MnATP (but not MgATP) induces a conformational change in GlnJ We hypothesized that, in the case of GlnJ, only the binding of MnATP would stabilize a protein conformation that allows the correct positioning of the T-loop for interaction Temsirolimus mw with GlnD, resulting in uridylylation. To analyze this possbility we used circular dichroism (CD) spectroscopy to evaluate changes in the secondary structure of GlnJ/GlnB upon incubation with either MgATP or MnATP. It is visible from our results that only MnATP induced a conformational

change in GlnJ, translated as a significant change in the CD spectrum (Figure 5A), while both Mg2+ and Mn2+ elicited a similar conformational change in GlnB (Figure 5B). These observations of divalent cation-induced conformational changes in the PII proteins correlate well with the conditions required for efficient uridylylation by GlnD. 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. The GlnJ and GlnB variants retain functionality To determine if the substitutions affected protein LY2603618 function we analyzed the functionality of the GlnJ and GlnB variants using an assay based on one of the cellular targets of PII proteins, the adenylyltransferase MK-0457 GlnE. We have previously used this assay as means to determine whether PII variants are still able to perform a PII dependent function [13]. GlnE is responsible for the regulation of GS activity by post-translational adenylylation [5]. PII proteins (in the unmodified form) interact with GlnE promoting adenylylation of GS, leading to lower GS activity (Figure 6A). 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. To analyze the functionality of all variants constructed, we tested the ability to activate GS adenylylation DCLK1 by GlnE, resulting in reduced GS activity. As shown in Figure 6B, all variants tested were able to activate the adenylylation activity of GlnE. Conclusions The two PII proteins GlnJ and GlnB from R. rubrum show different requirements in terms of divalent cations (Mg2+/Mn2+) for efficient uridylylation by GlnD. Specifically, the uridylylation of GlnJ requires the presence of Mn2+, with Mg2+ not being able to support this modification. Most likely this is due to the fact that only Mn2+ (or MnATP) is able to bind and induce a conformational change in GlnJ, as demonstrated here with CD spectroscopy.