Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

Abstract

PII constitutes a family of signal transduction proteins that act as nitrogen sensors in microorganisms and plants. Mycobacterium tuberculosis (Mtb) has a single homologue of PII whose precise role has as yet not been explored. We have solved the crystal structures of the Mtb PII protein in its apo and ATP bound forms to 1.4 and 2.4 Å resolutions, respectively. The protein forms a trimeric assembly in the crystal lattice and folds similarly to the other PII family proteins. The Mtb PII:ATP binary complex structure reveals three ATP molecules per trimer, each bound between the base of the T-loop of one subunit and the C-loop of the neighboring subunit. In contrast to the apo structure, at least one subunit of the binary complex structure contains a completely ordered T-loop indicating that ATP binding plays a role in orienting this loop region towards target proteins like the ammonium transporter, AmtB. Arg38 of the T-loop makes direct contact with the c-phosphate of the ATP molecule replacing the Mg 21 position seen in the Methanococcus jannaschii GlnK1 structure. The C-loop of a neighboring subunit encloses the other side of the ATP molecule, placing the GlnK specific C-terminal 3 10 helix in the vicinity. Homology modeling studies with the E. coli GlnK:AmtB complex reveal that Mtb PII could form a complex similar to the complex in E. coli. The structural conservation and operon organization suggests that the Mtb PII gene encodes for a GlnK protein and might play a key role in the nitrogen regulatory pathway.

A PII-Like Protein Regulated by Bicarbonate: Structural and Biochemical Studies of the Carboxysome-Associated CPII Protein

“…Based on sequence comparison methods (see Methods), the fourth member of the novel four-gene cluster reveals remote but statistically detectable homology to the ubiquitous PII signal-transducer proteins. An analysis of these carboxysome-associated PII-like proteins reveals divergent PII-like motifs (B-loop and C-loop) thought to be critical for nitrogen regulatory PII activity 71–74 , suggesting a distinct or specialized function. Estimates of evolutionary divergence place the carboxysome-associated PII-like proteins in a distinct clade (Fig.…”

“…2) is similar to structures reported for well-studied nrPII proteins and to a structure deposited in the PDB (2CZ4) for a PII-like protein from Thermus thermophilus . 74–78 PII proteins are homotrimeric with nucleotide binding sites at three identical subunit interfaces. The CPII secondary structure consists of two consecutive but interdigitated β-α-β motifs connected by a flexible loop that comprises the so-called “T-loop”.…”

Adenylylation of mycobacterial Glnk (PII) protein is induced by nitrogen limitation

“…The amtB gene is frequently co-transcribed with glnK , strongly suggesting that these proteins function together, 16 and direct evidence for the interaction of GlnK and AmtB has been provided by crystal structure studies 17,18 . Comparing the structure of M. tuberculosis GlnK (PII) (apo- and ATP-bound forms) with the E. coli GlnK:AmtB complex structure suggests that M. tuberculosis GlnK (PII) could form a complex with AmtB in a similar manner 14 . These studies have also shown that the M. tuberculosis GlnK (PII) folds similarly to other PII family proteins 19 and that ATP binding is vital for complex formation with AmtB 14 …”

“…Comparing the structure of M. tuberculosis GlnK (PII) (apo- and ATP-bound forms) with the E. coli GlnK:AmtB complex structure suggests that M. tuberculosis GlnK (PII) could form a complex with AmtB in a similar manner 14 . These studies have also shown that the M. tuberculosis GlnK (PII) folds similarly to other PII family proteins 19 and that ATP binding is vital for complex formation with AmtB 14 …”

“…In the Mycobacterium tuberculosis H 37 Rv genome, Rv2919c is currently annotated as a probable nitrogen regulatory protein PII (GlnB) based on homology to E. coli (61.1% identity to GlnB and 54% identity to GlnK) 13 . However, the recent crystal structure determination of M. tuberculosis PII showed structural conservation with other GlnK proteins, and the location of Rv2919c in an operon with amtB (Rv2920) and glnD (Rv2918), means it is now generally accepted that the PII protein in  M. tuberculosis is in fact a GlnK and not GlnB 14 . Currently M. tuberculosis GlnD is annotated as an uridylyl transferase enzyme, but to date there is no evidence that uridylylation occurs as a post-translational modification mechanism in the Actinomycetales.…”