Wang Lab Publishes in Nature Structural and Molecular Biology

Tue, 06/16/2015 - 4:00am

Dr. Nathan Karpowich in the laboratory of Da-Neng Wang of the Skirball Institute of Biomolecular Medicine at the NYU Lagone Medical Center have made a major advance in determining how some pathogenic bacteria “take their vitamins.”  Their work was published online on June 8 in Nature Structural and Molecular Biology as an article titled “ATP binding drives substrate capture in an ECF transporter by a release and catch mechanism.”

Vitamins are small organic molecules that function as essential enzymatic co-factors in all organisms.  Due to their complex chemical nature, vitamins are metabolically expensive for cells to synthesize de novo. As a result, some bacteria possess Energy Coupling Factor (ECF) transporters to import these essential micronutrients from the extracellular milieu. Previous work has shown that the active ECF transporter is composed of four unique subunits: a high affinity integral membrane substrate-binding subunit (EcfS) in complex with an energy coupling factor composed of a transmembrane coupling subunit (EcfT) and two homologous ABC ATPases (EcfA and EcfA’). Amazingly, in some bacteria, EcfS subunits for distinct substrates share a common ECF module for driving vitamin uptake.  For example, in L. lactis, EcfS proteins for eight different vitamins utilize the ECF transporter platform to import their substrates. 

For their work, Dr. Karpowich and his colleagues studied the mechanism of the purified ECF transport system from the human pathogen Listeria monocytogenes.  Using a combination of structural and biochemical approaches, the researchers found that ATP binding to the EcfAA¢ATPases drives a conformational change that dissociates the EcfS subunit from the EcfAA¢T ECF module.  Upon release from the ECF module, the isolated EcfS subunit tightly binds the transport substrate.  This “release and catch mechanism” explains how ECF transporters capture the transport substrate from the extracellular environment. 

This transport model involving EcfS release predicts that S subunits for different vitamins will compete for the ECF module.  Indeed, such competition has been suggested by vitamin uptake experiments and co-purification experiments from native hosts.  Through the development of a novel approach to examine transporter assembly, the authors found that EcfS subunits for distinct substrates compete for the ATP-bound state of the ECF module in vitro.  Specifically, the data indicated that the thiamine and riboflavin-binding EcfS proteins exclude binding of one another to the ECF complex. Thus, via this competition, cells can readily adapt to differences in substrate availability while utilizing a single transporter platform.  These results confirm the in vivo observations on S subunit competition for which the ECF transporter family was named.

 The research was performed by Dr. Nathan Karpowich, Jin Mei Song, and Nicolette Cocco. The American Heart Association and the U.S. National Institutes of Health (F32-HL091618) provided funding to Dr. Karpowich. Ms. Cocco is supported by a Biophysics training grant to NYU (T32-GM088118-05).  This work was also financially supported by the NIH grants R01DK099023,R01-DK073973, R01-GM093825 and R01- MH083840 to D.N.W.


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