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.
Laura Salguero,† Carrie Partch,‡ and Kevin Gardner‡
†New Mexico State University, Department of Physics
‡UT Southwestern Medical Center, Departments of Biochemistry and Pharmacology
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. 100m 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 protein-protein 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 AINT fragments
growth of solid tumors
bHLH AINT fragments
Card, P.B. et al (2005) J Mol Biol
Tom Scheuermann (2008)
ARNT domain structure
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.
1.) Identify minimal region of AINT that interacts with ARNT PAS-B
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
, AINT fragmentsHis-ARNT PAS-B 10% Input ,
GST-AINT: 4 8 6 7 9 10 11 4 8 6 7 9 10 11
Ni2+ resinIdentification of Minimal AINT Fragment
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.
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.
I AINT fragmentsFT Eluate ,
Time (m):0 2 5 10 20 60 180
MonomerPurification of AINT 10
AINT 10 - 5,473.3 g/mol
Input Eluate ,
Far UV CD is a spectroscopic technique that measures the arrangement of peptide bonds in secondary structure elements like helices and strandsCircular Dichroism (CD) Spectrum of AINT 10
AINT 10 is a parallel coiled-coil dimer
AINT 10 appears to bind on to the
helical face of ARNT PAS-B
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
This work could provide critical understanding of the transcriptional response to hypoxia and help develop chemotherapeutic intervention.