Fatty acids exchange on confined protein. X-ray evidence and MD explanation.

PODJARNY, ALBERTO D. ; ESPINOSA SILVA, YANIS RICARDO; PODJARNY, ALBERTO D. ; ESPINOSA SILVA, YANIS RICARDO; CARLEVARO, CARLOS MANUEL; ALVAREZ, H. ARIEL; CARLEVARO, CARLOS MANUEL; ALVAREZ, H. ARIEL; HOWARD, EDUARDO I.; COUSIDO-SIAH, ALEXANDRA ; HOWARD, EDUARDO I.; COUSIDO-SIAH, ALEXANDRA

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Sociedades de Biociencias de Argentina

The fatty-acid-binding proteins (FABPs) act as intracellular lipidchaperones. The structure is a beta barrel surrounding an inner cavitythat accommodates fatty acids, enabling their transport in anaqueous media inside the cell. But the mechanism of lipid bindingand release is still the topic of many studies. This exchange involvesat least three stages: opening a portal at the protein surface, ligandentry/release, and portal closure. It is challenging to obtain experimentalevidence on the functionality of the portal without modifyingthe amino acid sequence or the protein conformation. Studying theseprocesses in the crystallized protein opens a unique possibility,because when confined in the crystalline network the conformationalchanges are limited by the crystal packing. We soaked a FABP-Palmiticacid crystal with Br-palmitic acid, using the heavy Br atom asa marker. After solving the structure at 1Å resolution, we observed Br-palmitic acid inside the cavity, proving that ligand exchange doestake place in the crystal. However, the crystal structure does notdiffer significantly from the already resolved structures, and doesnot show details of the ligand exchange. Since the accuracy of ourstructure provides a reliable starting point for Molecular Dynamicssimulations, we build a model of 2 unit cells (8 proteins), and properlychoosing the simulation box, we model the whole crystal bymeans of the periodic boundary conditions. After 2 μs of simulationat constant volume (NVT ensemble), the computational results correctlydescribe experimental behavior. MD simulation shows details of possible pathways for lipid release that are not visible by diffractiontechniques, independently of their quality. Therefore, this simulationprovides a complementary methodology to fill in the gaps leftby the crystallographic images of the conformational average. Thejoint use of diffraction techniques at subatomic resolution, and moleculardynamics from reliable models, are consequently confirmedhere as a powerful set of tools.