Transcript Laura Salguero Presentation

Identification and
characterization of minimal
ARNT-binding AINT fragments
Laura Salguero,† Carrie Partch,‡ and Kevin Gardner‡
†New Mexico State University, Department of Physics
‡UT Southwestern Medical Center, Departments of Biochemistry and Pharmacology
Overview
The HIF (Hypoxia Inducible Factor) transcription factor is composed of two
proteins: HIF- and HIF- (a.k.a. ARNT ARyl hydrocarbon Nuclear
Translocator). These two proteins bind each other in response to specific signals
from the cell that it has encountered low oxygen levels; most of these interactions
occur via protein-protein interaction domains called PAS (Per-ARNT-Sim)
domains in each protein. Previously, NMR spectroscopy and X-ray
crystallography was used to solve the structures of a heterodimeric complex of
PAS domains from both HIF-2 and ARNT: This structure has provided insights
both into the nature of the interaction and information about how it might be
artificially controlled. Controlling HIF signaling is particularly important because
oxygen can only diffuse ca. 100m in living tissue, causing solid tumors to send
out a variety of biochemical signals that trigger new blood vessel formation and
other responses that let them adapt to low oxygen levels.
This project was focused on studying the next step in hypoxia signaling: how the
HIF- heterodimer recruits a coactivator protein, AINT (ARNT INTeracting
protein). This is of particular interest to us in that initial data suggests that AINT
interacts directly with the ARNT PAS-B domain, implicating a new type of proteinprotein interaction that PAS domains might be able to participate in. This also
firmly indicates that these PAS domains can actually simultaneously bind two
different protein partners at the same time. The structure of such a novel complex
is unknown at this point, and is of intense interest.
HIF-1 complex is an oxygen-sensitive
transcriptional switch
• promotion of angiogenesis, or
formation of new blood vessels
• activation of glucose transport
and glycolysis
• induction of erythropoeisis,
the formation of new red blood
cells
growth of solid tumors
Co-activator recruitment by ARNT
Transcription factors must recruit diverse co-activator proteins to initiate transcription
–Modification of local chromatin structure
–Recruitment of transcriptional machinery
AINT is a co-activator that interacts with nuclear Histone AcetylTransferases (HAT), that
modify histones to stimulate transcription. (Gangisetty, O et al (2004) Oncogene)
AINT binds to ARNT PAS-B and over expression of it stimulates the transcriptional
response to hypoxia by HIF- and ARNT. (Sadek, C et al (2000) Mech. of Dev.)
ARNT domain structure
bHLH
PAS-A
PAS-B
Hypothesis
Since the  sheet of ARNT PAS-B
is involved in binding to HIF-as
shown, the  helices on ARNT
PAS-B must be binding to AINT.
ARNT
AINT binding?
Hif-2
Card, P.B. et al (2005) J Mol Biol
Tom Scheuermann (2008)
Goals of Project
1.) Identify minimal region of AINT that interacts with ARNT PAS-B
– express GST-tagged AINT fragments in E.coli and test interaction with
His-tagged ARNT PAS-B by pulldown assay
– Map interaction site on 15N ARNT PAS-B by 2D protein NMR
The ARNT binding domain of AINT is the C-terminal
coiled-coil domain (Sadek, C et al (2000) Mech of Dev). Since
coiled-coil structures are highly elongated, it is
desirable to identify the smallest segment
representing the ARNT binding sequence while
keeping coiled-coil structure.
Sec2p coiled-coil (150 res.)
Sec4p (169 res.)
Dong, G. et al (2007) Mol Cell
2.)
Purify AINT minimal domain using HisGB1-tag and
monitor the coiled-coil structure by circular dichroism
spectroscopy
3.) Set up crystallization trays with an ARNT PAS-B:AINT complex for high
resolution structure determination
Identification of Minimal AINT Fragment
,
His-ARNT PAS-B
GST-AINT: 4 8 6 7 9 10 11 4 8
62
47.5
32.5
25
16.6
kDa
10% Input
,
6 7 9 10 11
A pulldown assay is a technique to assess
protein-protein interactions. Soluble
E.coli extract with GST-AINT fragments
was incubated with His-ARNT PAS-B
and Ni2+ resin. Protein binding was
assessed after pelleting the resin,
washing, and resolving the mix by
denaturing gel electrophoresis.
ARNT PAS-B
GST
His
Ni2+ resin
Conclusion:
AINT 10 is smallest fragment (40 amino acids) tested with binding to
ARNT PAS-B and the C-terminal 20 residues are essential for binding.
Purification of AINT 10
•His-GB1 AINT 10 was expressed in E.coli BL21(DE3) cells; soluble
extract was run over a Ni2+ column. The His-GB1 tag attaches to the
Ni2+ resin and debris is washed away in the flow through. Protein
was eluted with a gradient of imidazole.
•His-TEV (Tobacco Etch Virus) protease was added to cleave the
His-GB1 tag; protease and tag were removed by Ni2+ column.
Time (m):0
14.4
kDa
2
5
10
20 60 180
Dimer
Monomer
AINT 10 - 5,473.3 g/mol
EVLALQASLRKAQMQNHSLEMTLEQKTKEIDELTRICDDLISKMEKI
EVLALQASLRKAQMQNHSLEMTLEQKTKEIDELTRICDDLISKMEKI
Imidazole
I FT
Eluate
,
97
66
45
30
20.1
14.4
kDa
•Each monomer of AINT 10 has only one
cysteine suggesting that it forms a native
disulfide bond as a coiled-coil dimer,
therefore CuSO4 was used to oxidize the
cysteines and induce disulfide formation.
•1mM AINT10 was incubated with 1mM
CuSO4 at room temperature for three hours.
The reaction was quenched with 2mM EDTA.
NaCl
Input
•Dimer was run over a Mono S cation exchange column to
remove CuSO4 and further contaminates, and buffer exchanged
to dilute the high salt required to elute AINT 10 from the column.
97
66
45
30
20.1
14.4
kDa
Eluate
,
Circular Dichroism (CD) Spectrum of AINT 10
Far UV CD is a spectroscopic technique that measures the arrangement
of peptide bonds in secondary structure elements like  helices and 
strands
40000
 (deg*cm2/dmol*res)
30000
Ref: www.photophysics.com/circulardichroism.php
20000
10000
0
190
200
210
220
230
-10000
Acquired in Aviv 62D Spectropolarimeter at 25C
•15M disulfide-linked AINT 10 dimer
•10mM Sodium Phosphate pH 6.5, 17mM NaCl
-20000
Conclusion:
AINT 10 is a parallel coiled-coil dimer
Wavelength (nm)
240
250
260
Mapping AINT 10 binding to ARNT PAS-B
• 15N-1H Heteronuclear Single Quantum Coherence (HSQC) spectrum is a
SS Dimer
‘protein fingerprint’ where each crosspeak corresponds to a backbone amide
• This titration experiment monitors the perturbation of 15N ARNT PAS-B with
the addition of unlabeled AINT 10
Conclusions:
AINT 10 appears to bind on to the
 helical face of ARNT PAS-B
X-ray Crystallography
•
•
•
•
For determining protein structures X-ray
crystallography can be fast and provide
high resolution structural details if a protein
will crystallize nicely.
Crystallography is a technique that
determines crystalline structures using
Bragg’s Law, n=2dsin()
X-rays are generated and focused to
maximum intensity, here at 8keV, and
diffracted off a crystal at multiple angles in
order to see multiple planes within the
crystal.
Since x-rays will have both constructive
and destructive interference, the diffraction
pattern resembles dots in a ring around a
central position. These spots represent a
spacing between atoms which is inversely
proportional to the distance of the ring to
the center. The closer the spots are to the
center, the larger distance they represent.
http://www-structmed.cimr.cam.ac.uk/Course/Basic_diffraction/Diffraction.xht
Crystal Screens
•
•
All protein have different conditions at
which they want to crystallize. As there
is no way of knowing what these
conditions may be for a novel protein, a
screen is set up that tests the protein
under a wide variety of precipitants, pHs
and buffers.
All of our crystal screening trays, except
three of the optimization trays, were set
up using the Phoenix robot in the
Structural Biology Lab:
–
–
–
–
–
–
•
QIAGEN The Classics
QIAGEN The JCSG+ Suite
QIAGEN The Protein Complex Suite
QIAGEN The PEGs Suite
Hampton Salt Rx
Hampton Index
From these screens, over 170
conditions formed crystals.
Four
optimization trays and one streakseeding tray has been set up. This
effort will be continued into the future.
ARNT PAS-B-AINT 10 Crystals
Conclusions
•Found minimal domain of AINT necessary for binding ARNT
•Mapped the binding on ARNT PAS-B to the -helices
•Showed by CD that AINT 10 is parallel coiled-coil dimer
•Crystallography of the protein complex in process
This work could provide critical understanding
of the transcriptional response to hypoxia and
help develop chemotherapeutic intervention.
Acknowledgements
Thank you to:
The Gardner lab: Jason Key, Abby Nash, Matthew Evans, Kim
Shahi, Amy Zhou, Qiong Wu, Abby Nash, Lisa Ko, Tom
Scheuermann, Paul Card, Fernando Correa, Qun Du
Diana Tomchick and the Structural Biology lab
Charles Dann III
Carlos Amezcua and the Biomolecular NMR Facility
Quantitative & Physical Sciences Summer Training Program;
NIGMS 5R25GM072832-04
Minority Access to Research Careers; GMO7667-31