used to follow peptide binding in vivo. Using these probes we have tracked a
developmentally regulated cell-surface peptide-binding activity in primary human
monocyte-derived dendritic cells.
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Figure 1.
(a) Chemical structures of DANA, 4-DAPA and 6-DMNA. (b) In
the crystal structure of the DR1–HA peptide complex, a
tyrosine residue is buried deep into the hydrophobic P1
pocket^5. DANA, 4-DAPA and 6-DMNA were modeled in silico into
this pocket in place of tyrosine. Residues shown lining the
pocket are (clockwise from upper right) Phe  54,
Phe 32,
Trp 43
(behind), Ile 7,
Trp  153,
Phe 48,
Thr 90,
Val 91,
Tyr 83,
Gly 86
and Val 85.
The side chains of Asn 82
and His 81
(upper left) form hydrogen bonds with the peptide main chain at
the mouth of the P1 pocket. Other residues lining the pocket but
not shown are Phe 24,
Phe 26
and Phe 48.
(c) Binding of fluorogenic peptides to DR1 assessed using a
competitive binding assay. DR1 was incubated with biotin–HA
peptide and various concentrations of unlabeled inhibitor
peptides, and biotin-HA binding was quantified by a sandwich
ELISA assay using streptavidin alkaline phosphatase. Binding of
Fmoc-(4-DAPA) was assessed to evaluate nonspecific binding of
the fluorophore. IC[50] values for these and other peptides
(Supplementary Table 1) were determined as described
(Supplementary Methods).
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Figure 3.
(a) (4-DAPA)-HA was incubated with HLA-DR1 as described for
(4-DAPA)-RSMA[4]L, and the complex formed was purified by gel
filtration. Fluorescence of DR-(4-DAPA)-HA was compared with
that of the free peptide. (b) Activation of HA1.7 T-cell
receptor hybridoma by antigen-presenting cells pulsed with HA
(filled circles) or (4-DAPA)-HA (closed triangles) peptides.
T-cell activation reported as counts per minute (c.p.m.)
measured in a thymidine incorporation bioassay for IL-2 as
secreted by activated T cells. Error bars indicate s.d. of
triplicate measurements. (c–e) Crystal structure of
(4-DAPA)-HA bound to DR1. (c) (4-DAPA)-HA peptide shown with
surface representation of the DR1 peptide binding site, with the
4-DAPA side chain shown with yellow bonds extending down into
the P1 pocket (top); unmodified HA peptide from the crystal
structure of the DR1-HA-SEC (3B2) complex (PDB ID 1JWU) shown
after alignment of MHC peptide binding domain, with tyrosine
side chain at the P1 position shown with cyan bonds (bottom).
(d) 2F[o] - F[c] omit map of the region around the P1 pocket
with all residues shown removed from the model before map
calculation. (e) Section through the P1 pocket, showing the HA
peptide tyrosine side chain and the (4-DAPA)-HA fluorophore,
along with the corresponding ordered water molecules, colored as
in c. Panels c–e made using PyMOL^30.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Chem Biol
(2007,
3,
222-228)
copyright 2007.
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