Molecular basis for the distinct divalent cation requirement in the uridylylation of the signal transduction proteins GlnJ and GlnB from Rhodospirillum rubrum

Abstract

Background: PII proteins have a fundamental role in the control of nitrogen metabolism in bacteria, through interactions with different PII targets, controlled by metabolite binding and post-translational modification, uridylylation in most organisms. In the photosynthetic bacterium Rhodospirillum rubrum, the PII proteins GlnB and GlnJ were shown, in spite of their high degree of similarity, to have different requirements for post-translational uridylylation, with respect to the divalent cations, Mg(2+) and Mn(2+).

Results: Given the importance of uridylylation in the functional interactions of PII proteins, we have hypothesized that the difference in the divalent cation requirement for the uridylylation is related to efficient binding of Mg/Mn-ATP to the PII proteins. We concluded that the amino acids at positions 42 and 85 in GlnJ and GlnB (in the vicinity of the ATP binding site) influence the divalent cation requirement for uridylylation catalyzed by GlnD.

Conclusions: Efficient binding of Mg/Mn-ATP to the PII proteins is required for uridylylation by GlnD. Our results show that by simply exchanging two amino acid residues, we could modulate the divalent cation requirement in the uridylylation of GlnJ and GlnB.Considering that post-translational uridylylation of PII proteins modulates their signaling properties, a different requirement for divalent cations in the modification of GlnB and GlnJ adds an extra regulatory layer to the already intricate control of PII function.

Figure 1

Alignment of the amino acid sequence of the R. rubrum GlnB and GlnJ proteins, constructed using ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The loop regions are highlighted and the positions of the amino acid substitutions used in this study are marked with a star.

Figure 2

Uridylylation of GlnJ (A) and GlnB (B) variants. The reactions were performed as described in the Materials and methods in the presence of Mn²⁺, Mg²⁺ or without either divalent cation (control - C), and the uridylylation status analyzed by native PAGE. U – unmodified, M3- modified (fully modified trimmers).

Figure 3

Time-course uridylylation of GlnJ, GlnJ^Q42H, GlnJ^K85Rand GlnJ^Q42HK85R. At the time points indicated samples were withdrawn and analyzed by native PAGE. The number of uridylylated subunits (0–3) is indicated.

Figure 4

Cartoon representation of the structural model for GlnJ, constructed based on the determined structure of A. brasilense GlnZ, with ligands (PDB 3MHY). ATP is shown in gray, Magnesium ion in yellow, 2-OG in red and the residues K85 and Q42 are highlighted in blue and green respectively.

Figure 5

CD spectra for GlnJ (A) and GlnB (B); protein only (dashed), protein + MnATP (solid) and protein + MgATP (dotted). Proteins were at 100 μM trimer concentration, ATP at 10 mM and MgCl₂/MnCl₂ at 10 mM. Spectra were recorded at 24°C.

Figure 6

Analysis of PII protein function in the activation of GlnE.(A) Model representing the role of PII proteins in the regulation of GS activity, through GlnE in R. rubrum . (B) Glutamine synthetase activity after 30 minutes of incubation with GlnE and PII proteins (as indicated). Results are the average of three experiments and are shown as mean ± SD.