The Oxoglutarate Binding Site and Regulatory Mechanism Are Conserved in Ammonium Transporter Inhibitors GlnKs from Methanococcales

Abstract

Protein inhibition is a natural regulatory process to control cellular metabolic fluxes. PII-family signal-transducing effectors are in this matter key regulators of the nitrogen metabolism. Their interaction with their various targets is governed by the cellular nitrogen level and the energy charge. Structural studies on GlnK, a PII-family inhibitor of the ammonium transporters (Amt), showed that the T-loops responsible for channel obstruction are displaced upon the binding of 2-oxoglutarate, magnesium and ATP in a conserved cleft. However, GlnK from Methanocaldococcus jannaschii was shown to bind 2-oxoglutarate on the tip of its T-loop, causing a moderate disruption to GlnK–Amt interaction, raising the question if methanogenic archaea use a singular adaptive strategy. Here we show that membrane fractions of Methanothermococcus thermolithotrophicus released GlnKs only in the presence of Mg-ATP and 2-oxoglutarate. This observation led us to structurally characterize the two GlnK isoforms apo or in complex with ligands. Together, our results show that the 2-oxoglutarate binding interface is conserved in GlnKs from Methanococcales, including Methanocaldococcus jannaschii, emphasizing the importance of a free carboxy-terminal group to facilitate ligand binding and to provoke the shift of the T-loop positions.

Transcriptomics sheds light on N2-fixation strategies employed by a thermophilic member of theMethanococcales

“…The genome of M. thermolithotrophicus harbors two different amtB genes with their associated glnK regulators (40). Interestingly, amtB1 and glnK1 were not differentially expressed (Supplementary Table 2, Figure 4A-B), which might point to the existence of an internal regulator or an additional promotor.…”

“…Series of incubations with different NH4Cl concentrations, ranging from 0.1 to 16.8 mM, showed that M. thermolithotrophicus required a minimum of 10 mM NH4Cl for best growth (Fig. 1A) in our optimized medium (see Materials and Methods for composition) (39,40).…”

“…Series of incubations with different NH 4 Cl concentrations, ranging from 0.1 to 16.8 mM, showed that M. thermolithotrophicus required a minimum of 10 mM NH 4 Cl for best growth (Fig. 1A) in our optimized medium (see Materials and Methods for composition) (39, 40). Higher NH 4 Cl concentrations of 25-200 mM NH 4 Cl did not result in higher cell yields, and a 500 mM excess of NH 4 Cl led to a decrease (Fig.…”

“…The other genes upregulated at 3 h included the ammonium transporter amtB 2 and its P II -family regulatory protein glnK 2 with the log 2 FC of 6.2 and 5.8, respectively (Supplementary Table 2). The genome of M. thermolithotrophicus harbors two different amtB genes with their associated glnK regulators (40). Interestingly, amtB 1 and glnK 1 were not differentially expressed (Supplementary Table 2, Figure 4A-B), which might point to the existence of an internal regulator or an additional promotor.…”

“…The used minimal mineral medium was prepared as described in Müller et al . 2021 (40), but with replacement at equal final concentration of Fe(NH 4 ) 2 (SO 4 )2 x 12H 2 O by FeCl 2 x 4H 2 O, and of Na 2 SeO 3 x 5H 2 O by Na 2 SeO 4 . The used trace metal solution 100 fold concentrated contained 7.1 mM nitrilotriacetic acid, 0.45 mM MnCl 2 x 2H 2 O, 0.68 mM FeCl 3 x 6H 2 O, 0.41 mM CaCl 2 , 0.76 mM CoCl 2 , 0.66 mM ZnSO 4 x 6H 2 O, 0.28 mM CuSO 4 , 0.19 mM Na 2 MoO 4 x 2H 2 O, 0.38 mM NiCl 2 x 6H 2 O and 0.19 mM VCl 3 .…”