Mechanisms of Substrate Selectivity and Transport by a Bacterial Methionine ABC Importer
Project Number5SC3GM144189-04
Contact PI/Project LeaderYANG, JANET G.
Awardee OrganizationUNIVERSITY OF SAN FRANCISCO
Description
Abstract Text
Project Summary
All organisms must selectively regulate the uptake and extrusion of molecules from the environment. In addition
to identifying and importing crucial ions, nutrients, and transition metals, cells must also rigorously select and
export a wide range of substrates, including peptides, polysaccharides, and toxins. The cell employs ATP-binding
cassette (ABC) transporters to drive the unidirectional transport of substrates against their concentration
gradients. This proposal seeks to understand how prokaryotic ABC importers uptake nutrients that are crucial
for survival, and these findings could provide new targets for treatment against bacterial pathogens. Towards
this end, we will employ biochemical and biophysical methods to dissect how the E. coli MetNI transporter, an
established model system, transports methionine from the periplasm to the cytoplasm. While the most widely-
accepted model for import is based on structural studies, emerging functional studies suggest a substantially
different model for substrate translocation. These disparate models appear to conflict; however, we hypothesize
that the MetNI transporter can adopt multiple modes of transport in response to substrate features and
availability. We will use the tools of mechanistic enzymology to resolve these longstanding issues and to propose
new models that may merge previous findings or uncover entirely new mechanisms of action. In Aim 1, we will
test our hypothesis by measuring the kinetic and thermodynamic parameters of individual steps in the transport
cycle. Using different substrates (L-Met, D-Met, and larger methionine derivatives) and binding-impaired
mutants, we will dissect three steps: (a) binding of the MetQ cognate periplasmic protein to the MetNI transporter,
(b) ATP binding and hydrolysis, and (c) transport. Our findings could reveal that the MetNI-Q mechanism is
inherently versatile and could preferentially select different translocation pathways depending on the substrate.
For Aim 2, we have generated heterodimeric MetNI “chimeras” to decipher if the two identical ATP sites work
together to enable efficient and specific transport. Specifically, we will determine if one or two ATP molecules
are hydrolyzed per transport cycle, and if transport efficiency is affected by impairment of one ATP site. Overall,
our findings will yield critical insights into how ABC transporters select for different substrates, and how ATP
usage drives substrate translocation across membranes.
Public Health Relevance Statement
Project Narrative
The survival of pathogenic bacteria such as Bacillus anthracis, Yersinia pestis, and Salmonella enterica relies
on ATP-binding cassette (ABC) transporters for the acquisition of extracellular nutrients from the environment.
Despite this crucial role of ABC transporters, how these transporters identify substrates and utilize ATP is not
fully understood. The overall goal of this project is to dissect individual steps in the mechanisms of ABC
transporters which will aid in the development of new strategies to combat infection and attenuate virulence.
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