ABSTRACT:

1
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.
2
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. Thre
e iLBPs enhance transcriptional activity of
nuclear receptors with which they share a common
ligand CRABP II RAR? A-FABP
PPAR?/? K-FABP PPAR?
3
Problem
None of these iLBPs contains a NLS
Furthermore
Nuclear localization only occurs upon binding of
ligand
4
CRABP II story
Retinoic acid (RA) induces nuclear import of
CRABP II
Nuclear export signal (NES)
MDLCQAFSDVILAEF Leptomycin B (LMB) inhibits NES
mediated export
COS-7 cells transfected with denoted CRABP II
expression vectors
(Sessler Noy 2005)
5
CRABP II story
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)
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CRABP II story
colored by B-factor, non-linear NLS in spacefill
K30
K20
K20
R29
R29
K30
apo
holo
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CRABP II story
Mutating K20, R29, K30 to ala abolishes nuclear
import
(Sessler Noy 2005)
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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
9
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?
10
K-FABP 135AA, 1 disulfide
K34
C
R33
K24
N
1JJJ NMR structure, holo with stearic acid
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R33
K24
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.
K34
K34
K24
R33
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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
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Curiosity What is the difference between iLBPs
that do and dont localize to the nucleus
upon ligand binding?
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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 pHs result in sample aggregation
15
K-FABP 15N edited HSQC spectrum of stable sample
10mM HEPES pH 7.7, 40mM NaCl, 5mM DTT, 15C
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K-FABP Ongoing work Spin system
assignment 15N, TOCSY NOESY 15N 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-HNHA Side chain information rotomer ?1
angles, 3JH?? coupling 15N-HNHB Dipolor
coupling 15N and 13C HSQC NOESY Dynamic
analysis 15N - 1H NOESY