The crystal structure of the Escherichia coliAmtB–GlnK complex reveals how GlnKregulates the ammonia channelMatthew J. Conroy*, Anne Durand†, Domenico Lupo‡, Xiao-Dan Li‡, Per A. Bullough*, Fritz K. Winkler‡§,and Mike Merrick†

*Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom;†Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom; and ‡Biomolecular Research,Paul Scherrer Institut, CH-5232 Villigen, Switzerland

Communicated by Stephen C. Harrison, Harvard Medical School, Boston, MA, November 28, 2006 (received for review November 10, 2006)

Amt proteins are ubiquitous channels for the conduction of am-monia in archaea, eubacteria, fungi, and plants. In Escherichia coli,previous studies have indicated that binding of the PII signaltransduction protein GlnK to the ammonia channel AmtB regulatesthe channel thereby controlling ammonium influx in response tothe intracellular nitrogen status. Here, we describe the crystalstructure of the complex between AmtB and GlnK at a resolutionof 2.5 Å. This structure of PII in a complex with one of its targetsreveals physiologically relevant conformations of both AmtB andGlnK. GlnK interacts with AmtB almost exclusively via a longsurface loop containing Y51 (T-loop), the tip of which insertsdeeply into the cytoplasmic pore exit, blocking ammonia conduc-tion. Y51 of GlnK is also buried in the pore exit, explaining whyuridylylation of this residue prevents complex formation.

regulation � x-ray structure � PII protein

In conditions of nitrogen limitation, ammonium uptake isfacilitated by a family of integral membrane proteins known as

the ammonium transporter (Amt) family (1). They are almostubiquitous among archaea, eubacteria, fungi, and plants,whereas in animals they are represented by the closely relatedRhesus family.

The Escherichia coli AmtB protein has become the paradigmfor studies on the biology of these proteins. AmtB is a stablehomotrimer in the cytoplasmic membrane and retains thisstructure when purified and reconstituted in two-dimensionalcrystals (2, 3). Mature E. coli AmtB has 11 transmembranehelices with an Nout and Cin topology (4–7) that appears tocharacterize all members of the Amt family. The x-ray crystalstructures of E. coli AmtB (5, 6) and the related Archaeoglobusfulgidus Amt-1 (8) are very similar. Each subunit of the trimerhas a pseudo-twofold symmetry relating helices M1 to M5 andM6 to M10, with M11 across the lipid-accessible face of eachmonomer. In A. fulgidus, the cytosolic C-terminal region (CTR)of Amt-1 (8) has a defined structure, but in the E. coli structures(5, 6) this region is disordered. The Amt structures suggest thatsubstrate conductance occurs through a narrow, mainly hydro-phobic pore located at the center of each monomer and con-taining two highly conserved histidines (H168 and H318) each ofwhich is essential for conductance of the ammonium analoguemethylammonium (9). A potential high-affinity ammoniumbinding site was identified at the periplasmic entrance to thepore, and these structural features, together with complemen-tary data, suggest that ammonium is the substrate recognized butthat ammonia is the translocated species (5, 6, 10, 11). [In thisarticle, we use the term ammonium to refer to both the proton-ated (NH4

�) and unprotonated (NH3) forms, and we use theterm ammonia to refer specifically to NH3.] Therefore, aspreviously proposed (12), Amt proteins have been described asammonia channels (5, 6).

In almost all eubacteria and archaea, the amtB gene isinvariably linked to glnK, suggesting a functional relationship forAmtB and GlnK (4). GlnK belongs to the PII family of cytosolicsignal transduction proteins that act as sensors of cellularnitrogen status and regulate the activities of many differentproteins, including enzymes and transcription factors, by pro-tein–protein interaction (13–15). We therefore have suggestedthat AmtB activity might be regulated by GlnK (16). Indeed, invivo, GlnK binds rapidly and reversibly to AmtB in response toan extracellular ammonium shock (�50 �M extracellular am-monium) (17), and this interaction has also been shown in anumber of other eubacterial species (16, 18–20). Methylammo-nium uptake by AmtB is also inhibited by a GlnK variant thatforms an irreversible complex, supporting the concept that GlnKregulates ammonium flux through AmtB (17).

Like AmtB, GlnK is also a homotrimer; its three subunits forma compact barrel in which each monomer folds into a four-stranded �-sheet packed against two helices (21). Three notableloops, designated the B-, C-, and T-loops, are present in thestructure. A lateral cleft between each subunit is formed be-tween the T- and B-loops of one subunit and the C-loop ofanother. E. coli expresses two PII proteins, GlnK and GlnB,whose activities are regulated covalently and noncovalently. Aconserved residue Y51 within the T-loop is covalently modifiedby uridylylation in cells that are subjected to nitrogen starvationand this modification is reversed in nitrogen sufficiency (22–24).Only deuridylylated GlnK binds to AmtB (16, 17, 25). PIIproteins synergistically bind ATP and 2-oxoglutarate (2-OG), asmall-molecule signal of cellular nitrogen status (14, 15, 22) anda metabolite of the Krebs cycle. The crystal structures of E. coliGlnB–ATP and GlnK–ATP co-complexes revealed ATP boundin the lateral clefts between the subunits (21, 26).

Very little is known about the mechanism whereby PII proteinsinteract with their targets, although a number of studies haveimplicated the T-loop in such interactions (22, 27–29). In E. coli,the failure of uridylylated GlnK to interact with AmtB alsoimplicates the T-loop (16, 17). Conversely the CTR of AmtB isimportant for GlnK binding (16, 30). In vitro biochemical analysisrevealed a direct and stoichiometric interaction between the two

Author contributions: M.J.C. and A.D. contributed equally to this work; X.-D.L., P.A.B.,F.K.W., and M.M. designed research; M.J.C., A.D., and D.L. performed research; M.J.C., D.L.,X.-D.L., and F.K.W. analyzed data; and M.J.C., A.D., P.A.B., F.K.W., and M.M. wrote thepaper.

The authors declare no conflict of interest.

Abbreviations: 2-OG, 2-oxoglutarate; CTR, C-terminal region; PDB, Protein Data Bank.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID code 2NUU).

§To whom correspondence should be addressed. E-mail: fritz.winkler@psi.ch.

This article contains supporting information online at www.pnas.org/cgi/content/full/0610348104/DC1.

© 2007 by The National Academy of Sciences of the USA

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proteins (25). Furthermore, in vitro dissociation of GlnK fromAmtB does not require uridylylation of GlnK, and association/dissociation of the complex is sensitive to 2-OG in the presenceof ATP (25).

To date, there have been no structural data for any AmtB–GlnK complex or for any PII protein complexed to one of itstargets. Here, we describe the crystal structure of the E. coliAmtB–GlnK complex, which provides direct visualization of theeffect of GlnK in blocking ammonia conductance by AmtB.

ResultsOverall Structure of the Complex. In vitro reconstitution of theAmtB–GlnK complex is only achievable in specific conditions ofeffector composition and concentration (25). Consequently, wechose to purify the complex directly from cells grown in physi-ologically relevant conditions as described in Methods. Crystal-lization and structure determination are described in Methods,and crystallographic statistical data are given in Table 1. Twocopies of an AmtB–GlnK complex make up the asymmetric unit,stacked such that the periplasmic faces of AmtB interact. All sixcopies of each protein in the asymmetric unit have very similarstructures, with the few small differences being attributable tocrystal contacts.

AmtB and GlnK form a threefold symmetric complex in whichthe GlnK trimer binds to the cytoplasmic face of AmtB with astoichiometry of AmtB3:GlnK3 (Fig. 1 A and B), confirming thestoichiometry previously determined analytically (25). The over-all shape of the complex is that of a truncated cone with a heightof 85 Å and a width of 80 Å at the periplasmic face and 50 Å atthe base (Fig. 1 A and B). Each GlnK subunit inserts an orderedT-loop into the cytoplasmic pore exit of an adjacent AmtBsubunit (Fig. 1 A and B). GlnK does not pack tightly against thecytoplasmic surface of AmtB but displays a rather open inter-face. This is the first structure for which we can be confident thatthe observed conformations of the cytoplasmic loops of AmtB

and the T-loops of GlnK, both prone to disorder, are physio-logically relevant because they occur in a naturally formedcomplex. We discuss below the features that we observe in thestructures of the individual proteins and then consider how thesefeatures facilitate complex formation.

Structure of AmtB in the Complex. The structure of AmtB in thiscomplex differs significantly from the previously publishedstructures (5, 6) on its cytoplasmic face, whereas the periplasmicface and transmembrane part are essentially unchanged. Theside chains of the residues lining the periplasmic vestibule andthose forming the ammonia conducting channel, including H168and H318, are coincident between all structures. When com-pared with the previous structure with ordered cytoplasmicloops [Protein Data Bank (PDB) entry 1U7G (5)], three of them,M3–M4, M5–M6, and M9–M10, exhibit highly altered confor-mations (Fig. 2A). A remarkable salt-bridge interaction networkformed by the highly conserved residues R37, E121, R185, andD309 and not present in PDB entry 1U7G links four of the fiveloops. For the AmtB structures with disordered loops (6), thecrystals were grown at pH 4.5 and at such a low pH thisstabilizing network would be strongly perturbed.

Different conformations at the cytosolic end of M10 (residues310–316) have been previously noted in the free AmtB struc-tures and speculatively linked to different states of the cytoplas-mic pore exit (6). One conformation, observed in two E. coliAmtB structures (PDB entries 1U7G and 1XQF), has a narrowpore constriction on the cytoplasmic side of H318, the otherconformation, observed in A. fulgidus Amt-1 (PDB entry 2B2F),E. coli AmtB (PDB entry 1XQE) and also in the AmtB–GlnKcomplex, has a �-type N-terminal turn that generates a wider andmore polar cytoplasmic opening (see figure 1b of ref. 11). In thisconformation, the aromatic ring of F315 at the cytosolic end ofM10 forms a small hydrophobic cluster with residues at thecytosolic end of M9, influencing the orientation of the M9–M10loop, which is fully ordered.

Importantly, and in contrast to previous structures of E. coliAmtB, the CTR (residues 383–406) is ordered (Fig. 2 A) andadopts a structure very similar to that predicted by homologymodeling using the CTR of A. fulgidus Amt-1 (PDB entry 2B2F)as a template (30). It contains two short helices separated by atight turn centered on G393. There are a large number ofcontacts and specific interactions of the cytoplasmic loops witheach other and with the CTR that appear to cooperativelystabilize the fully ordered state of the cytoplasmic face of AmtB.

Fig. 1. Overview of the AmtB–GlnK complex. The surface of AmtB is shown,and GlnK is shown in cartoon representation. Each subunit of AmtB and GlnKis colored independently. (A) View from the cytoplasm along the threefoldaxis perpendicular to the membrane. (B) View in the membrane plane. Allfigures were made using PyMOL (40) .

Table 1. Data collection and refinement statistics

Space group P212121

Cell dimensions (a, b, and c), Å 99.69, 107.87, 280.16Resolution, Å*, and wavelength, Å 50–2.5 (2.6–2.5) and 0.97972Completeness, %* 99.8 (99.9)Rsym, %* 9.9 (53.6)I/�* 13.3 (3.0)Reflections, total/unique 438,641/105,009Rcryst, %* 17.1 (26.0)Rfree, %* 24.9 (32.0)rmsd bond lengths, Å 0.009rmsd bond angles, ° 1.425No. of atoms/average B-factors, Å2

Protein 23,314/29.9PDP 162/33.5Water 550/31.5

Rsym � �hkl �i�I(hkl;i) � �I(hkl)��/�hkl �i I(hkl;i); Rcryst, Rfree � �hkl�Fobs(hkl) �Fcalc(hkl)�/�hklFobs(hkl), with summation over working set and test set reflec-tions, respectively.*Values in parentheses are for the outermost-resolution shell.

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The CTR forms extensive interactions with the cytoplasmicsurface of the cognate AmtB chain; namely, the M1–M2, M3–M4, and M5–M6 loops (Fig. 2B). Furthermore, it contacts theneighboring AmtB molecule in the trimer; namely, helix M1 andthe M5–M6 and M7–M8 loops. In this way, the last six residuesof the CTR form part of the wall of the cytoplasmic pore exit inthe neighboring subunit (Fig. 5A). This interaction mode istightly linked to the cytoplasmic loop conformations, and fur-thermore helix M6 is one turn shorter than in PDB entry 1U7G(5) to accommodate the C-terminal residues from the neigh-boring subunit. We consider it possible that the ordered state ofthe cytoplasmic AmtB face can also exist in the absence of GlnK,although binding of the latter will certainly stabilize it.

Structure of GlnK and Its Nucleotide Binding Site in the Complex. Theoverall fold of GlnK is very similar to that of the uncomplexedprotein (21). However, the T-loop exhibits a conformation thathas not been seen previously in PII proteins (21, 31–33). It formsa short two-stranded antiparallel �-sheet (residues E44–Y46 andA49–Y51), the strands of which are separated by a �-turn. TheT-loop extends 28 Å from the core of the protein (Fig. 3A), a

completely different conformation from that previously re-ported for E. coli PII proteins (Fig. 3B). The tip of the T-loop isformed by R47 which, together with the adjacent G48, is totallyconserved.

Within the lateral cleft between adjacent GlnK subunits, wesee clear electron density corresponding to the base, sugar, andtwo phosphate groups of a bound nucleotide (Fig. 4). Thus, weobserve an ADP-bound state of E. coli PII, with a stoichiometryof 3ADP:GlnK3. Previous studies have shown ATP can bind tothis site in both E. coli GlnK (21) and GlnB (26). However, giventhe close contacts with neighboring amino acid main-chain andside-chain atoms all around the diphosphate moiety as verifiedthrough omit maps, we rule out that we are observing ATP witha disordered �-phosphate. The ADP molecule is almost com-pletely buried in the lateral cleft, and hydrogen bonding inter-actions with the base and sugar moieties of ADP (Fig. 4) are thesame as in GlnK–ATP and GlnB–ATP. Superposition of ourstructure on that of GlnK–ATP (21) shows significant differ-ences in the conformation and relative position of the twosugar-phosphate moieties. In contrast, superposition with GlnBcomplexed with ATP in two different crystal forms (26) revealsa strikingly close fit for the nucleotides apart from the extra

Fig. 2. Features of cytoplasmic face of AmtB in the complex. (A) Comparisonof cytoplasmic loop conformations observed in the complex with the corre-sponding loops in PDB entry 1U7G (5) [M3–M4 (red), M5–M6 (green), andM9–M10 (magenta) in bright and pale colors, respectively]. For clarity, the endpoints of the loops shown as C� traces have been centered on the helicalcylinders. CTR is shown in blue (not ordered in PDB entry 1U7G). (B) Interactionof one CTR with the cytoplasmic loops of two adjacent AmtB subunits, coloredpink and blue with the CTR of the pink subunit shown in yellow. Residuesinvolved in hydrogen bonding interactions are shown as sticks. The arrowpoints at the position of G393.

Fig. 3. Features of GlnK in the complex. (A) The GlnK trimer showing theT-loops extending from the main body of the protein. R47 and Y51 are shownas sticks. One molecule of ADP in the nearest lateral cleft (between the greenand red subunits) is shown in stick representation. Residues at the base of theT-loop and of the B-loop from the red subunit together with residues of theC-loop from the green subunit contribute to this binding site. (B) Comparisonof T-loop conformations observed in the complex (green) with the corre-sponding loops in E. coli GlnK (PDB entry 1GNK) (21) (pink) and E. coli GlnB(PDB entry 2PII) (26) (yellow). R47 and Y51 are indicated. Superimpositionswere carried out by using backbone atoms of residues 1–35 and 58–103.

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�-phosphates. In one case, the ATP �-phosphate is orientedtoward the guanidinium moiety of R103, and in the other it isoriented toward the guanidinium moiety of R101; these differ-ences being accommodated by local structural adjustments andin one case by the additional displacement of the four C-terminalGlnB residues. Thus, the mobility of the R101 and R103 sidechains and the very C-terminal chain segment (D108–L112) ofone subunit, together with that of the B-loop of the other subunitat the interface, appear to accommodate various nucleotidebinding modes of these PII proteins.

An important difference between the ADP complex observedhere and the E. coli GlnK–ATP (21) and E. coli GlnB–ATP (26)complexes is that in the latter cases the T-loop is disordered beyondresidue G37. However, in our structure the T-loop is fully ordered,and we observe the two hydrogen bonds of the main-chain NHgroups of residues R38 and Q39 with �-phosphate oxygens (Fig. 4).These bonds appear important to fix a 90° kink in the GlnK chainat R38 (see arrow Fig. 5A). The R38 side chain reaches over the�-phosphate and makes a salt-bridge interaction with the C-terminal carboxylate of the adjacent subunit. However, this contactappears somewhat labile because the corresponding density is weakin some of the six crystallographically independent copies.

The Interaction of GlnK with AmtB. Many of the features of the E.coli AmtB and GlnK structures described above are likely tocontribute directly or indirectly to the stabilization of thecomplex. The interface between the cytoplasmic face of AmtBand GlnK is a remarkably open one with a relatively smallsurface area buried between the two proteins. The dockinginteraction in the complex is formed almost exclusively by theT-loops of GlnK acting as extended pins plugging into thecytoplasmic pore exits of AmtB (Fig. 1B), delineated by cytosolicloops M5–M6, M7–M8, and M9–M10 of one subunit and theCTR of the adjacent subunit (Fig. 5A). The correspondingburied surface areas are 1,079 Å2 with the cytosolic loops and 439Å2 with the CTR. The importance of the CTR for shaping theinteraction surface with the T-loop and thus GlnK is stronglyindicated by our mutational analysis of the CTR showing thatvery minor changes abolish the interaction with GlnK (30). Forexample, mutation of R384 or Y404 in the CTR, which makehydrogen bonds to residues in loop M5–M6 (Fig. 2B), preventGlnK binding (30). There is one other, albeit rather weak,contact involving F11 of GlnK and R391 of AmtB and someneighboring side chains with partly ill defined density. Overall,GlnK is not bound rigidly to the face of AmtB, as also indicatedby its elevated B-factors.

Whereas previous studies have implicated the T-loop ininteractions of PII proteins with their targets (27–29), here wedescribe such an interaction in molecular detail. At the tip of theT-loop, the charged guanidinium moiety of R47 forms hydrogenbonds to the carbonyl of AmtB-C312 and hydroxyl of AmtB-S263as well as a salt-bridge interaction with AmtB-D313 (Fig. 5B).The guanidinium group of R47 dramatically constricts the exitto the ammonia conducting pore, giving it a minimum radius of0.6 Å and effectively blocking any possible movement of am-monia through the channel (Fig. 5C). Hence, we conclude thatwhen GlnK is sequestered to AmtB in response to an increase inextracellular ammonium levels (16, 17) it sterically blocks thepore and prevents ammonia flux into the cell.

In the complex, the side chain of Y51 is packed against the AmtBM5–M6 loop that forms one wall of the cytoplasmic pore exit. TheOH group of Y51 is within hydrogen bonding distance (2.8 Å) ofF193 in the M5–M6 loop, and the hydrophobic ring packs againstthe aliphatic portion of the side chain of K194 (Fig. 5B).

DiscussionThis work provides structural insight into the role of GlnKbinding to AmtB while also describing the structure of a PIIprotein bound to one of its targets. The extension of the GlnKT-loops deep into the cytoplasmic exit pores of each of the AmtBsubunits clearly indicates that the mode of action of GlnK is toblock sterically the conductance of substrate through the AmtBpore. In a previous publication, a complex between A. fulgidusAmt-1 and GlnB-1 was modeled but necessarily could not show

Fig. 4. Stereo representation of the ADP binding site of GlnK. The differenceelectron density, contoured at 3�, was obtained after 20 cycles of restrainedrefinement with the ADP molecule omitted. Residues of the two GlnK subunitsforming the binding site and ADP are shown in gray, cyan, and magentaball-and-stick models, respectively. Hydrogen bonding interactions includingone water molecule (pink sphere) are shown with dashed lines, and selectedresidues are labeled.

Fig. 5. Interactions of the GlnK T-loop with AmtB and consequent poreblockage. (A) Two AmtB subunits are shown in surface representation, andtwo GlnK molecules with ADP at their interface are in cartoon representation.The green T-loop (arrow marks kink at R38) inserts into the vestibule of thepink AmtB subunit. The surfaces of its cytoplasmic loops interacting with theT-loop are emphasized in red (M7–M8) and salmon (M5–M6), respectively, andthat of the C-terminal residues of the neighboring subunit are emphasized indark blue. (B) Hydrogen bonding interactions of the T-loop (magenta) in-serted into an AmtB subunit (blue) with part of the cytoplasmic vestibulebeing formed by the neighboring CTR (green). Interacting residues are shownas sticks. Helices are labeled in the gray circles. H318 in the channel is includedfor orientation. (C) Insertion of R47 into the cytoplasmic end of the ammoniatranslocating channel. The channel is shown in cross-section with R47 of GlnKshown in spacefill. The rest of the T-loop is shown as a cartoon (red). H168 andH318 in the channel are shown for orientation.

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any of the molecular details seen here for the E. coli AmtB–GlnKbinding (8). Furthermore, in the absence of any structure for A.fulgidus GlnB-1, the authors used E. coli GlnB as a template topredict the structure of GlnB-1, and it is now apparent that theT-loop in the E. coli GlnB structure has a totally differentconformation from that now determined in our structure(Fig. 3B).

The occurrence of the AmtB–GlnK pair is highly conserved inboth eubacteria and archaea, and the interaction of theseproteins has already been demonstrated in a number of cases (16,18–20). Hence, we might expect that the mode of binding is alsoconserved. Comparison of predicted AmtB and GlnK sequencesindicates that R47 at the tip of the GlnK T-loop is completelyconserved, as is the adjacent G48, which potentially mediates theturn. Of the residues in AmtB that make contact with R47 ofGlnK, S263 and D313 are highly conserved, whereas C312 is not,presumably because the interaction is with the backbone car-bonyl rather than with the side chain.

The structure of the GlnK T-loop observed in the complexis distinct from those seen in previous T-loop structures, noneof which was crystallized in a functional context. In each of thefour previous PII structures in which the T-loop was ordered,the ability to resolve the loop is a direct consequence ofstabilization of the structure by crystal-packing contacts (21,31–33). Although two of the previous structures exhibit anextended conformation, in each of those cases this is aconsequence of extension of �-strands 2 and 3 (32, 33). Neitherexhibits the short �-strands observed in our structure, whichare composed of distinctly different residues. We suggest thatthe observed T-loop structure is likely to be a consequence ofbinding to AmtB, but it is also conceivable that its conforma-tion would be inf luenced by nucleotide binding.

Although Y51 is not completely protected from solvent in thecomplex, it is clear that there is not sufficient room within thepore exit to accommodate a bound uridine monophosphategroup on this residue, and thus the uridylylated form would besterically prevented from binding to AmtB. It would also seemunlikely that uridylyltransferase is able to access Y51 to uridy-lylate it while the complex is intact. Hence, we conclude thaturidylylation of Y51 is not the driving force for dissociation of thecomplex, and this is consistent with our in vitro studies in whichdissociation can occur in the absence of uridylylation (25).

Another feature of the complex is the presence of ADP boundto each subunit of GlnK. Bound ADP has been reported on twoprevious occasions: in Thermus thermophilus GlnK where ADPwas added during crystallization (33), and in Thermotoga mari-tima where ADP was found in the crystal structure of a PIIprotein purified from cells where the growth conditions were notdefined (34), the latter case being the only one to date wherenucleotide was found to copurify with PII. Consequently for E.coli GlnB and GlnK, the only comparisons that can be made arewith bound ATP that was added during crystallization (21, 26).An important difference between the ADP complex observedhere and those GlnK–ATP and GlnB–ATP complexes is that inthe latter cases, the T-loops are disordered, whereas in ourstructure they are fully ordered (21, 26).

The role of ATP or ADP binding to PII proteins is presentlyunclear, but it has been proposed that in cyanobacteria andRhodospirillum rubrum, these ligands could play a role in me-diating signaling of the cellular energy status by PII proteins (15,35). In E. coli, the relationship between ATP levels and cellularnitrogen status has not been extensively studied, and the phys-iological role, if any, of ATP/ADP binding to PII proteins isunclear. However, ammonium shock of nitrogen-limited cellshas been shown to cause a 10-fold drop in intracellular ATPlevels within 15 sec (36), which might be expected to cause aconcomitant rapid decrease in the ATP/ADP ratio. Hence, ourobservation that ADP is bound to GlnK in the complex could

reflect the intracellular levels of ADP induced by the ammoniumshock before purification of the complex. However, in vitro, ATPcan promote complex formation (25), and we cannot exclude thepossibility that the complex actually binds ATP that has beenhydrolyzed to ADP during purification.

The conformation of the GlnK T-loop is clearly critical forcomplex formation, and this is likely to be significantly influ-enced by binding of nucleotide. It is notable that in our structure,the �-phosphate of ADP makes contact with the main chain ofR38 and Q39 and fixes a marked kink in the GlnK chain at thebase of the T-loop (Fig. 5A). However, because PII proteins canclearly accommodate a variety of nucleotide binding modes, it isnot possible to predict the likely conformation if ATP replacedADP in the present structure.

We have also shown that in vitro association/dissociation of thecomplex is sensitive to the concentration of 2-OG in the presenceof Mg-ATP (25). It has also been suggested that 2-OG could bindin the lateral cleft in the vicinity of the phosphate group of thenucleotide (37). This could be accommodated either by the pres-ence of Mg2� serving to balance their respective negative charges(21, 25) or by displacement of the C-terminal GlnK residues andsubstitution of the C-terminal carboxylate by a carboxylate of 2-OG.These considerations suggest that in vivo complex formation mightnot exclusively depend on the uridylylation state of GlnK but maybe also influenced by the intracellular pools of ATP/ADP and2-OG.

This structure of an AmtB–GlnK complex offers significantinsights into the interaction mode, the likely consequences of theinteraction for ammonium conduction, and the effect of GlnKuridylylation on complex formation. The structure emphasizes thatthe primary function of GlnK is almost certainly regulation ofammonium flux into the cell, and the almost ubiquitous linkage ofthe glnK and amtB genes suggests that regulation of the ammoniachannel may be the ancestral role of PII proteins (38). However, insome systems, complex formation might also serve other functionssuch as sequestration of cytoplasmic proteins involved in regulationof nitrogen metabolism as suggested by recent studies in Azospiril-lum brasilense (19) and Bacillus subtilis (39).

MethodsComplex Preparation and Crystallization. To optimize the AmtB–GlnK interaction, AmtB was His-tagged on the periplasmic face,leaving GlnK and the interacting cytosolic face of AmtB in theirnative forms. Complex formation was induced by ammoniumshock, and purification was carried out by using lauryldimethyl-amine-N-oxide as solubilizing detergent throughout the pur-ification as described (25). The N-terminal sequence of therecombinant AmtB is APAVAHHHHHHA(3)VADK corre-sponding to the wild-type sequence from residue 3. The purecomplex was concentrated to 40 mg�ml�1 in a buffer containing50 mM Tris�HCl (pH 8.0), 100 mM NaCl, 10% (vol/vol) glycerol,and 0.05% (wt/vol) lauryldimethylamine-N-oxide. Crystals weregrown in sitting drops by mixing equal volumes of protein andprecipitant solution [65% (vol/vol) 2-methyl-2,4-pentanediol in100 mM Tris�HCl at pH 8.0] at room temperature. The complexcrystallized in space group P212121 with cell constants a � 99.7Å, b � 107.9 Å, and c � 280.2 Å.

Data Collection and Structure Determination. For details of datacollection and structure determination, see supporting informa-tion (SI) Methods.

We thank J. Thornton for assistance with fermentation and membranepreparation, E. Kunji and J. Rafferty for the very considerable time andadvice given in helping us with crystal screening in the early stages of theproject, and R. Dixon and A. Javelle for helpful comments on themanuscript. Crystallographic data were collected at the Swiss LightSource (Paul Scherrer Institut, Villigen, Switzerland) with the excellent

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help of T. Tomizaki. We thank D. Ollis for sending the coordinates ofGlnB–ATP complexes. This work was supported by a grant from theBiotechnology and Biological Sciences Research Council (to P.A.B. and

M.M.). F.K.W. acknowledges support from the Swiss National ScienceFoundation within the framework of the National Center of Competencein Research in Structural Biology.

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