Formation of the Amt/GlnK Complex.  GlnK and PII proteins regulate Amt-dependent ammonia uptake in prokaryotes.  Size exclusion chromatography revealed that GlnK and Amt bind in a one-to-one ratio.  GlnK and Amt form a complex in the absence of Mg-ATP and 2-KG not when these effectors are present.  Electron microscope images of the GlnK1 trimer show that GlnK1 binds to the central region of the cytoplasmic side of the membrane spanning ammonia channel Amt1.  This, result is significant because the channel through which ammonia passes into the cell exists within this region of the Amt1 membrane protein.

T-loop Dynamic Conformation.  The T-loop was found in two different forms.  One form was compact and rigid, and the other had an extended flexible formation as can be seen in Figure 9 by the green loop in the loose formation.  In the absence of Mg-ATP bound to GlnK1, the T-loop assumes an extended shape, regardless of the presence of other nucleotide phosphates in the binding site.  Although in this conformation the T-loop is relatively disordered, it has a tendency to be oriented in a way that could maximize the binding ability of GlnK1 to other proteins, such as Amt.

Keeping Ammonia Out.  From the crystal structures and known features of the Amt and GlnK protein families, a mechanism was proposed to explain regulation of ammonia uptake by a prokaryotic cell.  One interesting aspect of the interactions between the two proteins is that an overall negative charge is found on the cytoplasmic side of Amt protein concentrated near its ammonia channel, and effector-free GlnK1 has an overall positive charge.  Positively charged extended T-loops of GlnK1 could interact with the negatively charged regions, sealing the channels, and effectively inhibiting ammonia uptake (Figure 10a).  These types of interactions would occur at low concentrations of Mg-ATP and 2-KG within the cells, indicating ammonia is not needed for biosynthesis.  Another reason to exclude ammonia from the cell is when ATP levels are low, and ammonia enters the cell it tends to become protonated, dissipating the proton gradient required for ATP synthesis.  This result could be especially harmful to a cell that already has insufficient ATP for its metabolic needs.

Letting Ammonia Flow.  When ATP levels are high, the Mg-ATP complex can bind to the GlnK1 trimer and the T-loops assume the compact form.  When in the compact form, GlnK1 is unable to bind Amt1 (Figure 10b).  When 2-KG is bound, a pronounced negative charge occurs at the interacting surface of GlnK1.  The combination of these two effects causes the GlnK1 to dissociate from the Amt and electrostatic repulsion occurs between the similarly negative charged proteins.  Dissociation of GlnK1 unblocks the Amt ammonia channels and ammonia is free to flow into the cell.  When cellular levels of 2-KG and ATP are high, this indicates a sufficient amount of carbon compounds and energy to initiate nitrogen requiring biosynthesis. These conditions induce dissociation of the Amt/Glnk1 complex and allow the necessary ammonia to enter the cell to be incorporated into proteins and nucleic acids.