Robust Production, Crystallization, Structure Determination, and Analysis of [Fe–S] Proteins: Uncovering Control of Electron Shuttling and Gating in the Respiratory Metabolism of Molybdopterin Guanine Dinucleotide Enzymes

Tsai, Chi Lin; Tainer, John A.

doi:10.1016/bs.mie.2017.11.006

Cited by 10 publications

(11 citation statements)

References 183 publications

(243 reference statements)

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“…Here, we have shown that there is a single Fe–S cluster in four-subunit Pol ϵ and that this cluster is bound to the Cys-motif (CysX) at the base of the P-domain in the catalytic subunit, Pol2. Fe–S clusters have been reported to stabilize protein structures and subunit interactions, but also to regulate substrate binding and/or catalysis in a redox state-dependent manner (27–33). In the case of the two replicative eukaryotic polymerases, Pol δ and Pol ϵ, there are clear differences when comparing the function of their Fe–S clusters.…”

Section: Discussionmentioning

confidence: 99%

Structural evidence for an essential Fe–S cluster in the catalytic core domain of DNA polymerase ϵ

Beek

Parkash

Bylund

et al. 2019

Nucleic Acids Research

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DNA polymerase ϵ (Pol ϵ), the major leading-strand DNA polymerase in eukaryotes, has a catalytic subunit (Pol2) and three non-catalytic subunits. The N-terminal half of Pol2 (Pol2 CORE ) exhibits both polymerase and exonuclease activity. It has been suggested that both the non-catalytic C-terminal domain of Pol2 (with the two cysteine motifs CysA and CysB) and Pol2 CORE (with the CysX cysteine motif) are likely to coordinate an Fe–S cluster. Here, we present two new crystal structures of Pol2 CORE with an Fe–S cluster bound to the CysX motif, supported by an anomalous signal at that position. Furthermore we show that purified four-subunit Pol ϵ, Pol ϵ CysA MUT (C2111S/C2133S), and Pol ϵ CysB MUT (C2167S/C2181S) all have an Fe–S cluster that is not present in Pol ϵ CysX MUT (C665S/C668S). Pol ϵ CysA MUT and Pol ϵ CysB MUT behave similarly to wild-type Pol ϵ in in vitro assays, but Pol ϵ CysX MUT has severely compromised DNA polymerase activity that is not the result of an excessive exonuclease activity. Tetrad analyses show that haploid yeast strains carrying CysX MUT are inviable. In conclusion, Pol ϵ has a single Fe–S cluster bound at the base of the P-domain, and this Fe–S cluster is essential for cell viability and polymerase activity.

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Section: Discussionmentioning

confidence: 99%

Structural evidence for an essential Fe–S cluster in the catalytic core domain of DNA polymerase ϵ

Beek

Parkash

Bylund

et al. 2019

Nucleic Acids Research

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“…To accelerate biochemical studies of [Fe-S] proteins, genes encoding proteins of interest are often heterologously expressed in engineered E. coli strains designed for overproduction of proteins. A major challenge in the field is to obtain large enough quantities of proteins at high concentrations that are also maximally occupied with [Fe-S] clusters (34). Increasing the expression levels of housekeeping Isc pathway imparts variable improvement in [Fe-S] cluster protein yields (8,13,20,34).…”

Section: Introductionmentioning

confidence: 99%

“…A major challenge in the field is to obtain large enough quantities of proteins at high concentrations that are also maximally occupied with [Fe-S] clusters (34). Increasing the expression levels of housekeeping Isc pathway imparts variable improvement in [Fe-S] cluster protein yields (8,13,20,34). However, to our knowledge, a similar approach has not been examined for the Suf pathway despite it being the sole pathway for [Fe-S] biogenesis in many organisms.…”

Section: Introductionmentioning

confidence: 99%

Elevated Expression of a Functional Suf Pathway in theE.coliBL21(DE3) Cell Line Enhances Recombinant Production of an Iron-Sulfur Cluster Containing Protein

Ei¹,

et al. 2019

Preprint

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Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that Escherichia coli BL21(DE3), a strain routinely used to overexpress [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an inframe fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but isinactive in [Fe-S] biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (~3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxido-reductase complex. These results show that this engineered 'SufFeScient' derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein. IMPORTANCE Large quantities of recombinantly overexpressed iron-sulfur cluster-containing proteins are necessary for their in-depth biochemical characterization. Commercially available E.coli strain BL21(DE3) and its derivatives have a mutation that inactivates the function of one of the two native pathways (Suf pathway) responsible for cluster biogenesis.Correction of the mutation, combined with sequence changes that increase Suf expression can increase yield and cluster occupancy of [Fe-S] cluster-containing enzymes, facilitating the biochemical analysis of this fascinating group of proteins.

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“…To accelerate biochemical studies of [Fe-S] proteins, genes encoding proteins of interest are often heterologously expressed in engineered E. coli strains designed for overproduction of proteins. A major challenge in the field is to obtain large enough quantities of proteins at high concentrations that are also maximally occupied with [Fe-S] clusters (17) to enable spectroscopic and structural studies. Increasing the level of the housekeeping Isc pathway imparts variable improvement in [Fe-S] cluster protein yields (17)(18)(19)(20).…”

mentioning

confidence: 99%

“…A major challenge in the field is to obtain large enough quantities of proteins at high concentrations that are also maximally occupied with [Fe-S] clusters (17) to enable spectroscopic and structural studies. Increasing the level of the housekeeping Isc pathway imparts variable improvement in [Fe-S] cluster protein yields (17)(18)(19)(20). However, to our knowledge, a similar approach has not been examined for the Suf pathway despite it being the sole pathway for [Fe-S] biogenesis in many organisms.…”

mentioning

confidence: 99%

Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein

et al. 2020

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Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that Escherichia coli BL21(DE3), a strain routinely used to overproduce [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an in-frame fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but is inactive in [Fe-S] cluster biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (∼3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxidoreductase complex. These results show that this engineered “SufFeScient” derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein. IMPORTANCE Large quantities of recombinantly overproduced [Fe-S] cluster-containing proteins are necessary for their in-depth biochemical characterization. Commercially available E. coli strain BL21(DE3) and its derivatives have a mutation that inactivates the function of one of the two native pathways (Suf pathway) responsible for cluster biogenesis. Correction of the mutation, combined with sequence changes that elevate Suf protein levels, can increase yield and cluster occupancy of [Fe-S] cluster-containing enzymes, facilitating the biochemical analysis of this fascinating group of proteins.

show abstract

Robust Production, Crystallization, Structure Determination, and Analysis of [Fe–S] Proteins: Uncovering Control of Electron Shuttling and Gating in the Respiratory Metabolism of Molybdopterin Guanine Dinucleotide Enzymes

Cited by 10 publications

References 183 publications

Structural evidence for an essential Fe–S cluster in the catalytic core domain of DNA polymerase ϵ

Structural evidence for an essential Fe–S cluster in the catalytic core domain of DNA polymerase ϵ

Elevated Expression of a Functional Suf Pathway in theE.coliBL21(DE3) Cell Line Enhances Recombinant Production of an Iron-Sulfur Cluster Containing Protein

Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein

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