Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
We are interested in determining the conformational changes induced by ligand binding in the intracellular lipid binding protein (iLBP) karitinocyte fatty acid binding protein (K-FABP). The source of this interest is the differential behavior of K-FABP when ligand bound. If it binds a non-activating ligand, such as stearic acid, K-FABP acts as a typical fatty acid binding protein, chaperoning the ligand in the aqueous environment of the cytosol. If, however, K-FABP binds an activating ligand such as linolenic acid, the protein is directed to the nucleus of the cell. The source of this differential behavior is proposed to be the formation of a non-linear nuclear localization sequence (NLS) through conformational changes induced by the binding of an activating ligand. By determining the structure of K-FABP in both the activated and non-activated states we will be able to understand the basis for this curious behavior.
Subcellular targeting of a protein to the nucleus via a NLS
“classical” NLS K(K/R)X(K/R)
Such an NLS is recognizable by adaptor proteins called -importins that subsequently interact with -importins to control nuclear localization.
Three iLBPs enhance transcriptional activity of nuclear receptors with which they share a common ligand:
CRABP II RAR
None of these iLBPs contains a NLS
Nuclear localization only occurs upon binding of ligand
Retinoic acid (RA) induces nuclear import of CRABP II
Nuclear export signal (NES)
Leptomycin B (LMB)
inhibits NES mediated export
COS-7 cells transfected with denoted CRABP II expression vectors
(Sessler & Noy 2005)
In the absence of a NLS, a conformational change upon RA binding must “create” a non-linear NLS
RA binding induces a basic patch at the end of helix 2
Resulting in a topology for K20, R29 and K30 that mimics a NLS
SV40 NLS peptide
(Sessler & Noy 2005)
colored by B-factor, non-linear NLS in spacefill
Mutating K20, R29, K30 to ala abolishes nuclear import
(Sessler & Noy 2005)
RA causes CRABP II to accumulate in the nucleus
This is due to nuclear import
RA causes CRABP II to interact with importin (DNS)
conformational change upon RA binding results in a basic patch involving residues K20, R29, K30
Mutation of these residues abolishes nuclear import
RA binding results in formation of a non-linear NLS
binds a wide spectrum of ligands with similar affinity
nuclear localization response only to certain ligands
activating (PPAR binding): linolenic acid
non-activating: stearic acid
135AA, 1 disulfide
1JJJ: NMR structure,
holo with stearic acid
Overlay of residues 20-38 of NMR models 1-20 of the human protein.
There appears to be considerable conformational flexibility in K34 and especially K24.
Suggests that dynamics are critical to the phenomenon.
How to answer the question:
Why does K-FABP respond differently
to different ligands?
Solve the structure and query the dynamics in the presence of both activating and non-activating ligands
Hypothesis: binding of an activating ligand results in the formation of or bias toward a non-linear NLS while a non-activating ligand does not
Curiosity: What is the difference between iLBPs that do and don’t localize to the nucleus upon ligand binding?
Generate stable samples at NMR concentration
The K-FABP samples are remarkably unstable
a variety oflow salt buffers at multiple concentrations and pH’s result in sample aggregation
15N edited HSQC spectrum of stable sample
10mM HEPES pH 7.7, 40mM NaCl, 5mM DTT, 15°C
Spin system assignment
15N, TOCSY & NOESY
15N 13C, H(CC)(CO)NH and (H)CC(CO)NH
HNCA, HN(CA)CO, HNCO, HN(CO)CA (as needed)
13C shift from random coil, HNCA
3JHN coupling constants,15N-HNHA
Side chain information
rotomer 1 angles, 3JH coupling 15N-HNHB
15N and 13C HSQC NOESY
15N - 1H NOESY