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Abstract 3344394

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.


Abstract 3344394

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 RAR

A-FABP PPAR/

K-FABP PPAR


Abstract 3344394

Problem:

None of these iLBPs contains a NLS

Furthermore…

Nuclear localization only occurs upon binding of ligand


Abstract 3344394

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)


Abstract 3344394

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)


Abstract 3344394

CRABP II story

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

K30

K20

K20

R29

R29

K30

apo

holo


Abstract 3344394

CRABP II story

Mutating K20, R29, K30 to ala abolishes nuclear import

(Sessler & Noy 2005)


Abstract 3344394

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


Abstract 3344394

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?


Abstract 3344394

K-FABP:

135AA, 1 disulfide

K34

C

R33

K24

N

1JJJ: NMR structure,

holo with stearic acid


Abstract 3344394

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


Abstract 3344394

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


Abstract 3344394

Curiosity: What is the difference between iLBPs that do and don’t localize to the nucleus upon ligand binding?


Abstract 3344394

K-FABP: don’t localize to the nucleus upon ligand binding?

Action:

Generate stable samples at NMR concentration

Problem:

The K-FABP samples are remarkably unstable

a variety oflow salt buffers at multiple concentrations and pH’s result in sample aggregation


Abstract 3344394

K-FABP: don’t localize to the nucleus upon ligand binding?

15N edited HSQC spectrum of stable sample

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


Abstract 3344394

K-FABP: don’t localize to the nucleus upon ligand binding?

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