To understand the mechanisms and interactions which govern the binding of GlnK1 to Amt, it is necessary to visualize the protein structure.  GlnK1 was crystallized under these three conditions:

GlnK1 with No Effectors
GlnK1 with Mg-ATP
GlnK1 with Mg-ATP and 2-KG 

Analysis of GlnK1 and Amt structures under these different conditions suggested how regulation of ammonia uptake by GlnK1 occurs.

GlnK1 with No Effectors.  Four trimers of the GlnK1 protein formed a cluster when crystallized without effectors bound.  Each trimer is represented with a different color red, yellow, green and blue (Figure 5).  Each trimer contained three T-Loops which differed in conformation.  Six out the 12 T-loops in the four trimers were disordered, the remaining six were well-defined and in the extended conformation.  The overall structures of the T-loops varied greatly indicating a high degree of flexibility of the loop.  Although no nucleotides were presented during crystallization, the AMP and ADP present in the binding sites had been purified with the proteins isolated from the cells.  An important conclusion from the crystal structure of GlnK1 relates to its relative charge at neutral pH.  An overall positive charge found on the interface was proposed to interact with Amt1.  Clusters of several positively charged arginines were found on the tips of the extended T-loops.  The conformational flexibility of the T-loop and its role in  binding to Amt1 were further elucidated in the structure of the Mg-ATP complex bound to GlnK1.

GlnK1 with Mg-ATP.  Crystals of Glnk1 with both ATP and Mg bound revealed some important clues to the function of the T-loops (Figure 6).  The structure has a single trimer as a unit cell, instead of the complex of four trimers in the unit crystals characterized with substrate-free Glnk1.  The T-loops in the trimer occurred in a compact conformation and not in the extended, flexible state seen in the substrate-free Glnk1 structure.  All three binding sites within the trimer contained both Mg and ATP, represented by the stick molecules in the figure.  The Mg coordinates with three water molecules which in turn form hydrogen bridges with several residues within the T-loop.  Numerous other interactions between the ATP-Mg complex and the T-loop of the protein, stabilize its compact conformation.  These interactions also contribute to the dissipation of positive charge on the tips of the T-loops and also cause a change in the overall charge of GlnK1 from positive to slightly negative.  The Mg-ATP complex inhibits binding of GlnK1 to Amt1, however 2-ketoglutarate can play a role in this process.

Glnk1 with Mg-ATP and 2-KG.  2-Ketoglutarate (2-KG) had a stabilizing effect on the compact conformation of GlnK1 by forming favorable interactions with the protein and the Mg-ATP complex (Figure 7).  When 2-KG was added to the crystallization buffer, the protein was found to contain ATP in all three binding sites but only one of the three sites contained Mg.  Only the T-loop with active site bound Mg-ATP complex was in the compact form.  The other two loops were similar to the form found on the GlnK1 structure with no ATP bound.  For the T-loop that had Mg-ATP bound, one molecule of 2-KG was bound near the compact loop.  A network of hydrogen bonds helps to stabilize the binding of 2-KG to the protein, with the only significant difference between the 2-KG bound and unbound form being a slightly more compact conformation of several amino acid residues.  The 2-KG also causes an increase in the overall negative charge of GlnK1 and completely neutralizes the positive charges on the tip of the T-loop.