Conformational stability, dynamics and function of human frataxin: Tryptophan side chain interplay

Espeche, Lucía D.; Sewell, Karl Ellioth; Castro, Ignacio Hugo; Capece, Luciana; Pignataro, María Florencia; Daín, Liliana; Santos, Javier

doi:10.1016/j.abb.2021.109086

Cited by 3 publications

(10 citation statements)

References 46 publications

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“…The last part of the protein, the C‐terminal region (residues 196–210, CTR), is essential for FXN stability (Faraj et al 2014, 2016, 2019) and it lacks a periodic structure (Figure 1a,b). Remarkably, some frataxin variants, that result in FRDA, exhibit a destabilized conformation, among them G130V (Correia et al, 2008), G137V (Faggianelli et al, 2015), W173G (Espeche et al, 2022) and L198R (Faraj et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

“…In fact, it is unclear whether frataxin remains in a bound state during the catalytic cycle. Based on molecular dynamics simulations performed by our laboratory, one of our working hypotheses is that wild‐type frataxin forms part of the assembly site but it is also able to constrain the molecular motions of NFS1 and ISCU (Espeche et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

“…Remarkably, some frataxin variants, that result in FRDA, exhibit a destabilized conformation, among them G130V (Correia et al, 2008), G137V (Faggianelli et al, 2015), W173G (Espeche et al, 2022) and L198R (Faraj et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

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Selection of synthetic proteins to modulate the human frataxin function

Pignataro

Herrera

Fernández

et al. 2022

Biotech & Bioengineering

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Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreich's Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigated the possibility of obtaining small proteins exhibiting a high affinity for frataxin. In this study, we applied the ribosome display approach, using human frataxin as the target. We focused on Affi_224, one of the proteins that we were able to select after five rounds of selection. We have studied the interaction between both proteins and discussed some applications of this specific molecular tutor, concerning the modulation of the supercomplex activity. Affi_224 and frataxin showed a K D value in the nanomolar range, as judged by surface plasmon resonance analysis. Most likely, it binds to the frataxin acidic ridge, as suggested by the analysis of chemical shift perturbations (nuclear magnetic resonance) and computational simulations. Affi_224 was able to increase Cys NFS1 desulfurase activation exerted by the FRDA frataxin variant G130V. Importantly, Affi_224 interacts with frataxin in a human cellular model. Our results suggest quaternary addition may be a new tool to modulate frataxin function in vivo. Nevertheless, more functional experiments under physiological conditions should be carried out to evaluate Affi_224 effectiveness in FRDA cell models.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Selection of synthetic proteins to modulate the human frataxin function

Pignataro

Herrera

Fernández

et al. 2022

Biotech & Bioengineering

Self Cite

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show abstract

“…In fact, it is unclear whether frataxin remains in a bound state during the catalytic cycle. Based on molecular dynamics simulations performed by our laboratory, one of our working hypotheses is that wild–type frataxin forms part of the assembly site but it is also able to constrain the molecular motions of NFS1 and ISCU [19].…”

Section: Introductionmentioning

confidence: 99%

“…The last part of the protein, the C-terminal region (residues 196-210, CTR), is essential for FXN stability [11, 12, 16] and it lacks a periodic structure ( Figures 1A and B ). Remarkably, some frataxin variants, that result in FRDA, exhibit a destabilized conformation, among them G130V [17], G137V [18], W173G [19] and L198R [12].…”

Section: Introductionmentioning

confidence: 99%

Selection of Synthetic Proteins to Modulate the Human Frataxin Function

Pignataro

Herrera

Fernández

et al. 2022

Preprint

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Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreich's Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigated the possibility of obtaining small proteins exhibiting a high affinity for frataxin. In this study, we applied the ribosome display approach, using human frataxin as the target. We focused on Affi_224, one of the proteins that we were able to select after five selection rounds. We have studied the interaction between both proteins and discussed some applications of this specific molecular tutor, concerning the modulation of supercomplex activity. Affi_224 and frataxin showed a KD value in the nanomolar range, as judged by surface plasmon resonance analysis. Most likely, it binds to the frataxin acidic ridge, as suggested by the analysis of chemical shift perturbations (NMR) and computational simulations. Affi_224 was able to increase Cys NFS1 desulfurase activation exerted by the FRDA frataxin variant G130V. Our results suggest quaternary addition may be a new tool to modulate frataxin function in vivo. Nevertheless, more functional experiments under physiological conditions should be carried out to evaluate Affi_224 effectiveness in FRDA cell models.

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Direct Cysteine Desulfurase Activity Determination by NMR and the Study of the Functional Role of Key Structural Elements of Human NFS1

et al. 2023

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The mitochondrial cysteine desulfurase NFS1 is an essential PLP-dependent enzyme involved in iron–sulfur cluster assembly. The enzyme catalyzes the desulfurization of the l-Cys substrate, producing a persulfide and l-Ala as products. In this study, we set the measurement of the product l-Ala by NMR in vitro by means of 1H NMR spectra acquisition. This methodology provided us with the possibility of monitoring the reaction in both fixed-time and real-time experiments, with high sensitivity and accuracy. By studying I452A, W454A, Q456A, and H457A NFS1 variants, we found that the C-terminal stretch (CTS) of the enzyme is critical for function. Specifically, mutation of the extremely conserved position W454 resulted in highly decreased activity. Additionally, we worked on two singular variants: “GGG” and C158A. In the former, the catalytic Cys-loop was altered by including two Gly residues to increase the flexibility of this loop. This variant had significantly impaired activity, indicating that the Cys-loop motions are fine-tuned in the wild-type enzyme. In turn, for C158A, we found an unanticipated increase in l-Cys desulfurase activity. Furthermore, we carried out molecular dynamics simulations of the supercomplex dedicated to iron–sulfur cluster biosynthesis, which includes NFS1, ACP, ISD11, ISCU2, and FXN subunits. We identified CTS as a key element that established interactions with ISCU2 and FXN concurrently; we found specific interactions that are established when FXN is present, reinforcing the idea that FXN not only forms part of the iron–sulfur cluster assembly site but also modulates the internal motions of ISCU2.

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Conformational stability, dynamics and function of human frataxin: Tryptophan side chain interplay

Cited by 3 publications

References 46 publications

Selection of synthetic proteins to modulate the human frataxin function

Selection of synthetic proteins to modulate the human frataxin function

Selection of Synthetic Proteins to Modulate the Human Frataxin Function

Direct Cysteine Desulfurase Activity Determination by NMR and the Study of the Functional Role of Key Structural Elements of Human NFS1

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