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PROTEINS: Structure, Function, and Bioinformatics 67:000–000 (2007)
The Structure of the Escherichia coli Nucleoside Diphosphate Kinase Reveals a New Quaternary Architecture for This Enzyme Family AQ1
Lucile Moynie´, Marie-France Giraud, Florian Georgescauld, Ioan Lascu, and Alain Dautant* Institut de Biochimie et Ge´ne´tique Cellulaires, UMR 5095 CNRS-Universite´ Victor Segalen Bordeaux 2, 33077 Bordeaux cedex, France
ABSTRACT Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of g-phosphate from nucleoside triphosphates to nucleoside diphosphates. The subunit folding and the dimeric basic structural unit are remarkably the same for available structures but, depending on species, dimers self-associate to form hexamers or tetramers. The crystal structure of the Escherichia coli NDPK reveals a new tetrameric quaternary structure for this protein family. The two tetramers differ by the relative orientation of interacting dimmers, which face either the convex or the concave side of their central sheet as in either Myxococcus xanthus (type I) or E. coli (type II), respectively. In the type II tetramer, the subunits interact by a new interface harboring a zone called the Kpn loop as in hexamers, but by the opposite face of this loop. The evolutionary conservation of the interface residues indicates that this new quaternary structure seems to be the most frequent assembly mode in bacterial tetrameric NDP kinases. Proteins 2007;67:000–000. VC 2007 Wiley-Liss, Inc.
homologues, it was often assumed that any tetrameric NDPK had the same quaternary structure than Mx-NDPK. We present here the X-ray structure of the Ec-NDPK ˚ resolution, the highest resolution of a NDPK at 1.6 A structure reported to date. The monomer folding and the strong dimer assembly are remarkably similar to all previously known structures. Two putative interfaces alternate between dimers in the Ec-NDPK crystal packing. At one interface, the Killer of prune (Kpn) loop and the helices a1 and a3 are buried with a likeness of hexameric assembly, whereas the other interface involves helix aA and the C-terminal turn of the protein, with a likeness of Mx-NDPK tetramer. The protein–protein interface analysis suggests that the biologically relevant tetramer is the one that buries the Kpn loop, revealing a new quaternary structure further referred to as type II tetramer. Nevertheless, in the hexameric and tetrameric type II NDPKs, the oligomerization interfaces are located on the opposite sides of the Kpn loop. This diversity of quaternary structure confirms the choice of NDPK family as a model for studying multimeric protein association.
Key words: crystal structure; quaternary structure; dimer; tetramer; hexamer
MATERIALS AND METHODS Overexpression and Purification of the Ec-NDPK
INTRODUCTION Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of g-phosphate from nucleoside triphosphates to nucleoside diphosphates. Biochemical studies have shown that the Escherichia coli NDPK1,2 (Ec-NDPK) like other NDPKs of some bacteria (Salmonella typhymurium3, Myxococcus xanthus4,5 (Mx-NDPK), Streptomyces coelicolor6) were tetrameric in contrast to all NDPKs from eukaryotes,7,8 archaea and other bacteria Bacillus subtilis,9 Mycobacterium tuberculosis10) which were hexameric. Up to now, the frontier between the two bacterial groups was not clear.11 The only known X-ray structure of a tetrameric form, noted further type I tetramer, was from M. xanthus with or without ADP5,12 or in complex with cAMP13 while fifty hexameric structures from fifteen different organisms have been solved (for a review see Ref. 7). Ec-NDPK consists of 142 amino acid residues2 shorter on the C-terminus by only 2 residues than Mx-NDPK and by 5–7 residues than the great majority of eukaryotic NDPKs but 7 residues longer than M. tuberculosis. Since Mx-NDPK sequence shares at least 55% identity with its bacterial
Ec-NDPK is the product of the ndk gene14. The protein Ec-NDPK was overexpressed in Escherichia coli BL21 cells transformed with the plasmid ndkec (pJC20 is a pET derivative constructed by Dr M. Konrad). Cells were grown in 2YT medium containing 100 lg/mL ampicillin and induced with 1 mM IPTG when OD600 reached 0.5. Harvested cells were resuspended in buffer A (50 mM Tris/HCl pH 7.4, 1 mM DTT) and disrupted by sonication on ice. Cell debris were removed by centrifugation at 12,000 g for 30 min and the supernatant was loaded onto a Q-Sepharose column (Pharmacia) equilibrated with buffer A and eluted with a Grant sponsors: The Re´gion Aquitaine and Ministe`re de l’Education Nationale de la Recherche et de la Technologie. *Correspondence to: Alain Dautant, Institut de Biochimie et Ge´ne´tique Cellulaires, UMR 5095 CNRS-Universite´ Victor Segalen Bordeaux 2, 1 rue Camille Saint-Sae¨ns, 33077 Bordeaux cedex, France. E-mail:
[email protected] Received 17 August 2006; Revised 26 October 2006; Accepted 31 October 2006 Published online 00 Month 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/prot.21316
C 2007 WILEY-LISS, INC. V
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linear gradient of 0–1M NaCl. The active fraction was precipitated by 80% saturated ammonium sulphate (AMS). The precipitate was dissolved in a minimum volume of 60% saturated AMS buffer A and applied to a Sepharose 6B column (Pharmacia) equilibrated with 60% saturated AMS buffer A. Enzymes were eluted with a decreasing linear gradient of AMS from 60% to 20% saturation (salting-out chromatography). Active fractions were dialysed against 100% saturated AMS buffer A and the suspension was stored at 48C for future use. For crystallization, protein solutions were desalted on a G25 Sephadex column equilibrated with buffer A. Enzyme concentration was estimated using an extinction coefficient of 0.25 at 280 nm for 1 mg/mL solution from amino acid composition. Nucleoside diphosphate kinase activity was routinely assayed as described previously.15 Crystallization Crystallization screening was carried out with a Honeybee 961 robot (Cartesian Technology). Crystals appeared at 208C, by the sitting drop vapor diffusion method, a few hours after mixing 200 nL of protein solution (15 mg/mL) with 200 nL of reservoir solution containing 0.2M ammonium sulphate, 0.1M sodium acetate/ acetic acid buffer, pH 4.6 with either 30% PEG 2000 monomethyl ether or 25% PEG 4000 (Nextal, The Classics screen). The crystallization conditions were similar to those previously reported for Ec-NDPK2 and close to those found for Mx-NDPK (pH 5.2).5,12 Crystals were cryoprotected with the mother liquor containing 20% (v/v) glycerol and flash-frozen in liquid nitrogen. Data Collection
T1
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˚ resolution A preliminary dataset was collected at 2.2 A ˚ at 110 K (IPBS, Toulouse). A complete dataset at 1.62 A resolution was collected at 107 K on a ADSC Q4R CCD detector at ID14-1 beamline (ESRF, Grenoble) and processed with MOSFLM16 and CCP4 program suite.17 Crys˚ , b ¼ 76.09 tals belong to space group C2 (a ¼ 118.90 A ˚ , c ¼ 104.59 A ˚ , b ¼ 112.998). The asymmetric unit conA tains 6 chains, resulting in a Matthew’s coefficient of 2.3 ˚ 3/Da. The corresponding solvent content of 47.5% is A lower than for the Mx-NDPK of 51%. Data collection and refinement statistics are listed in Table I. Refinement The initial phases were obtained by molecular replacement with the MOLREP18 program using the coordinates of one dimer of Mx-NDPK crystallized without nucleotide (pdb entry ˚ and 2NCK)5 as search model using a Patterson radius of 35 A ˚ . The three first dyad peaks of the data in the range 30–3.0 A rotation function with Rotf/r greater than 8.0 were selected for three successive translation searches. The best final solution results in a correlation factor of amplitudes of 0.37 and a crystallographic R of 0.53 (intermediate values with 1 model: correlation ¼ 0.12; R ¼ 0.58; and with 2 models: correlation ¼ 0.21; R ¼ 0.55). Crystallographic refinement and side chains PROTEINS: Structure, Function, and Bioinformatics
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TABLE I. Data Collection and Refinement Statistics DATA Space group Cell dimensions: ˚) a, b, c (A a, b, g (8) ˚) Resolution (A Rsym I/r(I) Completeness (%) Redundancy ˚ 2) B-factor from Wilson plot (A REFINEMENT ˚) Resolution (A No. reflections Rwork/Rfree No. atoms Protein Water Average B-factors ˚ 2) Protein (A ˚ 2) Water (A R.m.s deviations ˚) Bond lengths (A Bond angles (8) Dihedral angles (8) a
C2 118.90, 76.09, 104.59 90.00, 112.99, 90.00 38.1–1.62 (1.66–1.62)a 0.09 (0.38) 16.6 (2.1) 99.7 (97.1) 3.5 (2.8) 17.6 38.1–1.62 (1.66–1.62) 108,677 (6,226) 0.19(0.27)/0.21(0.29) 6492 717 16.2 27.7 0.007 1.4 22.6
Highest resolution shell is shown in parenthesis.
model building were carried out using CNS ver1.119 and XTALVIEW20 respectively. Initially, a bulk solvent correction and NCS restraints on selected zones of the six chains were applied. Refinement began ˚ with torsion including observed data between 38.0 and 2.2 A angle molecular dynamics simulated annealing steps followed by conventional energy-restrained positional refinement. The R and Rfree values decreased to 0.28 and 0.32, respectively. Pro˚ . Inspection of difference gressively data were extended to 1.6 A electronic density maps clearly shows the presence of sulphate ions at the bottom of the binding site. The PICKWATER procedure from CNS was employed to locate water molecules in the difference maps. Finally these molecules were sorted by their nearest protein chain and renamed to be NCS consistent with SORTWATER program. In last refinement stages, NCS restraints were progressively removed, leading to final R and Rfree factors of 0.19 and 0.21, respectively. All the average temperature factors Bave of selected residues and standard deviations indicated in parenthesis were calculated with MOLEMAN2. Only structurally equivalent Ca atoms were included in the rms deviation calculation using LSQMAN21. Figures were drawn using MOLSCRIPT22 and PYMOL.23 The coordinates and structure factors of Escherichia coli nucleoside diphosphate kinase have been deposited in the RCSB Protein Data Bank under ID code 2HUR. RESULTS Description of the Monomer In the crystal, the asymmetric unit is made of six identical chains (root mean square (rms) deviation is
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STRUCTURE OF E. COLI NUCLEOSIDE DIPHOSPHATE KINASE
Fig. 1. Ribbon diagram of a monomer of Ec-NDPK view along a noncrystallographic two-fold axis of the tetramer as indicated by the target symbol. The side chains of five residues relevant to classify between hexameric and tetrameric quaternary structures are drawn in ball and stick. The nomenclature of the secondary structure elements is consistent with that defined for Dd-NDK.24
F1
˚ ) labelled from A to F. Each chain of the final 0.5(0.2) A model includes amino acids 2-143 (Fig. 1). Throughout this paper the residue numbering used is the Ec-NDPK one beginning at Met1 to facilitate comparisons with Mx-NDPK although the N-terminal methionine is missing in the E. coli recombinant protein (SWISS-PROT entry: NDK_ECOLI). Thus 5 must be added to obtain the Dictyostelium discoideum cytosolic NDPK (DdNDPK) numbering, which has been taken as reference for hexameric structures.24 Like all other NDPKs, the overall subunit structure is made of seven a helices, which partially cover the two faces of a central 4-stranded anti-parallel b sheet (Fig. 1). The backbone dihedral angles of all the non-Gly residues in each monomer fall in the allowed regions of the Ramachandran plot. All main and side chains are unambiguously defined in the electron density maps except in two loops (53–61 and 134–137). The loop (53–61) that connects the helices aA and a2 is highly solvent exposed in the set of chains A, C, and E. Like in all NDPK structures in absence of ligand, this loop is partially absent ˚ 2, Bave is the from electronic density maps (Bave ¼ 50(4) A average temperature factor). The flexibility of this region is required for nucleotide binding. For the set of chains B, D, and F, the crystal packing prevents this flexibility and even side chains of this loop show unambiguous densities. This loop adopts similar conformation for D and F ˚ 2) that have similar surroundings, chains (Bave ¼ 24(4) A ˚ 2). On but is slightly different for B chain (Bave ¼ 33(4)A the opposite wall of the nucleotide binding pocket, at the beginning of the Kpn loop, the chains (93–97) deviate once more according to the previously defined sets but
all the atoms are clearly visible in density maps (Bave ¼ ˚ 2). 26(3) A The region, called the Kpn loop (93–112), that connects the helix a3 (82–90) to the strand b4 (116–118) is well structured and encompasses a single turn helix called a30 .(103–107) The Kpn loop is so named because it contains, in Drosophila, the Killer of prune (Kpn) mutation that leads to a conditional lethal phenotype.25 Three intra-molecular hydrogen bonds between O-89 and N103, O-90 and N-104, as well as O-91 and Nh1-Arg104 interconnect the helices a3 and a30 creating a discontinuous bent helix. The bend angle between these helices is more pronounced in Ec-NDPK (368) and Mx-NDPK (278) than in Dd-NDPK (158), leading to maximum backbone ˚ at the end of the helix a30 and as far displacement of 3 A ˚ as 4.5 A at position 109. The a3–a30 linker is a spiral loop successively made up of a single turn of 310 helix (95– 97), a polyproline II stretch (98–100), and a type II bturn (99–102). In NDPKs, the b-turn is stabilized by an internal hydrogen bond from the Og-102 hydroxyl group to O-99. In this loop, a proline and a glycine are conserved in all hexameric interfaces at positions 100 and 105, respectively. Firstly, the replacement of this proline by an alanine in Ec-NDPK or by a glutamine in MxNDPK (or K/E/S/C in other bacterial NDPKs assumed to ˚ from be tetrameric) leads to a local distortion of 1.4 A a the hexameric C trace. Note that the presence of Gly101 (u ¼ 938, w ¼ 218) in Ec-NDPK allows the Og102 to preserve the internal hydrogen bond described above. Secondly, the replacement of the conserved Gly105 by an alanine as well as a lysine in Mx-NDPK could create a steric hindrance with carbonyl 98-O. Thus the main chain moves back putting the alanine Cb in place of the glycine Ca. Although the Kpn loop is solvent-exposed in Mx-NDPK whereas it is buried at an interface in Ec-NDPK, it adopts similar conformations in the two structures. In all available NDPK structures, a b-branched residue at position 115 is the central residue of a classical gturn and is located in a disfavored but allowed area of the Ramachandran plot. This turn, at the end of the Kpn loop, is important for a correct functional orientation of the catalytic histidine. In contrast, in the EcNDPK, there is a glycine (u ¼ 778, w ¼ 338), the turn is thus slightly relaxed and the hydrogen bond between N-115 and the amide group of Asn13 found in hexameric structures is absent. Nevertheless the location of the catalytic histidine is unchanged. Surprisingly, the residues 134–137, involved in a type II b-turn, are weakly defined in density maps for C, D, ˚ 2) and take part in the E, and F chains (Bave ¼ 38(4) A CD/EF interface described later, whereas they are better ˚ 2), where the ordered in A and B chains (Bave ¼ 28(3) A turn is solvent accessible. In Mx-NDPK, the main chain adopts a short 310 helix conformation and Gly134 is replaced by an arginine, which is the essential residue responsible of the tetrameric assembly. Two hydrogen bonds between O-35 and N-139 along with O-34 and Nh2-Arg141 anchor the fifth residue
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Overall Description of the Dimer
Fig. 2. Best superimposed models, in the alpha carbon representation, of the dimeric structural basic units from hexameric D. discoideum24 (yellow), tetrameric M. xanthus5 (blue) and E. coli (red) NDP kinases. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
before the C-terminal end of the protein to the edge of the b2 strand after a classical b-bulge involving Val34, Gly35, and Val76. The C-terminal residues are essential for correct folding and assembly of the enzyme since overexpression in E. coli of C-terminally truncated variants lacking of 5, 9 or 14 residues led to inclusion bodies.26 As expected from the high level of sequence identity (56%), the structures of Ec- and Mx-NDPK monomers ˚ . The largare very similar with a rms deviation of 1.1 A est differences are found in the two disordered loops, at the link aA-a2 and at the C-terminal end of the protein. The Active Site For each chain, one sulphate ion was found at the bottom of the active site cleft. It tightly interacts with conserved Lys11, Arg104, Asn114, and catalytic His117 along with Tyr51 via a water molecule. The sulphate ion approximately occupies a position similar to the b-phosphate in ADP-NDPK complexes.7 Despite the presence of this ion, the binding site is identical to the free form of M. xanthus enzyme. There was also such an anion in ammonium sulphate grown crystals of Dd-NDPK but it occupied a shallow position equivalent to the a-phosphate in ADP-NDPK complexes.27 By Ec-NDPK tetramer, three out of four sites are freely accessible but, in the case of D and F chains, Leu111 side chain from a neighboring tetramer is wedged between the Phe55 and Leu111 flanking residues of the cleft. PROTEINS: Structure, Function, and Bioinformatics
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At the dimeric interface, two 2-fold non-crystallographic symmetry (NCS) related b2 strands pair side by side by two hydrogen bonds between O-37 and N-39 backbone atoms to form a continuous, anti-parallel 8stranded b-sheet spanning the whole dimer (Fig. 2; Table IIA). In the same way, non-polar interactions between two anti-parallel helices a1 contribute to the dimer stability. After dimerization, the Cys139 is no more accessible and a disulfide bridge cannot be easily formed. The importance of a thiol group on the enzyme stability of S. typhymurium NDPK (96% identical to EcNDPK having the single Cys139) has been evidenced.3 ˚ 2 of the solvent accessible Dimer contacts bury 1180 A surface area (asa) per isolated subunit, a value in the same range as equivalent interface areas observed in the Mx-NDPK or the hexameric NDPKs (Table III). The three dimers (AB, CD and EF) are similar (rms ˚ between them). Likewise, the deviations less than 0.5 A rms deviations for structurally equivalent Ca atoms of Mx-(2-139) and Dd-NDPK(4-139) with respect to Ec˚ respectively (Fig. 2). NDPK dimers are 1.1 and 1.4 A Therefore, whatever the quaternary structure (hexamer, type I or II tetramers), NDP kinases share the same dimeric basic structural unit.
F2 T2
T3
Crystal Packing The crystal lattice shows stacked layers made of parallel rods constituted by blocks of six dimers roughly lined up along their minor inertia axis (Fig. 3). These axes are perpendicular to the b-sheet planes. Two different interfaces alternate in these blocks, therefore two types of assembly could be selected. One kind of tetramer associates the dimers AB with CD (red envelope) or the dimers EF with E‘F’ (green envelope). The noted E‘F’ counterpart of the half tetramer is formed from the coordinates of the dimer EF by applying the crystallographic 2-fold symmetry (x, y, 1 z). These two tetramers (ABCD and EFE‘F’) are similar ˚ ). On (rms deviation between the two tetramers is 0.46 A the other hand, the dimers CD and EF could participate in a fully different assembly (white envelope) with isolated AB dimer at the edge. The Interface CD/EF Results From Crystal Packing The interface between dimers CD and EF involves polar (Table IIC) and nonpolar interactions between the helix aA and the C-terminal part (130–135) of the protein. The two dimeric axes are not collinear with a short˚ avoiding the D2 symest distance between them of 6 A metry and therefore the assembly simply has a C2 symmetry. Depending on the interacting chains, these interfaces ˚ 2 (C/E and D/F) or 460 A ˚2 bury asa by monomer of 140 A (C/F and D/E) so smaller and furthermore less hydrophobic than regular protein–protein interfaces (Table IIC). Moreover if the crystal had grown from such tetramers,
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STRUCTURE OF E. COLI NUCLEOSIDE DIPHOSPHATE KINASE
TABLE II. Contacts between Subunits in Ec-NDK A. Contacts within the dimers AB, CD or EF Polar interactions ˚) ˚) O Val15-Nh1 Arg141 (3.20 A Od1 Asn18-Nh2 Arg141 (2.98 A ˚) ˚) N Ile20-Oe2 Glu28 (3.13 A N Gly21- Oe1 Glu28-(2.71 A ˚) ˚) O Thr37-N Met39 (3.03 A O Ser69-Og1 Thr142 (2.74 A ˚) ˚) Oe1 Glu113-Nh2 Arg143 (3.23 A Og Ser69-Nh1 Arg143 (2.84 A Nonpolar interactions Asn18-Glu28 Gly21-Phe24 Ile34-Met39 Val35-Met39 Thr37-Lys38 His41-Thr142 Phe66-Arg143 Ser69-Arg143 Gly70-Thr142 Pro71-Cys139 B. Contacts between dimers within the tetramer ABCD or EFE0 F0 Polar interactions ˚) ˚) O Gly101-Nh2 Arg26 (2.74 A Nh1 Arg26- NeArg26 (3.13 A h2 h2 ˚ N Arg26-N Arg26 (2.65 A) Non-polar interactions Arg26-Arg26 Ala29-Asp106 Ala29-Tyr107 Ala30-Asp106 Arg85-Ala100 Asp88-Leu99 Leu89-Ala100 Leu89-Gly101 C. Polar interactions and buried surface area by monomer at the interface CD/EF ˚ 2) (none) C/E (140 A ˚) ˚) ˚ 2) N Glu45-O Tyr131 (3.14 A Oe1 Glu45-Oe1 Gln46 (3.20 A C/F (440 A e1 e2 ˚ ˚) O Glu45-Ne2 Gln46 (2.96 A O Glu45-N Gln46 (3.17 A) ˚) Od2 Asp55-Nh1 Arg48 (3.06 A 2 ˚) ˚) ˚ O Glu45- Nh1 Arg48 (3.19 A Ne2Gln46–O Glu45 (3.01 A D/E (460 A ) ˚) ˚) O Tyr131-N Glu45 (3.11 A Nh1 Arg48-Od2 Asp55 (3.06 A ˚) ˚ 2) O Glu135-O Gly136 (2.83 A D/F (150 A ˚ are Accessible and buried surface area are given by monomer. Polar interactions shorter than 3.2 A listed.
TABLE III. Accessible and buried surface area by subunit (Dd-NDPK from pdb file 1npk, Awd-NDPK from pdb file 1ndl28, Mx-NDPK from pdb file 2nck, Ec-NDPK from pdb file 2hur) ˚ 2) Surface area per subunit (A
Accessible surface Dimer interface a1b2 C-term part Others (*loop a2-b3) Subtotal Trimer or tetramer II interface a3 & Kpn loop C-term part a1 Subtotal Tetramer I interface Link b2-aA C-term part Total in interfaces
Dd-NDPK
Awd-NDPK
Mx-NDPK
Ec-NDPK
8030
8770
7560
7560
530 80 30 640
570 230 220 1020
550 290 230 1070
700 360 * 120 1180
680 290 480 1450
720 320 640 1680
2090
dimers (AB and A‘B’) would have been required in solution, whereas only tetramers exist at the concentration used for crystallization as mentioned below. Therefore such an interface is certainly a crystallization artifact.29
2700
345 – 125 470 50 450 1570
1650
The Tetramers ABCD and EFE0 F0 are the Oligomers Observed in Solution In the tetramers ABCD and EFE0 F0, the helices a0, a1, a3, and a30 run towards the core of the complex whereas
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C O L O R
Fig. 3. View along the b axis of a molecular layer of Ec-NDPK crystal. The six chains of the asymmetric unit labelled A–F and their 2-fold symmetry mates (A0 –F0 ) constitute crystal building blocks. The envelopes colored in red and green surround the tetramers ABCD and EFE0 F0 respectively while the white envelope contours one other putative assembly CD/EF.
F4 F5
the helices aA, a2, and a4 are exposed to the molecular surface (Fig. 4). One side of the Kpn loop interacts with the helices a1 and a3 at the tetrameric interface [Fig. 5(a)]. In hexamers,24 the trimeric interface occurs between the opposite side of the Kpn loop with the end of the helix a1 and also with the C-terminal extension of the protein [Fig. 5(b)]. The formation of such a tetramer buries an asa for ˚ 2 (6% of the asa of the monoeach subunit of about 470 A mer) with only one direct hydrogen bond between Arg26 side chain and Gly101 carbonyl [Fig. 5(a), Tables IIB PROTEINS: Structure, Function, and Bioinformatics
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and III]. A hydrogen bond network connects the carboxylate group Asp106, the hydroxyl group of Tyr107, and the guanidinium groups of Arg26 and Arg85 through three bound water molecules. The buried surface of the Kpn loop is mainly hydrophobic (60%), which alone contributes to half of the total buried surface. At the interface, the stacking of the guanidinium planes of Arg26 from 2-fold symmetry related subunits (Fig. 6) reminds the stacking between Arg134 and Arg38 at the tetrameric interface of Mx-NDPK [Fig. 5(c)]. Comparable arginine ‘‘stacking’’ has been also observed in glutathione-
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S-transferase family30 or in lumazine synthase31 and has been found buried in interfaces. The major inertia axes of two dimers make an angle of about 538 and the minor axes are lined up drawing a tetramer of D2 sym˚ 3 56 A ˚ 3 48 A ˚. metry and of maximum dimensions 71 A There is no direct cross interaction between A and D or B and C subunits. At the centre of the tetramer, there is ˚ 3 lined by Asn22, Ala25, a small hydrated cavity of 30 A Arg26, and Tyr107. This cavity is without comparison with the large central cavity of hexameric assembly ˚ 3. Finally, inspection of the crystal lattice of about 2000 A shows that the crystal requires only such tetramers as building blocks (red and green envelopes on Fig. 3). The hydrophobic character of the interaction and size, shape, symmetry criteria32 bear out the identification of this interface as the relevant biological interface. Thus, this tetramer represents the 60 kDa oligomer found in solution by size exclusion chromatography.2,26 DISCUSSION Diversity in Quaternary Structures
Fig. 4. The nucleoside diphosphate kinase of E. coli shows a tetrameric quaternary structure. Two dimers are facing the concave side of their central sheet to form a type II NDPK tetramer. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
While the monomeric and dimeric structures of the nucleoside diphosphate kinases are remarkably similar, their quaternary structures exhibit an amazing diversity (Fig. 7). The two tetramer types differ by the orientation of the two interacting dimers. In M. xanthus (type I), dimers face the convex side of their central sheet whereas in E. coli (type II) they face the concave side. In the type I tetramer, the overall shape with maxi˚ 3 58 A ˚ 3 54 A ˚ is less compact mum dimensions of 76 A than Ec-NDPK. Mx-NDPK dimers interact by a salt bridge between residues Arg134 and Glu137 and three hydrogen bonds [Fig. 5(c)]. The dimeric axes are collinear and the tetramer adopts a D2 symmetry. From these observations, Williams and his collaborators have con-
Fig. 5. Comparison of the interfaces in (a) tetrameric Ec-NDPK (pdb file 2hur), (b) hexameric DdNDPK24 (pdb file 1npk), and (c) tetrameric Mx-NDPK5 (pdb file 2nck). For sake of clarity, only dimers and a trimer have been drawn. All the Kpn loops are colored in yellow. The upper subunits are fitted on a similar orientation in (a) and (b). The water molecules that expand the dimeric interface have been included in (a). In Ec-NDPK, the dimeric interface occurs on one side of the Kpn loop and the central part of helix a1 while in hexameric Dd-NDPK the trimeric interface involves the opposite side of the Kpn loop, the C-term end of the helix a1 and the carboxy end of the protein. In Mx-NDPK dimers interact by a salt bridge and hydrogen bonds. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] PROTEINS: Structure, Function, and Bioinformatics
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Fig. 6. Stacking of the guanidinium planes (distance of 3.1 A˚) of the Arg26 residues from 2-fold symmetry related subunits (chains A and C) with the (2Fo-Fc) electron density map contoured at 3 sigma. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
cluded that it was unambiguously the biologically significant interface.5 In the type II tetramer, subunits interact by a new interface harboring the Kpn loop, as in hexamers but on the opposite face of this loop [Fig. 5(a,b)]. Diversity in quaternary architecture is not an exclusivity of NDPK family. For example, it is encountered in haemoglobin family33 and recently, the members of the S100 family of EF-hand calcium-binding proteins have been found to form dimers, tetramers, hexamers, and octamers.34 The Type II Quaternary Structure is More Prevalent in the Bacterial Tetrameric NDP Kinases F8
From the analysis of sequence alignment (Fig. 8) and available quaternary structure information, it appears that five residues at positions 30, 100, 105, 50, and 131 are relevant to classify the hexameric and tetrameric quaternary structures. Three of them (Lys30, Pro100, and Gly105) are important for the formation of hexamers. In PROTEINS: Structure, Function, and Bioinformatics
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that case, the NH3þ group of Lys30 protrudes from the end of a1 towards one side of the Kpn loop of a neighboring molecule, more precisely the carboxyl end of helix a30 , and makes no less than three hydrogen bonds with the carbonyls O-95, O-105, and O-108 [Fig. 5(b)]. However, it must be pointed out that Deinococcus radiodurans NDPK with Lys30 but Ala105 is hexameric in solution (Mocan, I., unpublished results) whereas S. coelicolor NDPK with these three key residues but with an alanine residue inserted after Lys30 has been shown to form tetramers6 In type II tetramers, the replacement of Lys30 by a nonpositively charged residue comes with the change of Gly105 by Ala/Lys. In Ec-NDPK, the side chain of Ala30 is buried within the Kpn loop as mentioned already and the three carbonyls (95, 105, and 108) are fully accessible to the solvent. The main interface residue (Arg26) is highly conserved in all NDPKs including hexamers and Mx-NDPK, where it participates in intra-subunit salt bridges. Finally, the concomitant presence of a lysine at both positions 30 and 105 forbids the formation of hexamer as well as type II tetramer and leads to the formation of a
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STRUCTURE OF E. COLI NUCLEOSIDE DIPHOSPHATE KINASE
C O L O R
Fig. 7. Diversity on the quaternary structure of nucleoside diphosphate kinase family: Views of the (a) hexamer as in Dd-NDPK24 (pdb file 1npk), (b) type II tetramer in Ec-NDPK (pdb file 2hur) and (c) type I tetramer in Mx-NDPK5 (pdb file 2nck). The basic dimeric structural unit, colored in red and blue, is always shown in the same orientation.
C O L O R
Fig. 8. Alignment of sequences of NDP kinases mentioned in the text. Important residues differentiating hexameric and tetrameric quaternary structures have been highlighted. The alignment was drawn using ESPript.35
type I tetramer. In Mx-NDPK, Lys30 is fully accessible to the solvent. Among 200 available bacterial NDPK sequences, this simultaneous occurrence is exclusively observed in sequences of two Myxococcaceae (M. xanthus and Anaeromyxobacter dehalogenans) and of one proteobacteria (Candidatus Pelagibacter ubique) and the Arg134 interface residue is present only in Mx-NDPK.
Then, two-thirds of the sequences can be predicted as type II tetramers, one-third as hexamers, and only a few as type I tetramer. Surprisingly, two aromatic side chains at both positions 50 and 131 are conserved in type I and II tetramers in place of His50 and Leu131 in hexamers. These two side chains are not directly engaged in the active
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site or in any oligomerization interface but reinforce the hydrophobic character of helices aA and a4. Size exclusion chromatography and low-resolution biophysical techniques are not resolutive enough to differentiate between tetramer types hence more bacterial NDP kinase crystal structures will be required to accurately predict the type to which a NDP kinase belongs. Mutational studies of conserved residues present at interfaces should be also informative on oligomeric diversity. Does NDP Kinase Exist as Stable Dimers in Some Organisms? Biochemical data on bacterial NDPKs need to be reconsidered on the basis of this new structure. The present structural analysis shows that subunits interact more tightly across the dimeric basic unit than at the tetramerization interface. This is consistent with denaturation–renaturation experiments26 and with differential scanning microcalorimetric studies, indicating that EcNDPK tetramer first dissociates at relatively low temperature (37.58C) and unfolds irreversibly at 55.78C.36 These data suggest that folding/unfolding pathways could go through a dimeric intermediate. Recently protein–protein interactions studies by optical biosensor and analytical ultracentrifugation have shown that Ec-NDP kinase in micromolar range is in equilibrium between dimeric and tetrameric forms.37 Moreover the tetramer dissociation constant is decreased by an order of magnitude in the presence of 1 mM ADP or ATP. These results are in agreement with the loose association observed in the crystal state between two dimers. It has to be pointed out that the protein concentration used for crystallization was around 0.5 mM. Whether or not a nucleoside diphosphate kinase of some organisms may exist as a stable and active dimer in vivo is an interesting question. ACKNOWLEDGMENTS The authors thank the staffs of IPBS (Toulouse, France), BM30A and ID14-1 (ESRF, Grenoble, France) for X-ray diffraction facilities. Thanks are due to Dr. Manfred Konrad (Max-Planck Institut, Goettingen, Germany) for the ndkec expression vector, to Dr Roger L. Williams (MRC LMB, Cambridge, UK) for space group I222 coordinates of Mx-NDPK before PDB release, to Pr Joe¨l Janin and Dr Jean Velours for helpful comments and suggestions. REFERENCES 1. Ohtsuki K, Yokoyama M, Koike T, Ishida N. Nucleoside diphosphate kinase in Escherichia coli: its polypeptide structure and reaction intermediate. Biochem Int 1984;8:715–723. 2. Almaula N, Lu Q, Delgado J, Belkin S, Inouye M. Nucleoside diphosphate kinase from Escherichia coli. J Bacteriol 1995;177: 2524–2529. 3. Ginther CL, Ingraham JL. Nucleoside diphosphokinase of Salmonella typhimurium J Biol Chem 1974;249:3406–3411. PROTEINS: Structure, Function, and Bioinformatics
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4. Munoz-Dorado J, Inouye S, Inouye M. Nucleoside diphosphate kinase from Myxococcus xanthus. II. Biochemical characterization. J Biol Chem 1990;265:2707–2712. 5. Williams RL, Oren DA, Munoz-Dorado J, Inouye S, Inouye M, Arnold E. Crystal structure of Myxococcus xanthus nucleoside diphosphate kinase and its interaction with a nucleotide sub˚ resolution. J Mol Biol 1993;234:1230–1247. strate at 2.0 A 6. Brodbeck M, Rohling A, Wohlleben W, Thompson CJ, Susstrunk U. Nucleoside-diphosphate kinase from Streptomyces coelicolor. Eur J Biochem 1996;239:208–213. 7. Janin J, Dumas C, More´ra S, Xu Y, Meyer P, Chiadmi M, Cherfils J. J Bioenerg Biomembr 2000;32:215–225. 8. Giraud MF, Georgescauld F, Lascu I, Dautant A. Crystal structures of S120G mutant and wild type human of Nucleoside Diphosphate Kinase A in complex with ADP. J Bioenerg Biomembr 2006;38:261–264. 9. Sedmak J, Ramaley R. Purification and properties of Bacillus subtilis nucleoside diphosphokinase. J Biol Chem 1971;246: 5365–5372. 10. Chen Y, More´ra S, Mocan J, Lascu I, Janin J. X-ray structure of Mycobacterium tuberculosis nucleoside diphosphate kinase. Proteins: Struct Funct Genet 2002;47:556–557. 11. Lascu I, Giartosio A, Ransac S, Erent M. Quaternary structure of nucleoside diphosphate kinases. J Bioenerg Biomembr 2000;32:227–236. 12. Williams RL, Munoz-Dorado J, Jacobo-Molina A, Inouye S, Inouye M, Arnold E. Crystallization and preliminary X-ray diffraction analysis of nucleoside diphosphate kinase from Myxococcus xanthus. J Mol Biol 1991;220:5–7. ˚ crystal 13. Strelkov SV, Perisic O, Webb PA, Williams RL. The 1.9 A structure of a nucleoside diphosphate kinase complex with adenosine 30 ,50 -cyclic monophosphate: evidence for competitive inhibition. J Mol Biol 1995;249:665–674. 14. Hama H, Almaula N, Lerner CG, Inouye S, Inouye M. Nucleoside diphosphate kinase from Escherichia coli; its overproduction and sequence comparison with eukaryotic enzymes. Gene 1991;105:31–36. 15. Lascu I, Pop RD, Porumb H, Presecan E, Proinov I. Pig heart nucleosidediphosphate kinase. Phosphorylation and interaction with Cibacron blue 3GA. Eur J Biochem 1983;135:497– 503. 16. Leslie AGW. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 þ ESF-EAMCB Newsletter on Protein Crystallography, No. 26, 1992. 17. Collaborative Computational Project, Number 4. Acta Crystallogr D Biol Crystallogr 1994; 50:760–763. 18. Vagin A, Teplyakov A. MOLREP: an Automated Program for Molecular Replacement. J Appl Cryst 1997;30:1022–1025. 19. Bru¨nger AT, Adams PD, Clore GM, DeLano WL, Gros P, GrosseKunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL. Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 1998;54:905–921. 20. MacRee DE. XtalView/Xfit–A versatile program for manipulating atomic coordinates and electron density. J Struct Biol 1999;125:156–165. 21. Kleywegt GJ, Zou JY, Kjeldgaard M, Jones TA. Around O. In: Rossmann MG, Arnold E, editors. International tables for crystallography, Vol. F: Crystallography of biological macromolecules. Dordrecht: Kluwer Academic; 2001. pp 353–356, 366–367. 22. Kraulis PJ. MOLSCRIPT. a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr 1991; 24:946–950. 23. DeLano WL. The PyMOL molecular graphics system user’s manual. San Carlos, CA: DeLano Scientific; 2002. 24. More´ra S, LeBras G, Lascu I, Lacombe ML, Ve´ron M, Janin J. Refined X-ray structure of Dictyostelium discoideum nucleoside ˚ resolution. J Mol Biol 1994;243: diphosphate kinase at 1.8 A 873–890. 25. Dumas C, Lascu I, More´ra S, Glaser P, Fourme R, Wallet V, Lacombe ML, Ve´ron M, Janin J. X-ray structure of nucleoside diphosphate kinase. EMBO J 1992;11:3203–3208. 26. Erent M. Nucle´oside diphosphate kinases. Stabilisation de la prote´ine par la structure quaternaire. PhD Thesis, Bordeaux: Bordeaux 2 University, 1997.
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32. Bahadur RP, Chakrabarti P, Rodier F, Janin J. A dissection of specific and non-specific protein–protein interfaces. J Mol Biol 2004;336:943–955. 33. Royer WE, Jr, Knapp JE, Strand K, Heaslet HA. Cooperative hemoglobins: conserved fold, diverse quaternary assemblies and allosteric mechanisms. Trends Biochem Sci 2001;26:297–304. 34. Moroz OV, Dodson GG, Wilson KS, Lukanidin E, Bronstein IB. Multiple structural states of S100A12: a key to its functional diversity. Microsc Res Tech 2003;60:581–592. 35. Gouet P, Courcelle E, Stuart DI, Metoz F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 1999;15:305–308. 36. Giartosio A, Erent M, Cervoni L, More´ra S, Janin J, Konrad M, Lascu I. Thermal stability of hexameric and tetrameric nucleoside diphosphate kinases. Effect of subunit interaction. J Biol Chem 1996;271:17845–17851. 37. Shen R, Wheeler LJ, Mathews CK. Molecular interactions involving Escherichia coli nucleoside diphosphate kinase. J Bioenerg Biomembr 2006;38:255–259.
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