Transcript PIZZA AND IT’S POPULARITY AMONG ALL THE COOL PEOPLE …

The Role of Zinc in the Interaction of TAX1BP1 and Itch
Studied by Fluorescence Anisotropy and Structural Modeling
Ashley F. Cowan, Joel E.P. Brandis, and Barbara T. Amann
Department of Biology and Chemistry, Goucher College, 1021 Dulaney Valley Rd., Baltimore, MD 21204
Modeling
Abstract
Results
• Zinc deficiency leads to a suppressed immune system
although the mechanism is not fully understood.
• WW domain is a small three beta strand structure found in many
proteins.
• The Nuclear Factor kappa B (NF-kB) signaling pathway
turns inflammation on and off.
• Many key proteins in this pathway bind to zinc.
• Each TAX1BP1 zinc finger contains a PPXY domain and binds
zinc with two conserved cysteines and histidines.
• The tightest interaction occurs between of the second
TAX1BP1 zinc finger (TAXF2) and the first Itch WW
domain (WW1) domain when zinc is present.
• Through mutational studies, we have determined that
four amino acids residues located in the TAXF2 are
responsible for this increase affinity in the presence of
zinc.
WW domain
Background
• Zinc deficiency affects 2 billion people worldwide and
leads to major health problems including a suppressed
immune system.
• The NFkB signaling pathway has many zinc binding
proteins, which may explain why zinc leads to a
suppressed immune system.
• Zinc binding proteins are necessary for turning on and
off inflammation.
• Within the NFkB pathway, the A20 complex shuts off
inflammation through negative feedback loop.
• TAX1BP1 and Itch are integral parts of the A20
complex.
Classic TFIIA zinc
finger and PPXY region
unattached
Model of TAXF2 zinc
finger with PPXY
region in red
• TAX1BP1 fingers were modeled using a classical zinc finger.
This causes the PPXY sequence to curve.
Goal
TAXF1
• To determine what amino acids in TAXF2 allows it to bind the
strongest to WWI when zinc is present.
Methods
• Mutations were made to specific amino acids to turn TAXF1 into
TAXF2:
TAXF1 KKCPLCELMFPPNYDQSKFEEHVESHFK
TAXF1a KKCPMCELQFPPNYDQQKFEEHVETHFK
TAXF1b1 KVCPMCSLQFPPDYDQQVFEEHVETHFK
ITCH
RNF11
TAXF1b2 KKCPMCEEQFPPNYDQQKFERHVQTHFD
TAX1BP1
A20
TAXF2
A20 Complex includes the
proteins RNF11, Itch, TAX1BP1,
and A20
• The TAX1BP1 and Itch proteins interact with one another
through two PPXY zinc finger domains and four WW
domains, respectively.
PPXY PPXY
CC1
CC2
CC3
ZF1
C2
WW1 WW2
WW3
WW4
ZF2
HECT
Figure 1. Anisotropy compared to the concentrations of TAXF2 or
mutants of TAXF2 when added to WWI. Graph produced through the
use of KaleidaGraph.
KVCPMCSEQFPPDYDQQVFERHVQTHFD
Synthetic peptides
purified by HPLC.
452
TAXF1A
TAXF1B1
TAXF1B2
281
371
130
Table 1. Kd values in mM of TAX1BP1 domains TAXF2 and TAXF1
with mutations bond to Itch WW1. All TAX1BP1 domains contain
zinc. Kd values are averaged from 2-3 different trials.
Conclusion
• Of the TAXF1 mutants, TAXF1B2 binds the strongest to WWI and
has the most similar Kd value to TAXF2.
TAXF1
TAXF1b2
TAXF2
KKCPLCELMFPPNYDQSKFEEHVESHFK
KKCPMCEEQFPPNYDQQKFERHVQTHFD
KVCPMCSEQFPPDYDQQVFERHVQTHFD
• The amino acids in red are those
determined to be important to the
interaction of TAXF2 and WWI.
Experimental Procedure
Binding activity of
cobalt to protein
quantified by uv/vis
spectroscopy.
Cobalt then
substituted by zinc.
WW1
TAXF2
BTA
AFC
51
98
Kd values of zinc
fingers bond to WWI
determined from
fluorescence
anisotropy.
• Future experiments could lead to us
discovering the specific amino acid(s)
responsible for the increased bonding.