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Abstract
GlnK proteins regulate the active uptake of ammonium by Amt transport proteins by inserting their regulatory T-loops into the transport channels of the Amt trimer and physically blocking substrate passage. They sense the cellular nitrogen status through 2-oxoglutarate, and the energy level of the cell by binding both ATP and ADP with different affinities. The hyperthermophilic euryarchaeon Archaeoglobus fulgidus possesses three Amt proteins, each encoded in an operon with a GlnK ortholog. One of these proteins, GlnK2 was recently found to be incapable of binding 2-OG, and in order to understand the implications of this finding we conducted a detailed structural and functional analysis of a second GlnK protein from A. fulgidus, GlnK3. Contrary to Af-GlnK2 this protein was able to bind both ATP/2-OG and ADP to yield inactive and functional states, respectively. Due to the thermostable nature of the protein we could observe the exact positioning of the notoriously flexible T-loops and explain the binding behavior of GlnK proteins to their interaction partner, the Amt proteins. A thermodynamic analysis of these binding events using microcalorimetry evaluated by microstate modeling revealed significant differences in binding cooperativity compared to other characterized PII proteins, underlining the diversity and adaptability of this class of regulatory signaling proteins.
Highlights
The survival and growth of an organism in a competitive environment depends on the precise regulation and availability of its natural resources
The crucial structural feature is the loop connecting beta strands 2 and 3, the T-loop. It spans 21 amino acid residues from G35 to L56 and undergoes conformational changes upon binding of effector molecules that directly affect the affinity of the protein for its interaction partner, AfAmt3
The thermostable ortholog Af-GlnK3 does allow for the observation of a defined conformation for the T-loop with the bound effectors MgATP and 2-OG (PDB code 3TA2) that prohibits binding to Af-Amt3, as well as with bound ADP (PDB code 3TA1), in a conformation that promotes this complex formation
Summary
The survival and growth of an organism in a competitive environment depends on the precise regulation and availability of its natural resources. Ammonium (pKa = 9.25) is readily incorporated into glutamate by glutamate dehydrogenase (GDH) or the ATP-dependent glutamine synthetase (GS). Glutamate:oxoglutarate amidotransferase (GOGAT) closes this ammonium assimilation cycle by transferring the amido group of glutamine to 2-oxoglutarate (2-OG) to yield two molecules of glutamate [8]. In this process, the action of GS is tightly regulated at both a post-transcriptional and translational levels [9,10]. Central regulators of GS and Amt are trimeric cytoplasmic proteins of the PII family, termed GlnB or GlnK, respectively [11]. The distinction between the two is not unambiguous and according to current nomenclature glnK is the gene that is located in an operon together with the amt gene encoding the membraneintegral ammonium transporter, while GlnB is the main regulator of GS and is encoded elsewhere in the genome [11,12,13,14]
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