# Metal Ion Transport by the Cation Diffusion Facilitator Family

> **NIH NIH R01** · NEW YORK UNIVERSITY SCHOOL OF MEDICINE · 2022 · $434,162

## Abstract

ABSTRACT
This project addresses fundamental mechanisms by which secondary transporters in the Cation
Diffusion Facilitator (CDF) family carry transition metal ions such as Zn2+, Mn2+, Fe2+ and Co2+
across the membrane. These ions serve as cofactors for a diverse array of enzymes and
regulatory proteins. The ions play a role in many different physiological processes and, as a
result, CDF transporters are widespread. CDF transporters are involved both in uptake of ions,
which are normally trace elements in the environment, and in export of ions, thus providing
tolerance to extreme environments. We propose to combine structural, functional and
computational studies to generate a detailed mechanistic understanding of the bacterial Zn2+
transporter YiiP and to extend this understanding to other branches of the family represented by
specific bacterial and eukaryotic homologs displaying different in ion selectivities and having
unique structural domains. Aim 1 will focus on defining conformational changes in YiiP that
characterize the alternating access mechanism, a paradigm for the transport of substrates
across biological membranes. For this first aim, we will use cryo-EM to characterize the
structure of the outward-facing state as well as Zn2+-free states of YiiP in a lipid environment.
We will also use Molecular Dynamics to characterize the dynamics of conformational changes
between these states as well as the energetics of the transport cycle. Aim 2 will investigate
functional determinants of transport. In particular, we will study energy coupling of YiiP using in
vitro transport assays to characterize the coupling of Zn2+ transport to the proton motive force,
will explore potential roles of Zn2+ binding sites in the cytoplasmic domain as either structural
elements stabilizing the homodimer or as functional elements that regulate activity, and will
characterize cooperativity between the two molecules that compose the dimer. In Aim 3, we will
expand our studies on YiiP to related CDF family members from a diverse array of organisms,
thus sampling all three branches of the family tree. We have identified from previous
publications a number of bacterial and eukaryotic homologs have been heterologously
expressed in either E. coli or S. cerevisiae and used for cell-based assays. We will screen these
homologs for high expression levels and stability and use the best behaved to explore the basis
for ion selectivity, to compare mechanisms of energy coupling, and to evaluate the functional
role of histidine-rich loops. These loops have been postulated to bind metal ions, suggesting
potential roles in regulation or activation of transport.

## Key facts

- **NIH application ID:** 10319967
- **Project number:** 5R01GM125081-04
- **Recipient organization:** NEW YORK UNIVERSITY SCHOOL OF MEDICINE
- **Principal Investigator:** David L. Stokes
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $434,162
- **Award type:** 5
- **Project period:** 2019-01-01 → 2022-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10319967

## Citation

> US National Institutes of Health, RePORTER application 10319967, Metal Ion Transport by the Cation Diffusion Facilitator Family (5R01GM125081-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10319967. Licensed CC0.

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