A dynamic protein interactome drives energy conservation and electron flux in Thermococcus kodakarensis

Abstract

Life is supported by energy gains fueled by catabolism of a wide range of substrates, each reliant on the selective partitioning of electrons through redox (reduction and oxidation) reactions. Electron flux through tunable and regulated protein interactions provides dynamic routes for energy conservation, but how electron flux is regulated in vivo, particularly for archaeal metabolisms that support rapid growth at the thermodynamic limits of life, is poorly understood. Identification of bona fide in vivo protein assemblies and how such assemblies dictate the totality of electron flux is critical to our understanding of the regulation imposed on metabolism, energy production, and energy conservation. Here, 25 key proteins in central metabolic redox pathways in the model, genetically accessible, hyperthermophilic archaeon Thermococcus kodakarensis, were purified to reveal an extensive, dynamic, and tightly interconnected network of protein interactions that responds to environmental cues (such as the availability of various reductive sinks) to direct electron flux to maximize energetic gains. Interactions connecting disparate functions suggest many catabolic and anabolic activities occur in spatial proximity in vivo, and while protein complexes have been historically defined under optimal conditions, many of these complexes appear to maintain alternative partnerships in changing conditions. The totality of the results obtained redefines our understanding of in vivo assemblies driving ancient metabolic strategies supporting the growth of modern Archaea.IMPORTANCEGiven the potential for rational genetic manipulations of biofuel- and biotech-promising archaea to yield transformative results for major markets, it is a priority to define how the metabolisms of such species are controlled, at least in part, by in vivo protein assemblies, and from such, define routes of energy flux that can be most efficiently altered toward biofuel or biotechnological gains. Proteinaceous electron carriers (PECs, such as ferredoxins) offer the potential for specific protein-protein interactions to coordinate selective reductive flow. Employing the model, genetically accessible, hyperthermophilic archaeon, Thermococcus kodakarensis, we establish the metabolic protein interactome of 25 key redox proteins, revealing that each redox active protein has a dynamic partnership profile, suggesting catabolic and anabolic activities may occur in concert and in temporal and spatial proximity in vivo. These results reveal critical importance in evaluating the newly identified partnerships and their role and utility in providing regulated redox flux in T. kodakarensis.

Keywords: affinity purification coupled to mass spectrometry (AP-MS); archaea; electron flow; ferredoxin; oxidoreductase; protein-protein interaction; redox metabolism.

Fig 1

AP-MS identifies in vivo partnerships of tagged proteins at native expression levels. (A) Spectral counts of peptides are mapped to the T. kodakarensis proteome. Proteins identified in tagged samples are compared to those identified in the parent strain (a near isogenic control, TS559). A Fisher’s exact test is used to determine statistically significant, meaningful differences between tagged and control samples (P value < 0.05, log₂ fold change ≥ 2). Proteins that pass all statistical thresholds are ranked (column at far right), such that proteins that appear at a P value < 0.01 and a log₂ fold change >4, with spectral counts in all tagged replicate samples, earn a rank of 5. The rank score decreases by one point for each category not met. (B) Tagged proteins retained, on average, ~30 (range of 3–95) highly reproducible protein partnerships with changes introduced under different metabolic strategies induced by the absence (blue) or presence (purple) of the preferred terminal electron acceptor S˚. Protein partnerships are ranked and tallied for each target protein identified by the encoding gene number (e.g., TK0135) and complex acronym (e.g., IOR). The total protein associations copurified for each tagged protein are noted at the top of each bar, and the rank score of each partner is indicated by increasing shade color. At far right, a total of 870 and 725 proteins were identified as significant in +S˚ and –S˚ conditions, respectively, from these 25 tagged proteins. The majority of proteins (~44%) were identified with a rank score of 4, while ~34% were identified with a rank score of 5, followed by ~18% identified with a rank score of 3. Associations observed for GAPOR in S˚ conditions lacking pyruvate (–P) are depicted using a gray scale. (C) Proteins (ellipses) copurified with the tagged target protein (rectangle at center) in sulfur (+S˚, purple connecting lines) and non-sulfur (–S˚, blue connecting lines) conditions are visualized, such that associations with higher rank are shown with thicker lines and associations with lower rank scores are shown with thinner lines. Significant proteins are shown in colored ellipses (identified by gene number: TKxxxx) and generally arranged from lower gene numbers on the left to higher gene numbers moving right. While the majority of copurifying proteins are shown in blue, redox-associated proteins of special interest are displayed in varying colors, which match for proteins of similar annotation or within the same operon.

Fig 2

The redox protein metabolic network of T. kodakarensis is highly interconnected. The global redox protein network of rank 5 protein associations is shown. Tagged proteins in colored rectangles copurified proteins in colored ellipses in +S˚ (purple lines) or –S˚ (blue lines) conditions. Tagged proteins are arranged on the periphery, and subunits that are known or predicted to form complexes are colored to match: beginning at the top and moving clockwise, FDHs (fuchsia), GGR (dark blue), GAPOR (seafoam green), OGORs (mustard), FNORs (green), MBS (red), RBRs (gray), PORs (dark purple), VORs (plum), and IORs (orange). The majority of copurified proteins are displayed as bright blue ellipses, but subunits of some metabolically relevant complexes have been colored differently to stand out (e.g., MBH, TK2080-2093, lime green). Proteins that were copurified with only a single tagged protein are placed radiating outward from the network, while proteins that were independently copurified with multiple tagged proteins are located in the interior of the matrix (Cytoscape v3.10.1.).

Fig 3

POR and VOR complexes dynamically associate in vivo. (A) At the left, branched-chain amino acids are deaminated by an amino transferase (AT) to produce α-keto acids (or 2-oxoacids). VOR (plum ellipse) preferentially catalyzes the oxidation of α-keto acids into acyl-CoA while generating a reduced Fd. Acyl-CoA synthase (ACS) generates ATP, recycles CoA, and releases excreted acids. At the right, T. kodakarensis utilizes a modified EMP pathway in glycolysis, generating a net gain of just two ATP and two reduced Fds for the conversion of glucose into pyruvate. POR (dark purple) catalyzes the conversion of pyruvate into acetyl-CoA and CO₂ coupled to reduction of a Fd. Note that pyruvate is homologous to an α-keto acid from a deaminated alanine (box at the top), thus POR can additionally act on substrates from amino acid fermentation pathways. (B) POR and VOR share a γ subunit encoded by the first gene (purple, TK1978) in the vor/por operon, TK1978-1984. The remaining three subunits that make up the POR heterotetramer (dark purple, TK1982-TK1984) are encoded directly downstream of vor (plum, TK1978-TK1981). Three primary transcription start sites (TSS) appear upstream of TK1978, TK1979, and TK1984. (C) All seven POR/VOR subunits were tagged, and most tagged subunits identified all three of the other subunits of their respective complexes in both +S˚ (purple lines) and –S˚ (blue lines) conditions. (D) Intra-complex associations have been removed, and instead, associations between complexes (excluding VORg, TK1978) are shown. Eight inter-complex associations are observed in –S˚ while just two inter-complex associations are observed in +S˚.

Fig 4

Presumptive in vivo redox complex assemblages are redefined by AP-MS. (A) Seven genes encode the proposed OGOR heterooctamer: TK1123–TK1126 and TK1129–TK1131. When the OGORα₂ (TK1130), OGORβ₂ (TK1129), and OGORγ₁ (TK1123) subunits were tagged (mustard rectangles), AP-MS identified all seven subunits of the proposed 2α2β2δ2γ heterooctamer. While OGORα₁ was only copurified with OGORγ₁, all other subunits were copurified by multiple OGOR-tagged subunits, suggesting that while there could be 2α₁2β₁2δ2γ₁ or 2α₂2β₂2δ2γ₂ (an octamer composed of homodimers) complexes, the full heterodimeric (α₁α₂β₁β₂2δγ₁γ₂) complex is likely abundant. While OGORa₂ may associate with OGOR₁ subunits more than the other OGOR₂ subunits, OGORγ₁ identified OGORβ₂ and γ₂ only in –S˚ conditions, suggesting that there may be a preference for the heterooctameric confirmation in –S˚ conditions. (B) IOR subunits α₁ and β₁ (TK0136 and TK0135, respectively) reproducibly and reciprocally copurify, supporting regular association of protein products of these operonic encoded genes. Two non-operonic genes encoding an additional IOR α and β protein are annotated in the genome (TK1643, IORα₂ and TK2244, IORβ₂), and while both IORα₁ and IORβ₁ identified IORβ₂, no tagged IOR proteins identified IORα₂, suggesting that the functional in vivo IOR tetramer(s) may be composed as a 2α₁2β₁, 2α₁2β₂, or 2α₁β₁β₂ complex and that IORα₂ does not associate with the other IOR subunits. (C) TK2075, TK2077, and TK2078 are annotated as [4Fe-4S] cluster-binding proteins associated with FDH and are considered FDHγ_2-4, while TK2076 is annotated as FDHα. FDHγ_2-4 reciprocally copurified with one another, and FDHγ₂ and FDHγ₃ also copurified with TK2074, which is annotated as a glutamate synthase but homologous to To-FDH3B (31). While the FDHγ subunits copurify FDHα (predominantly in +S˚ conditions), FDHα did not copurify any FDHγ subunits, suggesting that FDHα may function independently, especially in –S˚ conditions. Associations seen in +S˚ are shown in purple, while associations seen in –S˚ are shown in blue (Cytoscape v3.10.1). Line thickness corresponds to the rank observed for each association, with a thicker line representing a higher rank score.

Fig 5

OGOR, FDH, and MBS form a highly interconnected network, linking disparate metabolic functions in sulfur conditions. OGOR (mustard rectangles), MBS (red rectangle), and FDH (fuchsia rectangles) copurify the same eight proteins (bright blue ellipses), including Fd3 (TK2012, yellow ellipse) and MBH-N (TK2093, lime green ellipse) in sulfur conditions. Line thickness corresponds to the rank observed for each association, with a thicker line representing a higher rank score. Gene annotations for associated proteins are as follows: TK0574, metallophosphoesterase, calcineurin superfamily; TK0789, glycerol-1-phosphate dehydrogenase [NAD(P)⁺]; TK0930, uncharacterized protein; TK0953, predicted ATPase, AAA superfamily, containing PIN and KH nucleic acid-binding domains; TK1328, tRNA (1-methyladenosine) methyltransferase; TK2012, ferredoxin 3; TK2093, membrane-bound hydrogenase, [4Fe-4S] cluster-binding subunit, MBH-N; TK2106, enolase. Line thickness corresponds to the rank observed for each association, with a thicker line representing a higher rank score (Cytoscape v3.10.1).

Fig 6

PECs are copurified with interacting partners and likely help maintain redox balance. (A) Tagged proteins (rectangles) copurify Fd2 (TK1087, bright yellow ellipse) and Fd3 (TK2012, yellow ellipse). Fd2 (TK1087) was copurified by GGR (TK1088, blue rectangle) in both +S˚ (purple line) and –S˚ (blue line) conditions. GGR also copurified Fd3 in –S˚ conditions. Both RBR1 (TK0650, gray rectangle) and RBR2 (TK0826, light gray rectangle) copurified Fd3, although the associations were observed in different redox conditions: RBR1-Fd3 was observed in +S˚, and RBR2-Fd3 was observed in –S˚. OGORβ (TK1129, mustard rectangle), FNOR2β (TK1685, green rectangle), and FDHγ₃ (TK2077, fuchsia rectangle) copurified Fd3 in +S˚ conditions, while MBH-L (TK1215) was the only protein to copurify Fd3 in both +S˚ and –S˚ conditions. (B) T. kodakarensis encodes three rubrerythrins, RBR1 (TK0650, dark gray ellipse), RBR2 (TK0826, gray ellipse), and RBR3 (TK1056, light gray ellipse), which were found to be significant interacting partners in the AP-MS data by 14 different tagged proteins (colored rectangles). All four subunits of VOR (TK1978-TK1981, purple rectangles) copurified RBR1. All four subunits of POR (TK1978, TK1982-TK1984, dark purple rectangles) copurified RBR2. Additionally, FDHγ₃, FDHγ₄ (TK2077 and TK2078, respectively, fuchsia rectangles), and GGR (TK1088, blue rectangle) copurified RBR2. Three subunits of OGOR (TK1123-TK1129, mustard rectangles) and MBS-L (TK1215, red rectangle) copurified RBR3. Associations seen in +S˚ are shown in purple, while associations seen in –S˚ are shown in blue (Cytoscape v3.10.1). Line thickness corresponds to the rank observed for each association, with a thicker line representing a higher rank score.