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 RAR  A-FABP PPAR  /  K-FABPPPAR 

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 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 (Sessler & Noy 2005) SV40 NLS peptide

colored by B-factor, non-linear NLS in spacefill apoholo CRABP II story K20 R29 K30

CRABP II story Mutating 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?

K-FABP: 135AA, 1 disulfide 1JJJ: NMR structure, holo with stearic acid N C K24 R33 K34

K-FABP: Overlay of residues 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 K24 R33 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: 15 N edited HSQC spectrum of stable sample 10mM HEPES pH 7.7, 40mM NaCl, 5mM DTT, 15°C

K-FABP: Ongoing work: Spin system assignment 15 N, TOCSY & NOESY 15 N 13 C, 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 13 C  shift from random coil, HNCA 3 J HN  coupling constants, 15 N-HNHA Side chain information rotomer  1 angles, 3J H  coupling 15 N-HNHB Dipolor coupling 15 N and 13 C HSQC NOESY Dynamic analysis 15 N - 1 H NOESY