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

colored by B-factor, non-linear NLS in spacefill

apo holo

CRABP II story

K20 K20

R29

R29

K30

K30

CRABP II storyMutating K20, R29, K30 to ala abolishes nuclear import

(Sessler & Noy 2005)

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:135AA, 1 disulfide

1JJJ: NMR structure, holo with stearic acid

N

CK24 R33

K34

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:15N edited HSQC spectrum of stable sample

10mM HEPES pH 7.7, 40mM NaCl, 5mM DTT, 15°C

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