Substrate Specificity and Assembly of the Catalytic Center Derived from two Structures of Ligated Uridylate Kinase

JMB—MS 343 Cust. Ref. No. RH 61/94

[SGML] J. Mol. Biol. (1995) 246, 522–530

Substrate Specificity and Assembly of the Catalytic Center Derived from two Structures of Ligated Uridylate Kinase Hans-Joachim Mu¨ller-Dieckmann and Georg E. Schulz* Institut fu¨r Organische Chemie und Biochemie Albert-Ludwigs-Universita¨t Albertstr 21, 79104 Freiburg im Breisgau, Germany

*Corresponding author

Two crystal structures of ligated uridylate kinase from Saccharomyces cerevisiae were determined by X-ray analyses. The ligands were ADP and AMP. Cocrystallization with ATP yielded crystals with ADP at the ATP site and a mixture of AMP and ADP at the NMP site. Cocrystallization with ADP gave rise to a distinct crystal type with ADP at the ATP site, but only AMP at the NMP site. In both cases, the substrates are kept in place by favorable crystal contacts. The structures have been refined to R-factors of 17.8% and 19.6% at resolutions of 2.1 Å and 1.9 Å, respectively. A comparison with the related cytosolic adenylate kinase from pig disclosed large induced-fit movements on substrate binding and the disassembly of the catalytic center in the absence of substrates. The relatively high side-activity of uridylate kinase for AMP is explained by the finding that the binding pocket is sized for an AMP, but constructed to bind UMP together with a water molecule. Keywords: refined X-ray structure; uridylate kinase; substrate specificity; induced-fit; catalytic center

Introduction Uridylate kinase from yeast (UKyst , EC 2.7.4.14) catalyzes the phosphorylation of nucleoside monophosphates at the expense of ATP according to: ATP·Mg2+ + NMP F ADP + Mg2+ + NDP. UKyst requires a divalent cation and accepts UMP, AMP and CMP as nucleoside monophosphates with relative reaction rates of 100%, 30% and 10%, respectively (Mu¨ller-Dieckmann, 1990), whereas GMP is rejected (Schricker et al., 1992). The enzyme is monomeric and consists of 204 amino acid residues (Liljelund et al., 1989) with an Mr of 22,933 as derived from the cDNA. A sequence analysis of the ten N-terminal amino acid residues of the purified enzyme showed that the recombinant UKyst used in our analysis lacks Met1. UKyst is vital as it is the only yeast enzyme that can phosphorylate UMP (Liljelund & Lacroute, 1986). Its residual activity for AMP, on the other hand, explains why Abbreviations used: UKyst , uridylate kinase from Saccharomyces cerevisiae; r.m.s., root-mean square; UKdic , uridylate kinase from Dictyostelium discoideum; B-factor, crystallographic temperature factor; AKeco , adenylate kinase from Escherichia coli; AK1, cytosolic adenylate kinase from vertebrates (here, pig); Ap5 A, P1, P5bis(5'-adenosyl)-pentaphosphate; PEG, polyethylene glycol. 0022–2836/95/090522–09 $08.00/0

yeast survives the deficiency of the proper adenylate kinase (Schricker et al., 1992). UKyst resembles the adenylate kinases, several structures of which are known (Egner et al., 1987; Dreusicke et al., 1988; Diederichs & Schulz, 1991; Mu¨ller & Schulz, 1992; Berry et al., 1994). The homology between UKyst and the adenylate kinases ranges from 43% identity for a short cytosolic to 25% for a long bacterial variant. The two structures described here are enzyme:substrate complexes. In both complexes the ATP site is occupied by an ADP while the NMP site accommodates a mixture of ADP and AMP in one case (named UKyst :ADP:ADP/AMP; Mu¨ller-Dieckmann & Schulz, 1994), and merely an AMP in the other (named UKyst :ADP:AMP). The two reported structures were compared with the substrate-free homologous structure of cytosolic adenylate kinase from pig (AK1; Dreusicke et al., 1988) revealing large induced-fit motions and disassembly of the catalytic center.

Results and Discussion Quality of the models The co-ordinate error estimates according to ˚ for both final Luzzati (1952) were about 0.23 A models, while sA plots (Read, 1986) yielded values 7 1995 Academic Press Limited

JMB—MS 343 523

Ligated Structures of Uridylate Kinase

(a)

(b)

(c)

˚ for UKyst :ADP:AMP and 0.23 A ˚ for of 0.21 A UKyst :ADP:ADP/AMP. The real space correlation plots (Jones et al., 1991) showed weak points in the poorly defined N-terminal regions and in the loop

Figure 1. Accuracy and mobility of UKyst :ADP:AMP (thick lines) and UKyst :ADP:ADP/AMP (thin lines). (a) Real space density correlation as calculated for all atoms (Jones et al., 1991). (b) r.m.s. difference between the main-chain atoms of the 2 complexes after the best superposition of all backbone atoms. (c) B-factors of main-chain atoms, the locations of a-helices (—) and bstrands (QQQ) are marked.

regions around positions 95 and 144 (Figure 1(a)). The average correlation was 0.92 for UKyst : ADP:AMP and 0.90 for UKyst :ADP:ADP/AMP. As a further quality check we superimposed the two

Table 1 Quality of the final models Total reflections Unique reflections in ˚) resolution range (A Completeness (%) Completeness (%) in the ˚) outermost shell (A R-factor for all data (%) ˚) Bond length deviations† (A Bond angle deviations† (deg) Protein atoms Substrate atoms Water molecules ˚ 2) Mean B-factor of main chain‡ (A ˚ 2) Mean B-factor of substrates (A ˚ 2) Mean B-factor of water (A

UKyst :ADP:AMP

UKyst :ADP:ADP/AMP

84,238 17,480 10–1.93 97 96 1.95–1.93 19.6 0.009 1.5 1556 50 106 20 14 35

32,483 12,091 10–2.13 91 64 2.19–2.13 17.8 0.018 3.1 1556 54 101 19 15 34

† r.m.s. deviations from the geometry described by Engh & Huber (1991). ˚ 2 (3.5 A ˚ 2 ) for 2 ‡ In the main chain the average r.m.s. B-factor difference was set to 1.5 A ˚ 2 (5.0 A ˚ 2 ) for 2 atoms connected via a third atom, where directly connected atoms and to 2.0 A the values in parentheses refer to side-chain atoms.

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Ligated Structures of Uridylate Kinase

Figure 2. Secondary structure assignment for UKyst :ADP:AMP and alignment with AK1 from pig. First line, primary structure of UKyst with numbering (Liljelund et al., 1989); 2nd line, manually assigned secondary structure with names of a-helices and b-strands; 3rd line, borders of domains NMPbind and LID as defined in AK1 (see the text). The 99 Ca ˚ distance cut-off are marked atoms of UKyst and AK1 that remained in the superposition of CORE domains with a 1 A (- - -); 4th line, residues (*) conserved between UKyst and AK1; 5th line, secondary structures of AK1 manually assigned by Dreusicke et al. (1988); 6th line, sequence and numbering of AK1.

polypeptides (Figure 1(b)). Except for the N-terminal ˚ with region the residual deviations are below 0.3 A ˚ an average of about 0.2 A, which agrees with the other estimates. The main quality parameters of the final models are given in Table 1. Chain conformation In both models, all main chain dihedral angles of non-glycine residues are found in favorable regions. The Ramachandran plots show only two residues, Asn96 and Ser178, in the left-handed a-helical region around (+60°, +40°). As in all adenylate kinases the

Figure 3. Ribbon representation (Kraulis, 1991) of the complex UKyst :ADP:ADP/AMP. The ADP molecules at left and right occupy the ATP and NMP sites, respectively. The partially occupied b-phosphoryl group is marked by light balls as atoms. Some residues are labeled, the disulfide bridge is shown as a broken line. Domain NMPbind and peptide segment LID are indicated.

conserved Pro106 of UKyst adopts a cis-conformation. This proline participates in a loop involved in NMP binding. Among the 13 glycine residues, 5 are in regions forbidden for residues with side-chains. The secondary structures were identical for both complexes. They are given in Figure 2 together with those of AK1 (Dreusicke et al., 1988). A comparison between UKyst and AK1 shows that all a-helices and b-strands agree with each other, except for deviations at some of their ends and except for the N-terminal a-helix of AK1, which has no counterpart in UKyst . For an overview, a ribbon plot is depicted in Figure 3. The N terminus of UKyst has an extension of eight residues when compared to AK1. This extension is disordered in the crystal, its function is not known. Like all other members of the NMP kinase family, UKyst consists of a central 5-stranded parallel b-sheet surrounded by a-helices (Figure 3). The b-sheet and some adjacent a-helices remain unaffected by the movements during catalysis and have been named the CORE domain, whereas the moving parts have been named NMPbind and LID (Schulz et al., 1990). For the small variants like UKyst and AK1, LID is merely an 11-residue peptide segment, whereas it is a 38-residue domain in the long variants. The domains are defined in Figure 2. A superposition of the 153 equivalenced Ca atoms of the CORE domains of UKyst and AK1 using program OVERLAY (Kabsch, 1978) with a cut-off distance of ˚ retained 99 Ca atoms, with an r.m.s. deviation of 1A ˚ . This superposition has been used for all UKyst 0.6 A versus AK1 comparisons in this report. Chain mobility and solvent structure In Figure 1(c) the main-chain B-factors of both ˚2 complexes are plotted, the averages being about 20 A

JMB—MS 343 525

Ligated Structures of Uridylate Kinase

Table 2 Crystal contacts of UKyst :ADP:AMP Contact area‡ ˚ 2) (A

Contacting residues§

I-II I-III I-IV

630 495 490

aI :aII bI :cIII cI :bIV

I-V I-VI

470 310

dI :dV eI :eVI

I-VII I-VIII

130 120

fI :gVII gI :fVIII

Contact†

Polar interactions> Atom 1:Atom 2

Distance ˚) (A

Lys67-NZ:Lys144-O Glu132-N:Asp39-O Glu132-OE1:Arg201-NH1 Glu132-OE2:Arg201-NH1 Asp133-OD1:Ser41-N Asp133-OD2:Lys99-NZ Lys157-NZ:Asp198-OD1 Lys157-NZ:Asp202-OD1 Lys157-NZ:Asp202-OD2 Asn161-OD1:Arg201-NH2 Tyr62-OH:Glu79-OE1 Arg117-NE:Thr176-O Arg117-NH2:Thr176-O

2.96 2.83 3.02 3.31 2.91 2.76 3.04 3.10 2.79 2.74 2.57 3.14 3.29

† Enzyme molecules II through VIII are related to the reference molecule I by the following symmetry operators in fractional co-ordinates: II, (1,0,0=1,-1,0=0,0,-1), (0,1,1/6); III, (1,-1,0=0,-1,0=0,0,-1), (1,1,0); IV, (1,-1,0=0,-1,0=0,0,-1), (0,1,0); V, (0,1,0=1,0,0=0,0,-1), (0,0,1/3); VI, (1,0,0=1,-1,0=0,0,-1), (0,0,1/6); VII, (1,-1,0=1,0,0=0,0,1), (0,0,1/6); VIII, (0,1,0=-1,1,0=0,0,1), (0,0,-1/6). ‡ The contact area is half of the two buried surface areas, as calculated with X-PLOR ˚ ). (probe radius 1.4 A ˚ cut-off for any atom pair. The list of § As a distance criterion we took a 4.5 A contacting residues are: a, 31, 55-56, 58, 67, 95, 97, 144-146, 189, adenosine; b, 131-134, 136-137, 140, 153, 157, 160-161, 164; c, 39-41, 99, 197-198, 201-202, 204; d, 61-62, 64-65, 68-69, 74-75, 77, 79; e, 109, 113, 117, 173, 176-177; f, 94, 96; g, 129, 184. ˚ and a > The criterion for a hydrogen bond was a distance below 3.5 A donor–H · · · acceptor angle above 120°. Contacts I-III and I-IV have a common list of polar interactions.

(Table 1). The highest mobility is observed at the chain termini, relative maxima occur around positions 95, 144 and 177. Residues 95 and 177 are in loops connecting an a-helix with a following b-strand. Residue 144 is in the LID segment, which is in a closed-down conformation in both complexes. The substrate molecules have rather low average ˚ 2 (Table 1), indicating tight B-factors of about 15 A binding to the enzyme.

The average B-factors for the 100 odd water mole˚ 2 (Table 1). cules in the two complexes are about 35 A The water molecules are ordered according to their electron densities. Among the 12 water molecules ˚ ), 9 are at close to the substrates (distance <3.5 A conserved positions when compared with the structure of the complex between the two-substrate mimicking inhibitor Ap5 A and the adenylate kinase from Escherichia coli (AKeco :Ap5 A, Mu¨ller & Schulz, 1992).

Figure 4. Stereo view of the crystal contacts at domain NMPbind and at segment LID. Reference molecule I is located at the center, while molecule II is at the right-hand and molecule V at the upper left-hand side. The 2-fold axes are represented by straight lines. The contacting NMPbind domains (I-V) and LID segments (I-II) are emphasized by thick lines. Their borders are labeled with residue numbers. All residues in these contacts are marked by a dotted Ca atom.

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Ligated Structures of Uridylate Kinase

Figure 5. Deviation of the Ca atoms in a superposition of UKyst :ADP:AMP and AK1 using the 153 equivalenced Ca atoms of the CORE domains with a distance ˚ , which left the basic cut-off of 1 A superposition set of 99 Ca atoms that are marked in Figure 2. The average deviation of the 194 equivalenced Ca atoms of the whole chains of UKyst ˚ . The 2 and AK1 amounts to 2.3 A insertions in UKyst (residues 58 and 95) are marked by asterisks.

Crystal contacts Every UKyst molecule in the crystal has contacts to seven neighbors as specified in Table 2. Three of these contacts (I-II, I-V, I-VI) are across 2-fold axes. The total solvent-accessible surfaces of UKyst in complexes UKyst :ADP:AMP and UKyst :ADP:ADP/ ˚ 2 and 9000 A ˚ 2, respectively. In AMP are 8900 A both complexes, the enzyme does not cover the ADP at the ATP site completely; about 20% of the ADP surface (mostly at the adenosine) remains solvent-accessible. Atom O2RA of this ADP contributes to contact I-II by non-specific van der Waals’ interactions. In contrast, the nucleotide at the NMP site is virtually completely covered. ˚ 2 for The sum of all contact areas is 2645 A ˚ UKyst :ADP:AMP (Table 2) and 2520 A2 for UKyst :ADP:ADP/AMP, which are 30% and 28% of the total accessible surfaces, respectively. These values are comparatively high. Enzymes I, II and V are depicted in Figure 4. Contact I-II juxtaposes the

LID segments and contact I-V the NMPbind domains, both of which are in their closed-down conformation. The packing arrangement prevents the dissociation of the substrates. This is one of the rare cases where a crystalline enzyme can be analyzed with its actual substrates in place. The absence of Mg2+ excludes catalysis. Induced-fit motions Induced-fit movements have been reported for the NMPbind and LID domains of large variants among the adenylate kinases (Schulz et al., 1990). Now we present the motions of a small variant, where LID is merely an 11-residue segment. A superposition of ligated UKyst with substrate-free AK1 (Dreusicke et al., 1988) quantified the conformational changes as shown in Figure 5. Using program OVERLAY ˚ ), the NMPbind motion is (distance cut-off 1 A approximated as a 37° rotation. As illustrated in

Figure 6. Interactions between the closed domain NMPbind and helix a7 as observed in UKyst :ADP:AMP. All hydrogen ˚ and angles above 120°. The B-factor of the trapped water molecule is 40 A ˚ 2. bonds (broken lines) have lengths below 3.5 A

JMB—MS 343 Ligated Structures of Uridylate Kinase

527

Figure 7. Stereo view of the 11-residue segment LID plus Lys29 and Arg107 of UKyst :ADP:ADP/AMP (thick lines) as superimposed on the equivalent parts of AK1 (thin lines). Both LID segments are given as Ca backbones elongated by 2 Ca atoms (positions 152 and 153 in UKyst ). The superposition is the same as in Figure 5. On LID closure in UKyst , Asp150 and Asp151 form salt bridges to Arg148 and Arg142, respectively. The corresponding residues of substrate-free AK1 (Asp140, Asp141, Arg138 and Arg132) dangle around, indicating that the catalytic center is disassembled when the substrates are absent. Chain cuts are marked by dots, not all hydrogen bonds (broken lines) are given.

Figure 6, NMPbind of UKyst has assumed a completely closed conformation (see also Figure 3). In its motion, NMPbind hits the vertical a-helix (Figure 3) like a road block and forms hydrogen bonds. The movement of segment LID is smaller, but seems to be more important. It is depicted in Figure 7. On LID closure in ligated UKyst , Asp150 and Asp151 form salt bridges to Arg148 and Arg142, respectively, which are crucial for fixing the phosphoryl groups of the substrates. In substratefree AK1, the LID is open, the respective aspartate

residues are directed backwards towards the solvent in a very mobile part of the chain, the two arginine residues dangle around and the catalytic center is disassembled. Obviously, these kinases found the ultimate solution for preventing ATP hydrolysis, as they completely disassemble their active center during each catalytic cycle, reassembling it only on substrate binding. Both aspartate and arginine residues are strongly conserved within the adenylate kinase family except for guanylate kinase (Stehle & Schulz, 1990).

Figure 8. Schematic drawing of all hydrogen bonds between the 2 ADP molecules and the polypeptide in the complex ˚ and angles above 120°. The modeled uracil UKyst :ADP:ADP/AMP. Hydrogen bonds are defined by distances below 3.5 A at the NMP site with its putative interactions is shown as an inset. See the text for the rotation of Gln111.

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Ligated Structures of Uridylate Kinase

Table 3 Course of structure refinements with program X-PLOR Round

Resolution ˚) limit (A

UKyst :ADP:ADP/AMP 1 3.0

R-factor (%) 35.3

6

2.13

24.6

15

2.13

17.8

UKyst :ADP:AMP 1 1.9 7

1.9

20.0 19.6

Comment Complete backbone (residues 11 to 204), 40% of side-chains Complete real model (residues 9 to 204), 2 ADP molecules Final model with 101 water molecules Refined final model of UKyst :ADP:ADP/AMP with water molecules Final model with 106 water molecules

Substrate binding sites The substrate sites in the adenylate kinase family have been clarified by structure (Egner et al., 1987; Mu¨ller & Schulz, 1988; Stehle & Schulz, 1990; Diederichs & Schulz, 1991; Berry et al., 1994) and mutational (Tsai & Yan, 1991) analyses. In ligated UKyst both substrates are involved in a dense hydrogen bonding network, mostly between their phosphoryl groups and five arginine residues. Every oxygen atom of the phosphoryl groups participates in at least one hydrogen bond to the polypeptide. The phosphoryl groups are ideally positioned for an SN 2 in-line associative mechanism (Richard & Frey, 1978; Buchwald et al., 1982; Mu¨ller & Schulz, 1992; Mu¨ller-Dieckmann & Schulz, 1994; Berry et al., 1994). There is a remarkable asymmetry of the bound adenosines best recognized in Figure 8. While the adenosine at the ATP site forms only one hydrogen bond with the polypeptide, the other adenosine is fixed by as many as six. Furthermore, there is a sandwich interaction between the ATP site adenosine and Arg138, which is conserved in all members of the adenylate kinase family. The differences in binding mode between the small variant UKyst and the large variant AKeco are mostly due to differing LID sizes. In AKeco the 38-residue LID domain covers the ATP site adenosine completely, whereas the small 11-residue LID segment of UKyst leaves 20% of the surface of the respective adenosine accessible to solvent. The strong interactions of the NMP site adenosine in UKyst (Figure 8) resemble closely those of AKeco :Ap5 A. The hydrogen bonding pattern of O3RA, Wat3, Asp150 and the main-chain carbonyl of residue 74 (UKyst numbering) is exactly the same as in AKeco . Only the Thr31-OG1 of AKeco has been substituted by Arg107-NH1 in the weak hydrogen bond to adenosine N7. It should be noted that UKyst contains the classical mononucleotide binding motif (Schulz, 1992), consisting of a b-strand and a following a-helix connected by a glycine-rich loop with the strongly conserved finger-print sequence -G23-X-X-G-X-G-K-. This loop forms a giant anion hole that accommo-

dates the b-phosphate at the ATP site by donating hydrogen bonds from as many as five backbone amides (Dreusicke & Schulz, 1986; and Figure 8). Tight association of the ATP b-phosphate together with weak contacts to the adenosine appear to be general properties of this type of ATP site, which is usually not very specific for the nucleotide base. It has also been found at the crucial locations of myosin (Rayment et al., 1993) and F1-ATPase (Abrahams et al., 1994). UMP specificity UKyst shows side-activities of about 30% and 10% for AMP and CMP, respectively, whereas the analyzed AMP-kinases possess virtually no activity for UMP and CMP. A comparison of the two known UMP-kinase sequences with those of 28 AMPkinases shows that Ala47 and Ile75 are specific for UMP, and it shows a unique exchange Gln111 : Asn in UKdic (always UKyst numbering). In the AMPkinases, Ala47 is a Thr (79%) or a Ser (11%) and Ile75 is a Leu (93%). This sequence comparison agrees well with the observation that mutant Thr47 : Ala/ Leu75 : Ile (UKyst numbering) of chicken AMPkinase has appreciably increased side-activities for UMP and CMP and decreased AMP activity (Okajima et al., 1993). Surprisingly, the characteristic UMP-kinase residues Ala47, Ile75 and Asn111 (UKdic ) enlarge the binding pocket volume relative to the AMP-kinases instead of reducing it for the smaller substrates UMP and CMP. This riddle would be explained, if the induced-fit movement of domain NMPbind could adjust itself to the size of the bound nucleotide. This possibility can be excluded, however, because the motion of NMPbind runs up to a block (Figures 3 and 6) for the largest substrate AMP, i.e. no further motion on UMP or CMP binding is conceivable. As a second possibility, UMP and CMP may bind together with a water molecule. This agrees with the following observations: exchanging AMP for UMP in our model provides space for a water molecule bound deep in the pocket as sketched in Figure 8. This water molecule can form favorable hydrogen bonds to uracil-N3, Thr81-OG1, Val76-O and the carboxamide ˚ ). Water needs of Gln111 (all distances around 2.9 A somewhat more space than the volume difference between adenine and uracil (difference = two nonhydrogen atoms), which agrees well with the puzzling pocket enlargement for UMP binding. Moreover, residue Thr81, which participates in water binding, is a valine (64%) in most adenylate kinases. This valine excludes water and thus diminishes UMP and CMP binding. It should be noted that the model suggests a 180° rotation of the Gln111 carboxamide on binding UMP in its dominant tautomeric form (keto form, inset of Figure 8), but no rotation on binding the enol tautomer, which has an abundance of 0.01% in a physiological environment (Katritzky & Waring, 1962). The carboxamide rotation would break the hydrogen bond to Arg107-N (Figure 8), while the

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Ligated Structures of Uridylate Kinase

tautomer shift would require energies of similar magnitude. Further experiments are needed before a decision can be made. Modeling uracil coplanar to adenine causes a bad contact between its O4a-atom ˚ ), which, however, can and Gly104-O (distance 2.3 A be relaxed by a displacement towards Ala47. This relaxation is prevented by the equivalent threonine in the adenylate kinases. In conclusion, we find that all the presented arguments agree with the suggestion that the NMP site of UKyst accommodates either UMP or CMP together with a water molecule or AMP without one. This is an example of a nucleotide binding pocket with multiple specificity in spite of rather defined interactions.

Materials and Methods Crystallization and data collection The gene of UKyst was kindly provided by Drs Liljelund and Lacroute (Strasbourg). After creating new restriction sites by the polymerase chain reaction (PCR), the UKyst gene was cloned in a 750 bp fragment into vector pT7-7, which was then transformed into E. coli strain JM109-DE3 (plasmid and strain were a gift from H. Ko¨ssel, Freiburg) yielding a highly effective expression system (Hollender, 1990). After working out a purification procedure we obtained 200 mg pure protein per 10 l culture. Crystals were grown by the hanging drop method using drops of 15 ml and reservoirs of 1 ml. The drops contained 5 mg/ml UKyst , 10% (w/v) PEG-3350, 0.02% n-octyl-b-Dglucoside, 1.5 to 2.0 mM ATP or ADP and 50 mM potassium phosphate at pH 6.5. The reservoir contained 15% (w/v) PEG-3350 and 50 mM potassium phosphate at pH 6.5. Crystals grew within 6 to 8 weeks to 80 mm × 80 mm × 80 mm and were then used as seeds, giving rise to crystal sizes up to 1500 mm × 400 mm × 400 mm within 2 weeks. Since no storage buffer could be found, the crystals were kept in mother liquor. Cocrystallizing with ATP yielded the complex UKyst :ADP:ADP/AMP, which contained a mixture of ADP and AMP at the NMP site (Mu¨ller-Dieckmann & Schulz, 1994). Knowing these ligands we then cocrystallized UKyst with ADP. Thin-layer chromatography of these crystals showed equal amounts of ADP and AMP whereas the amount of ATP was below 5%. In agreement, the electron density showed an ADP at the ATP site and only an AMP at the NMP site. This crystal type was named UKyst :ADP:AMP. While the native data of UKyst :ADP:ADP/AMP were collected using a multi-wire detector (model X1000, Xentronics/Siemens) on a rotating anode (model RU200B, Rigaku), those of UKyst :ADP:AMP were obtained with an image plate at a synchrotron (beam line X11 at wavelength ˚ , EMBL outstation at DESY Hamburg). The new 0.92 A UKyst :ADP:AMP crystals have unit cell parameters of ˚ and c = 183.4 A ˚ as compared to 64.2 A ˚ and a = b = 63.4 A ˚ for the earlier ones. They are isomorphous in space 185.0 A group P61 22, but distinct. The unit cell differences may be partly due to inaccuracies in crystal–detector distances and/or wavelengths. Structure refinement The structure of UKyst :ADP:ADP/AMP has been ˚ elucidated by multiple isomorphous replacement at 3.0 A resolution and subjected to a preliminary refinement (Mu¨ller-Dieckmann & Schulz, 1994). The isomorphous

complex UKyst :ADP:AMP was derived from a differenceFourier map. Both complexes have now been refined to convergence by simulated annealing using the program package X-PLOR (Bru¨nger et al., 1987). After each round, the models were corrected manually on a graphics system (ESV station, Evans & Sutherland). Model building was based on (Fo − Fc ) and (2Fo − Fc ) electron density maps. After round 6, a complete model of UKyst :ADP:ADP/ AMP without water molecules yielded an R-factor of 24.6%. Water molecules were then introduced at sites where the (Fo − Fc ) map showed positive difference density above 3.5s. After each round, water molecules with densities below 1s in the (2Fo − Fc ) map or B-factors above ˚ 2 were deleted. The refinement was halted at an 55 A R-factor of 17.8% (Table 3). The complex UKyst :ADP:AMP was refined along the same lines using the final model of UKyst :ADP:ADP/AMP with water as the starting model. The refinement converged after seven rounds at an R-factor of 19.6% (Table 2).

Acknowledgements We thank Drs P. Liljelund and F. Lacroute for providing us with the gene for the enzyme and Dr Schiltz for discussions. Furthermore, we thank Dr K. S. Wilson and his team at the EMBL Outstation at DESY (Hamburg) for their help in data collection. The project was supported by the Land Baden-Wu¨rttemberg. The co-ordinates and structure factors have been deposited in the Protein Data Bank, Brookhaven, U.S.A. under the file names 1UKY and 1UKZ.

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Edited by R. Huber (Received 22 July 1994; accepted 25 November 1994)