Journal of Molecular Biology
Volume 402, Issue 3, 24 September 2010, Pages 552-559
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Crystal Structure of the Cyanobacterial Signal Transduction Protein PII in Complex with PipX

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Abstract

PII proteins are highly conserved signal transducers in bacteria, archaea, and plants. They have a large flexible loop (T-loop) that adopts different conformations after covalent modification or binding to different effectors to regulate the functions of diverse protein partners. The PII partner PipX (PII interaction protein X), first identified from Synechococcus sp. PCC 7942, exists uniquely in cyanobacteria. PipX also interacts with the cyanobacterial global nitrogen regulator NtcA. The mutually exclusive binding of PII and NtcA by PipX in a 2-oxoglutarate (2-OG)-dependent manner enables PII to indirectly regulate the transcriptional activity of NtcA. However, the structural basis for these exclusive interactions remains unknown. We solved the crystal structure of the PII–PipX complex from the filamentous cyanobacterium Anabaena sp. PCC 7120 at 1.90 Å resolution. A homotrimeric PII captures three subunits of PipX through the T-loops. Similar to PII from Synechococcus, the core structure consists of an antiparallel β-sheet with four β-strands and two α-helices at the lateral surface. PipX adopts a novel structure composed of five twisted antiparallel β-strands and two α-helices, which is reminiscent of the PII structure. The T-loop of each PII subunit extends from the core structure as an antenna that is stabilized at the cleft between two PipX monomers via hydrogen bonds. In addition, the interfaces between the β-sheets of PipX and PII core structures partially contribute to complex formation. Comparative structural analysis indicated that PipX and 2-OG share a common binding site that overlaps with the 14 signature residues of cyanobacterial PII proteins. Our structure of PipX and the recently solved NtcA structure enabled us to propose a putative model for the NtcA–PipX complex. Taken together, these findings provide structural insights into how PII regulates the transcriptional activity of NtcA via PipX upon accumulation of the metabolite 2-OG.

Graphical Abstract

Research Highlights

►The PII trimer forms a complex with three PipX molecules. ►PipX and 2-oxoglutarate share the same binding site on the T-loop of PII.

Introduction

PII signaling proteins are highly conserved in bacteria, archaea, and plants. Genes coding for PII proteins are divided into three subfamilies: glnB, glnK, and nifI.1 GlnB and GlnK function as homotrimers to modulate nitrogen assimilation. Heterotrimeric NifI is distinct from the members of the other two subfamilies and is found only in nitrogen-fixing archaea and some anaerobic bacteria.2 PII proteins sense carbon-related, nitrogen-related, and energy-related signals, and interact with diverse target proteins, most of which are involved in the regulation of nitrogen metabolism. A series of structures of PII proteins from bacteria,3, 4, 5, 6, 7 archaea,8 and plants9 have been solved. All exist as trimers, with each subunit sharing a highly similar core structure of an antiparallel β-sheet and two α-helices at the lateral surface. The three subunits form a short cylinder, with the functional large flexible loop (T-loop) of each subunit protruding towards the solvent as an antenna to interact with the partner proteins. Most PII proteins sense signals in two modes: through effector-triggered conformational changes and through covalent modifications.10 Both modes are primarily dependent on the variable T-loop. The binding pattern of the effectors ATP/ADP and 2-oxoglutarate (2-OG) is universal and conserved among PII proteins.1, 11, 12, 13 These effectors enable the T-loop to adopt different conformations for interacting with diverse PII partners.10 ATP is stabilized at the intersubunit cleft of PII,14 and its binding is synergistic with the association of 2-OG.15 In contrast, the covalent modification patterns are less conserved. Residue Ser49 of PII from the unicellular cyanobacterium Synechococcus elongatus PCC 7942 (referred to as Synechococcus) or Synechocystis PCC 6803 (referred to as Synechocystis) is phosphorylated under poor nitrogen conditions,11 while Tyr51 is nitrificated in the heterocystous cyanobacterium Anabaena sp. PCC 7120 (referred to as Anabaena).16 In Escherichia coli, PII is regulated by uridylylation at the conserved Tyr51 residue of the T-loop.17

To date, only GlnB-type PII has been found in all sequenced cyanobacteria.18 Because of the absence of 2-OG dehydrogenase in cyanobacteria,19 the 2-OG produced by the Krebs cycle serves mainly as a carbon skeleton for nitrogen assimilation through the glutamine synthetase–glutamate synthase pathway. The direct link of 2-OG level to nitrogen assimilation makes the metabolite 2-OG an important signal of the carbon/nitrogen balance in cyanobacteria, and this signal is sensed by PII protein.

Three PII partner proteins have been reported in cyanobacteria: N-acetyl glutamate kinase (NAGK)20 and PipX (PII interaction protein X) in Synechococcus,20, 21 and the membrane protein PamA of unknown function in Synechocystis.22 Of these, only the molecular mechanism of NAGK regulation by PII has been clearly illustrated from the structural point of view.23 After being first identified in Synechococcus,20, 21 PipX has been found exclusively in all cyanobacteria. It specifically interacts only with cyanobacterial PII, and not with E. coli GlnB or GlnK, or with Arabidopsis thaliana PII.21 PipX also interacts with the global nitrogen regulator NtcA,21 which belongs to the Crp/Fnr transcription factor family and regulates a group of cyanobacterial nitrogen assimilation genes. PipX switches between binding PII and binding NtcA, depending on the cellular 2-OG concentration.21, 24 At lower 2-OG concentrations, PipX is bound to PII. Upon 2-OG accumulation, PipX binds to NtcA.21 2-OG impairs the interaction between PII and PipX in the presence of ATP, but facilitates the formation of the NtcA–PipX complex. The three proteins PII, PipX, and NtcA are highly conserved in all sequenced cyanobacterial genomes, and NtcA and PipX are exclusively encoded by cyanobacteria, indicating that the 2-OG-dependent swapping of PipX might be a universal mechanism of cyanobacteria.

Here, we present the structure of the PII–PipX complex from Anabaena. After the structures of the PII–NAGK complex23, 25 and the AmtB–GlnK26 complex, our structure represents the third complex of PII with its partner. PII assembles into a trimer as reported and interacts with PipX through the T-loops. The homotrimer of PII resembles an upside-down tripod that holds three PipX molecules, which have only slight interactions with each other. Through comparative structural analysis, we deduced how 2-OG and ATP/ADP affect the interaction between PII and PipX. Further structural simulation provides insight into the swapping of PipX from PII to NtcA.

Section snippets

Overall structure of the PII–PipX complex

The structure of the PII–PipX complex was solved and refined to 1.90 Å resolution. It belongs to space group P321, with unit cell dimensions of a = 70.65 Å, b = 70.65 Å, c = 88.54 Å, α = 90.00°, β = 90.00°, and γ = 120.00°. The asymmetric unit contains one molecule each of PII and PipX, which form a 3-fold symmetric complex with a stoichiometry of 3 PII:3 PipX (Fig. 1a and b). The overall structure of the complex resembles a triangular prism that is 43 Å in height and 64 Å (for the PipX layer) or 40 Å (for

The interface between PII and PipX is highly conserved

Most cyanobacterial PII proteins share a sequence identity of 50%, and comparison with PII from other bacteria reveals a signature of 14 residues that are exclusively conserved in cyanobacteria.29 Of these 14 residues, the hydrogen-bonded residues Thr52/Val53 and the hydrophobic Leu59 are critical for stabilizing the PII–PipX complex. The contribution of these signature residues to the PII–PipX interface might explain why PipX specifically binds only to cyanobacterial PII proteins.21 Multiple

Protein expression, purification, and crystallization

PII was cloned into the NdeI/XhoI restriction sites of the pET29 expression vector (Novagen). PipX was cloned into the pET28 expression vector with an N-terminal His6-tag. Plasmids were transformed into E. coli strain BL21(DE3) (Novagen) and induced by isopropyl β-d-1-thiogalactopyranoside for overexpression. Cells were harvested after 4 h at 37 °C. Cells containing PII and PipX were mixed and sonicated. After centrifugation, target proteins in the supernatant were purified with a HiTrap

Acknowledgements

We are grateful to the developers of the CCP4 Suite, ESPript, TreeView, and PyMOL, as well as to the Shanghai Synchrotron Radiation Facility. This work was supported by the Ministry of Science and Technology of China (Projects 2006CB910202 and 2006CB806501), the National Natural Science Foundation of China (Program 30870490), and the Agence Nationale de la Recherche (Physico-Chimie du Vivant Program), France.

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    M.-X.Z. and Y.-L.J. contributed equally to this work.

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