# Biogenesis of human mitochondrial iron-sulfur proteins

> **NIH NIH R01** · UNIVERSITY OF WISCONSIN-MADISON · 2020 · $359,393

## Abstract

PROJECT SUMMARY/ABSTRACT
Defects in iron-sulfur cluster assembly or delivery underlie many human diseases and aging processes, yet the
detailed mechanisms for these processes are still unknown. Organisms have evolved machinery consisting of
specialized proteins that operate together to assemble Fe-S clusters efficiently in a way that minimizes cellular
exposure to their toxic constituents (iron and sulfide ions). Many of these proteins are dynamic and participate
in weak complexes that have resisted structural analysis. We are studying these proteins and their interactions
in solution by a combination of NMR spectroscopy, small angle X-ray scattering, chemical crosslinking,
isothermal titration calorimetry, and functional biochemical assays. We are applying this approach to address
important questions regarding the mechanism of assembly of Fe-S clusters in human mitochondria. We are
building on the results of recent X-ray structures that provide a framework for our investigations. Last year, two
independent groups published X-ray structures of the (NFS1-ISD11-Acp)2 complex that exhibited very different
quaternary structures. To determine if these two different structures exist in solution, our approach is to
introduce 19F into NFS1 at a site that is different in the two structures. If two structures are present in solution,
we expect to see separate 19F NMR signals for each. By using 2D NMR experiments, we can investigate the
local environment of the probe and, if separate signals are observed may be able to determine the lifetimes of
each state. To date, all structural and functional studies of the mitochondrial cysteine desulfurase complex
have utilized complexes produced by overexpressing the human proteins NFS1 and ISD11 in E. coli cells. The
resulting (NFS1-ISD11-Acp)2 complex contains the holo-form of E. coli acyl carrier protein (Acp) in place of
human mitochondrial acyl carrier protein (ACP). We will determine structural and functional properties of
(NFS1-ISD11-Acp)2 and (NFS1-ISD11-ACP)2 without an acyl chain and with acyl chains of different lengths to
identify differences between Acp and ACP and to test the published hypothesis that acyl carrier protein in the
cysteine desulfurase complex provides a regulatory link coordinating mitochondrial fatty acid synthesis with
iron sulfur cluster biogenesis. Another aim is to characterize the conformational changes in the cysteine
desulfurase complex that accompany different steps in cluster assembly and the transfer of the assembled
cluster on ISCU to the co-chaperone HSC20. Although it is known that ISCU populates two interconverting
conformations in solution (one structured and one intrinsically disordered), the functional roles of these two
states remain to be elucidated. By separately labeling Trp residues in ISCU and NFS1 with fluorine, we have
determined that 19F NMR enables the observation of different conformational states. These preliminary results
set the stage for experiments designe...

## Key facts

- **NIH application ID:** 10001537
- **Project number:** 5R01GM127666-02
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** JOHN LUTE MARKLEY
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $359,393
- **Award type:** 5
- **Project period:** 2019-09-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10001537

## Citation

> US National Institutes of Health, RePORTER application 10001537, Biogenesis of human mitochondrial iron-sulfur proteins (5R01GM127666-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10001537. Licensed CC0.

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