Xiao-Dan Li

The crystal structure of the Escherichia coli AmtB–GlnK complex reveals how GlnK regulates the ammonia channel

Amt proteins are ubiquitous channels for the conduction of ammonia in archaea, eubacteria, fungi, and plants. In Escherichia coli, previous studies have indicated that binding of the PII signal transduction protein GlnK to the ammonia channel AmtB regulates the channel thereby controlling ammonium influx in response to the intracellular nitrogen status. Here, we describe the crystal structure of the complex between AmtB and GlnK at a resolution of 2.5 Å. This structure of PII in a complex with one of its targets reveals physiologically relevant conformations of both AmtB and GlnK. GlnK interacts with AmtB almost exclusively via a long surface loop containing Y51 (T-loop), the tip of which inserts deeply into the cytoplasmic pore exit, blocking ammonia conduction. Y51 of GlnK is also buried in the pore exit, explaining why uridylylation of this residue prevents complex formation.

The 1.3-Å resolution structure of Nitrosomonas europaea Rh50 and mechanistic implications for NH3 transport by Rhesus family proteins

The Rhesus (Rh) proteins are a family of integral membrane proteins found throughout the animal kingdom that also occur in a number of lower eukaryotes. The significance of Rh proteins derives from their presence in the human red blood cell membrane, where they constitute the second most important group of antigens used in transfusion medicine after the ABO group. Rh proteins are related to the ammonium transport (Amt) protein family and there is considerable evidence that, like Amt proteins, they function as ammonia channels. We have now solved the structure of a rare bacterial homologue (from Nitrosomonas europaea) of human Rh50 proteins at a resolution of 1.3 Å. The protein is a trimer, and analysis of its subunit interface strongly argues that all Rh proteins are likely to be homotrimers and that the human erythrocyte proteins RhAG and RhCE/D are unlikely to form heterooligomers as previously proposed. When compared with structures of bacterial Amt proteins, NeRh50 shows several distinctive features of the substrate conduction pathway that support the concept that Rh proteins have much lower ammonium affinities than Amt proteins and might potentially function bidirectionally.

A New P II Protein Structure Identifies the 2-Oxoglutarate Binding Site

P(II) proteins of bacteria, archaea, and plants regulate many facets of nitrogen metabolism. They do so by interacting with their target proteins, which can be enzymes, transcription factors, or membrane proteins. A key feature of the ability of P(II) proteins to sense cellular nitrogen status and to interact accordingly with their targets is their binding of the key metabolic intermediate 2-oxoglutarate (2-OG). However, the binding site of this ligand within P(II) proteins has been controversial. We have now solved the X-ray structure, at 1.4 A resolution, of the Azospirillum brasilense P(II) protein GlnZ complexed with MgATP and 2-OG. This structure is in excellent agreement with previous biochemical data on 2-OG binding to a variety of P(II) proteins and shows that 2-oxoglutarate binds within the cleft formed between neighboring subunits of the homotrimer. The 2-oxo acid moiety of bound 2-OG ligates the bound Mg(2+) together with three phosphate oxygens of ATP and the side chain of the T-loop residue Gln39. Our structure is in stark contrast to an earlier structure of the Methanococcus jannaschii GlnK1 protein in which the authors reported 2-OG binding to the T-loop of that P(II) protein. In the light of our new structure, three families of T-loop conformations, each associated with a distinct effector binding mode and characterized by a different interaction partner of the ammonium group of the conserved residue Lys58, emerge as a common structural basis for effector signal output by P(II) proteins.

Two Alternative Conformations of a Voltage-Gated Sodium Channel.

Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4-S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged C-terminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures.

Crystal structure of the GlnZ-DraG complex reveals a different form of PII-target interaction

Nitrogen metabolism in bacteria and archaea is regulated by a ubiquitous class of proteins belonging to the PIIfamily. PII proteins act as sensors of cellular nitrogen, carbon, and energy levels, and they control the activities of a wide range of target proteins by protein-protein interaction. The sensing mechanism relies on conformational changes induced by the binding of small molecules to PII and also by PII posttranslational modifications. In the diazotrophic bacterium Azospirillum brasilense, high levels of extracellular ammonium inactivate the nitrogenase regulatory enzyme DraG by relocalizing it from the cytoplasm to the cell membrane. Membrane localization of DraG occurs through the formation of a ternary complex in which the PII protein GlnZ interacts simultaneously with DraG and the ammonia channel AmtB. Here we describe the crystal structure of the GlnZ-DraG complex at 2.1 Å resolution, and confirm the physiological relevance of the structural data by site-directed mutagenesis. In contrast to other known PII complexes, the majority of contacts with the target protein do not involve the T-loop region of PII. Hence this structure identifies a different mode of PII interaction with a target protein and demonstrates the potential for PII proteins to interact simultaneously with two different targets. A structural model of the AmtB-GlnZ-DraG ternary complex is presented. The results explain how the intracellular levels of ATP, ADP, and 2-oxoglutarate regulate the interaction between these three proteins and how DraG discriminates GlnZ from its close paralogue GlnB.

Large scale expression and purification of the mouse 5-HT3 receptor

Receptors of the Cys-loop family are central to neurotransmission and primary therapeutic targets. In order to decipher their gating and modulation mechanisms, structural data is essential. However, structural studies require large amounts of pure, functional receptors. Here, we present the expression and purification of the mouse serotonin 5-HT3 receptor to high purity and homogeneity levels. Inducible expression in human embryonic kidney 293 cells in suspension cultures with orbital shaking resulted in yields of 6-8mg receptor per liter of culture. Affinity purification using a strep tag provided pure protein in active form. Further deglycosylation and removal of the purification tag led to a pentameric receptor after size-exclusion chromatography, at the milligram scale. This material is suitable for crystallography, as demonstrated by X-ray diffraction of receptor crystals at low resolution.

Crystal Structure of Dinitrogenase Reductase-activating Glycohydrolase (DRAG) Reveals Conservation in the ADP-Ribosylhydrolase Fold and Specific Features in the ADP-Ribose-binding Pocket

Protein-reversible ADP-ribosylation is emerging as an important post-translational modification used to control enzymatic and protein activity in different biological systems. This modification regulates nitrogenase activity in several nitrogen-fixing bacterial species. ADP-ribosylation is catalyzed by ADP-ribosyltransferases and is reversed by ADP-ribosylhydrolases. The structure of the ADP-ribosylhydrolase that acts on Azospirillum brasilense nitrogenase (dinitrogenase reductase-activating glycohydrolase, DraG) has been solved at a resolution of 2.5 A. This bacterial member of the ADP-ribosylhydrolase family acts specifically towards a mono-ADP-ribosylated substrate. The protein shows an all-alpha-helix structure with two magnesium ions located in the active site. Comparison of the DraG structure with orthologues deposited in the Protein Data Bank from Archaea and mammals indicates that the ADP-ribosylhydrolase fold is conserved in all domains of life. Modeling of the binding of the substrate ADP-ribosyl moiety to DraG is in excellent agreement with biochemical data.

Time-resolved structural studies with serial crystallography: A new light on retinal proteins

Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron microscopy. However, these methods do not allow for the structure determination of most intermediate conformations. Recently, the potential of X-Ray Free Electron Lasers (X-FELs) for tracking the dynamics of light-triggered processes by pump-probe serial femtosecond crystallography has been demonstrated using 3D-micron-sized crystals. In addition, X-FELs provide new opportunities for protein 2D-crystal diffraction, which would allow to observe the course of conformational changes of membrane proteins in a close-to-physiological lipid bilayer environment. Here, we describe the strategies towards structural dynamic studies of retinal proteins at room temperature, using injector or fixed-target based serial femtosecond crystallography at X-FELs. Thanks to recent progress especially in sample delivery methods, serial crystallography is now also feasible at synchrotron X-ray sources, thus expanding the possibilities for time-resolved structure determination.

Structure and Thermodynamics of Effector Molecule Binding to the Nitrogen Signal Transduction PII Protein GlnZ from Azospirillum brasilense

The trimeric PII signal transduction proteins regulate the function of a variety of target proteins predominantly involved in nitrogen metabolism. ATP, ADP and 2-oxoglutarate (2-OG) are key effector molecules influencing PII binding to targets. Studies of PII proteins have established that the 20-residue T-loop plays a central role in effector sensing and target binding. However, the specific effects of effector binding on T-loop conformation have remained poorly documented. We present eight crystal structures of the Azospirillum brasilense PII protein GlnZ, six of which are cocrystallized and liganded with ADP or ATP. We find that interaction with the diphosphate moiety of bound ADP constrains the N-terminal part of the T-loop in a characteristic way that is maintained in ADP-promoted complexes with target proteins. In contrast, the interactions with the triphosphate moiety in ATP complexes are much more variable and no single predominant interaction mode is apparent except for the ternary MgATP/2-OG complex. These conclusions can be extended to most investigated PII proteins of the GlnB/GlnK subfamily. Unlike reported for other PII proteins, microcalorimetry reveals no cooperativity between the three binding sites of GlnZ trimers for any of the three effectors under carefully controlled experimental conditions.

Regulation of Nitrogenase by Reversible Mono-ADP-Ribosylation

Posttranslational modification of proteins plays a key role in the regulation of a plethora of metabolic functions. Protein modification by mono-ADP-ribosylation was first described as a mechanism of action of bacterial toxins. Since these pioneering studies, the number of pathways regulated by ADP-ribosylation in organisms from all domains of life expanded significantly. However, in only a few cases the full regulatory ADP-ribosylation circuit is known. Here, we review the system where mono-ADP-ribosylation regulates the activity of an enzyme: the regulation of nitrogenase in bacteria. When the nitrogenase product, ammonium, becomes available, the ADP-ribosyltransferase (DraT) covalently links an ADP-ribose moiety to a specific arginine residue on nitrogenase switching-off nitrogenase activity. After ammonium exhaustion, the ADP-ribosylhydrolase (DraG) removes the modifying group, restoring nitrogenase activity. DraT and DraG activities are reversibly regulated through interaction with PII signaling proteins . Bioinformatics analysis showed that DraT homologs are restricted to a few nitrogen-fixing bacteria while DraG homologs are widespread in Nature. Structural comparisons indicated that bacterial DraG is closely related to Archaea and mammalian ADP-ribosylhydrolases (ARH). In all available structures, the ARH active site consists of a hydrophilic cleft carrying a binuclear Mg(2+) or Mn(2+) cluster, which is critical for catalysis.