ABSTRACT:
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ABSTRACT:
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.
Nuclear localization
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 RARA-FABP PPAR/K-FABP PPAR
Problem:
None of these iLBPs contains a NLS
Furthermore…
Nuclear localization only occurs upon binding of ligand
COS-7 cells transfected with denoted CRABP II expression vectors(Sessler & Noy 2005)
Nuclear export signal (NES) MDLCQAFSDVILAEF
Leptomycin B (LMB)inhibits NES mediated export
Retinoic acid (RA) induces nuclear import of CRABP II
CRABP II story
In the absence of a NLS, a conformational change upon RA binding must “create” a non-linear NLS
CRABP II story
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
RA binding induces a basic patch at the end of helix 2
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
Resulting in a topology for K20, R29 and K30 that mimics a NLS
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
(Sessler & Noy 2005)SV40 NLS peptide
CRABP II Results:
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
Conclusion:
RA binding results in formation of a non-linear NLS
K-FABP: Displays an even more complex behavior
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
WHY?
OH
O
OH
O
K-FABP: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.
K24 R33
K34
K24R33
K34
K-FABP: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?
K-FABP:Action:
Generate stable samples at NMR concentration
Problem: The K-FABP samples are remarkably unstable
a variety of low salt buffers at multiple concentrations and pH’s result in sample
aggregation
K-FABP:Ongoing work:
Spin system assignment15N, TOCSY & NOESY15N 13C, H(CC)(CO)NH and (H)CC(CO)NH
Coming soon:Sequential assignment
HNCA, HN(CA)CO, HNCO, HN(CO)CA (as needed)Backbone information
13C shift from random coil, HNCA 3JHN coupling constants,
15N-HNHASide chain information rotomer 1 angles, 3JH coupling 15N-HNHBDipolor coupling
15N and 13C HSQC NOESYDynamic analysis
15N - 1H NOESY