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Heliorhodopsin binds and regulates glutamine synthetase activity [1]

['Shin-Gyu Cho', 'Department Of Life Science', 'Institute Of Biological Interfaces', 'Sogang University', 'Seoul', 'Research Institute For Basic Science', 'Myungchul Song', 'Kimleng Chuon', 'Jin-Gon Shim', 'Seanghun Meas']

Date: 2022-10

Photoreceptors are light-sensitive proteins found in various organisms that respond to light and relay signals into the cells. Heliorhodopsin, a retinal-binding membrane protein, has been recently discovered, however its function remains unknown. Herein, we investigated the relationship between Actinobacteria bacterium IMCC26103 heliorhodopsin (AbHeR) and an adjacent glutamine synthetase (AbGS) in the same operon. We demonstrate that AbHeR binds to AbGS and regulates AbGS activity. More specifically, the dissociation constant (K d ) value of the binding between AbHeR and AbGS is 6.06 μM. Moreover, the absence of positively charged residues within the intracellular loop of AbHeR impacted K d value as they serve as critical binding sites for AbGS. We also confirm that AbHeR up-regulates the biosynthetic enzyme activity of AbGS both in vitro and in vivo in the presence of light. GS is a key enzyme involved in nitrogen assimilation that catalyzes the conversion of glutamate and ammonia to glutamine. Hence, the interaction between AbHeR and AbGS may be critical for nitrogen assimilation in Actinobacteria bacterium IMCC26103 as it survives in low-nutrient environments. Overall, the findings of our study describe, for the first time, to the best of our knowledge, a novel function of heliorhodopsin as a regulatory rhodopsin with the capacity to bind and regulate enzyme activity required for nitrogen assimilation.

Funding: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2020R1D1A1A2070892 and NRF-2020R1A2C2008197 to K-H.J., https://www.nrf.re.kr/index ). This research was also supported by Korea Basic Science Institute (National research Facilities and Equipment Center) grant funded by the Ministry of Education (NRF-2020R1A6C101A192 to K-H.J., https://www.kbsi.re.kr ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Herein, we investigate the relationship between AbGS and AbHeR. Notably, protein–protein interactions (PPIs) were observed between AbHeR and AbGS, which was associated with the regulatory function of AbHeR for AbGS. Here, we report, for the first time, to the best of our knowledge, one of the functional roles for AbHeR in regulating AbGS activity.

To further explore the function of heliorhodopsin, we analyzed SRs that interact with other proteins called transducers, which genes are located adjacent to those of SRs, suggesting that a single promoter can transcribe both SR- and transducer-encoding genes [ 22 ]. We hypothesized that heliorhodopsin may also interact with other proteins encoded by adjacent genes, similar to SRs. In particular, Actinobacteria bacterium IMCC26103 heliorhodopsin (AbHeR) is flanked by glutamine synthetase (GS; AbGS, EC 6.3.1.2) [ 23 ]. GS is a central enzyme associated with nitrogen assimilation and is distributed in bacteria as well as plant and animal tissues. GS catalyzes the conversion of glutamate and ammonia to glutamine [ 24 – 27 ]. In fact, GS absence or mutation can lead to reduced growth in bacteria and plants, low grain yield in plants, as well as multi-organ failure and neonatal death in humans [ 27 – 29 ].

Recently, a new family of microbial rhodopsins, termed heliorhodopsin, has been identified. Heliorhodopsin can be differentiated from type I rhodopsins based on its sequence. That is, the inclusion of heliorhodopsins in the phylogenetic tree of microbial rhodopsins forms distinct clades from type I rhodopsins [ 17 ]. Although, heliorhodopsin is also a 7-transmembrane retinal-binding protein, it lacks critical residues involved in pumping ions. Unlike type I and type II rhodopsins, the topology of heliorhodopsin is invertedly embedded; i.e., the N- and C-termini are located on the cytoplasmic and extracellular sides, respectively [ 17 ]. Moreover, the oligomeric heliorhodopsin crystal structures revealed a dimer containing several hydrophobic residues, which is not conducive to ion transport [ 18 , 19 ]. In other studies, the binding of divalent cations to heliorhodopsin was studied using attenuated total reflectance-Fourier transform infrared spectroscopy, wherein the binding of Zn 2+ to heliorhodopsin was detected [ 20 ]. Heliorhodopsin photocycle was slow, a feature that is widespread in sensory rhodopsins (SRs). Accordingly, Pushkarev and colleagues suggested that heliorhodopsins function as a distinct type of signaling photoreceptors similar to SRs [ 17 ], whereas the N-termini of heliorhodopsins might participate in enzyme function [ 21 ].

Light-driven type I rhodopsins have been described as H + , Cl - , and Na + pumps, light sensors, and channelrhodopsins. They typically contain conserved residues that are specific for the ion that they transport [ 6 – 12 ]. Microbial rhodopsins contain various critical residues, including those within the retinal-binding pocket, retinal covalent linkage, and counterions associated with retinal [ 13 ]. N- and C-termini are located on the extracellular and cytoplasmic sides, respectively, in type I rhodopsins. Oligomeric forms of the ion pumps and channelrhodopsins are tri- to penta/hexamers, and dimers, respectively [ 14 – 16 ].

Organisms from all domains of life use photoreceptor proteins to sense and respond to light. Most rhodopsins, one of the groups of photoreceptor proteins, are light-driven 7-transmembrane retinal-binding proteins found in prokaryotic and eukaryotic organisms. Rhodopsin comprises opsin as apoprotein and retinal as a covalently linked chromophore that absorbs photons for energy conversion or signal transduction [ 1 ]. Indeed, rhodopsins play important roles in organism survival. More specifically, type II rhodopsins, which are found mostly in animals, participate in visual and non-visual phototransduction (circadian rhythms, sensing dawn/dark, and determining the horizon) [ 2 – 4 ]. In contrast, type I rhodopsins in microbes participate in the active or passive transport of ions and signal transduction [ 5 ].

Results

Growth rate test of cells with AbGS and AbHeR We hypothesized that co-expression of AbHeR and AbGS in a cell would up-regulate AbGS activity in the presence of light. E. coli K-12 MG1655 and JW3841 strains (glnA knockout (KO) strain derived from K-12 MG1655) were used as the positive control and experimental group to study the influence of the absence of glnA encoding GS alone, respectively. Cellular growth rate was assessed in E. coli K-12 MG1655 and JW3841 strains to compare the complementation of AbGS with AbHeR. We added L-glutamate to 3-morpholinopropane-1-sulfonic acid (MOPS) minimal medium to induce AbGS activity. The K-12 MG1655 strain was transformed with the all-in-one (AIO) plasmid containing ampicillin and kanamycin as the glnA KO strain exhibited kanamycin resistance. The glnA KO strain was transformed with pKA001 vector containing AbGS and AbHeR [46], which were regulated by P lac and P araBAD , respectively. The growth rate of cells was then measured in the absence or presence of L-glutamate, which is required for the catalytic activity of AbGS. Finally, we assessed the growth rate of cells upon complementation of AbGS with AbHeR. The K-12 MG1655 strain was grown in the absence of L-glutamate and saturated for 48 h. In the presence of L-glutamate, the cells were saturated at a higher cell density, indicating that supplementary L-glutamate influenced cell density and growth rate (Fig 4A); i.e., the MOPS minimal medium in place of Luria–Bertani (LB) medium is sufficient to permit cell growth. The glnA gene in E. coli is the key enzyme involved in nitrogen fixation; glnA-deficient E. coli strain reportedly does not grow in minimal mediums [47–49]. Therefore, we postulated that if AbGS catalyzes L-glutamate to L-glutamine in the glnA KO strain in the presence of L-glutamate, it would exhibit cell growth similar to that of the K-12 MG1655 strain. As expected, the glnA KO strain containing the null vector (glnA KO + pKA001) did not grow in MOPS minimal medium. Upon the addition of L-glutamate, cell density began to decrease after 72 h (Fig 4B). Similarly, the glnA KO strain expressing AbGS (glnA KO + AbGS) did not grow in the absence of L-glutamate. Meanwhile, the cells showed slow growth and became saturated at 72 h in the presence of L-glutamate, indicating that AbGS successfully converted L-glutamate to L-glutamine (Fig 4C). However, the complementation was not recovered to the level exhibited by the K-12 MG1655 strain, suggesting that AbGS activity is lower than that of the well-known GS. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. In vivo growth rate of E. coli glnA KO strains with AbGS and AbHeR. Cells are grown in LB medium and washed to remove components of the LB medium. The cells are then transferred to MOPS minimal medium. The growth of transferred cells was recorded at OD 600 at different time points; growth is normalized to the initial recording. The E. coli K-12 MG1655 strain transformed with AIO vector (A), glnA KO strain transformed with pKA001 null vector (B), and glnA KO strain transformed with pKA001-GS vector (C) were incubated in the absence and presence of L-glutamate in MOPS minimal medium containing IPTG, which regulates AbGS expression. (D–G) The E. coli glnA KO strain transformed with pKA001 null or pKA001-GS vector was incubated in MOPS minimal medium containing L-glutamate and glycerol (MOPS-GG). The E. coli glnA KO strain transformed with pKA001-GS-AbHeR WT (F) and K216Q (G) with and without ATR in the absence (dark) and presence of light (light, 532 nm) conditions at 40 μmol m-2 s-1 were incubated in MOPS-GG medium. (A–E) The tests were performed in an independent experimental group (n = 3). (F and G) The tests were performed in an independent experimental group (n = 9). (E) Statistical significance between the 2 groups (pKA001 and AbGS) was analyzed using the t test; p-values are indicated with asterisks. (F and G) Statistical significance between the 2 groups (dark and light) was analyzed using the t test; p-values are indicated with asterisks. The data are expressed as mean ± standard deviation. Based on data (F) and (G), half-life (t 1/2 ) for growth rate of cells was analyzed to quantify the effect of AbGS binding in the absence and presence of light by exponential decay. Nonlinear fitted lines are shown for AbHeR WT (H) and K216Q (I). Quantified t 1/2 values are indicated by the same color as that of each rectangle, circle, and triangle. The underlying data of the graph can be found in S3 Data. AIO, all-in-one; KO, knockout; LB, Luria–Bertani; WT, wild type. https://doi.org/10.1371/journal.pbio.3001817.g004 L-glucose, as a carbon source in the MOPS minimal medium, can interfere with isopropyl β-D-1-thiogalactopyranoside (IPTG), which regulates AbGS expression on the lac operon; therefore, we used glycerol as the carbon source in the MOPS minimal medium. In addition, we assumed that the medium supplemented with glycerol provides a limited source of energy for growth. The synthesis of recombinant proteins expressed by IPTG may affect cell growth rate. Therefore, we added different concentrations of IPTG to the medium. The glnA KO + AbGS showed no significant difference in cell growth across different IPTG concentrations in MOPS minimal medium containing glycerol (Fig 4D), implying that cells transferred into the medium do not require IPTG for complementation through AbGS. Next, we conducted experiments with MOPS minimal medium containing L-glutamate and glycerol in the absence of glucose and IPTG (MOPS-GG medium). We compared glnA KO + pKA001 and AbGS to confirm complementation of AbGS in MOPS-GG medium. AbGS showed complementation in the glnA KO strain; the cells exhibited reduced growth in the absence of AbGS and complementation with AbGS statistically significantly slowed down the decay of cells (Fig 4E). We designed a strain in which AbGS and AbHeR were co-expressed in the pKA001 vector, and their expression was induced by IPTG and L-arabinose, respectively. We assumed that AbHeR without ATR cannot regulate AbGS in the absence or presence of light, whereas AbHeR with ATR regulates AbGS in the presence of light. Therefore, the growth of cells may be altered. However, the cells co-expressing AbHeR and AbGS showed decreased growth levels similar to that of glnA KO + pKA001 cells (Fig 4F). AbGS expression levels in the cells co-expressing AbHeR and AbGS were lower than those in the cells expressing AbGS after induction, indicating that a specific amount of AbGS is essential for complementation (S4D–S4F Fig). Although the cells co-expressing AbHeR and AbGS showed reduced growth rate, AbHeR may influence the growth of cells in the presence of light. Surprisingly, the growth of cells co-expressing AbGS and AbHeR with ATR in the presence of light was statistically significantly higher than that in the absence of light. In addition, the growth of cells expressing AbGS and AbHeR without ATR was not significantly different from that of cells expressing AbGS and AbHeR with ATR in the absence of light. That is, AbHeR could not regulate AbGS in the absence of light harvesting (Fig 4F). The low binding affinity of AbGS in the AbHeR K216Q mutant was measured using the same experimental method as that for AbHeR WT. AbHeR K216Q did not influence cell growth significantly (Fig 4G). We quantified the growth levels because it was difficult to compare the down decay of cells expressing AbGS as well as AbHeR WT or K216Q. Using exponential decay, the t 1/2 of growth rate of cells containing AbHeR WT in the presence of light was 1.58 times greater than that in the absence of light (Fig 4H); however, t 1/2 values of growth rates of cells containing AbHeR K216Q in presence of light were not altered when compared with those in the absence of light (Fig 4I). These t 1/2 values also suggest that AbGS can be partially regulated by AbHeR in the presence of light and that AbGS does not bind well to AbHeR K216Q.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001817

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