Cosubstrate-induced dynamics of D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida Karthik S Paithankar1, Claudia Feller2, E Bartholomeus Kuettner1, Antje Keim1, Marlis Grunow2 and Norbert Strater1 ă Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Germany Institute of Biochemistry, Faculty of Biosciences, Pharmacy, and Psychology, University of Leipzig, Germany Keywords crystal structure; loop closure; protein dynamics; SDR Correspondence M Grunow, Institute of Biochemistry, Faculty of Biosciences, Pharmacy, and Psychology, University of Leipzig, Bruderstraòe 34, D-04103 Leipzig, Germany ă Fax: +49 341 9736998 Tel: +49 341 9736907 E-mail: gru@rz.uni-leipzig.de N Stra ăter, Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Deutscher Platz 5, D-04103 Leipzig, Germany Fax: +49 341 9731319 Tel: +49 341 9731311 E-mail: strater@bbz.uni-leipzig.de (Received July 2007, revised August 2007, accepted 10 September 2007) doi:10.1111/j.1742-4658.2007.06102.x D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida belongs to the family of short-chain dehydrogenases ⁄ reductases We have determined X-ray structures of the D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida, which was recombinantly expressed in Escherichia coli, in ˚ three different crystal forms to resolutions between 1.9 and 2.1 A The socalled substrate-binding loop (residues 187–210) was partially disordered in several subunits, in both the presence and absence of NAD+ However, in two subunits, this loop was completely defined in an open conformation in the apoenzyme and in a closed conformation in the complex structure with NAD+ Structural comparisons indicated that the loop moves as a rigid body by about 46° However, the two small a-helices (aFG1 and aFG2) of the loop also re-orientated slightly during the conformational change Probably, the interactions of Val185, Thr187 and Leu189 with the cosubstrate induced the conformational change A model of the binding mode of the substrate D-3-hydroxybutyrate indicated that the loop in the closed conformation, as a result of NAD+ binding, is positioned competent for catalysis Gln193 is the only residue of the substrate-binding loop that interacts directly with the substrate A translation, libration and screw (TLS) analysis of the rigid body movement of the loop in the crystal showed significant librational displacements, describing the coordinated movement of the substrate-binding loop in the crystal NAD+ binding increased the flexibility of the substrate-binding loop and shifted the equilibrium between the open and closed forms towards the closed form The finding that all NAD+-bound subunits are present in the closed form and all NAD+-free subunits in the open form indicates that the loop closure is induced by cosubstrate binding alone This mechanism may contribute to the sequential binding of cosubstrate followed by substrate Short-chain dehydrogenases ⁄ reductases (SDRs) constitute a large protein family that now includes more than 1000 enzymes in humans, mammals, insects and bacteria [1] The dehydrogenases act on a wide variety of substrates, including steroids, retinoids, prostaglandins, sugars and alcohols The name SDR is based on their smaller subunit size of about 250 residues compared with the medium-chain dehydrogenase ⁄ reductase family that has a subunit size of about 350 residues The SDR enzymes exhibit a sequence identity of 15–30% and share two signature motifs: a GxxxGxG motif involved in coenzyme binding; and a YxxxK Abbreviations PfHBDH, Pseudomonas fragi D-3-hydroxybutyrate dehydrogenase; PpHBDH, Pseudomonas putida D-3-hydroxybutyrate dehydrogenase; SDR, short-chain dehydrogenase ⁄ reductase FEBS Journal 274 (2007) 5767–5779 ª 2007 The Authors Journal compilation ª 2007 FEBS 5767 D-3-Hydroxybutyrate dehydrogenase K S Paithankar et al motif in the active site Crystal structures are now known for about 60 SDR members and it is now clear that SDRs are single-domain enzymes; by contrast, medium-chain dehydrogenases ⁄ reductases consist of a cosubstrate-binding domain and a substrate-binding domain [2,3] SDR enzymes exist as monomers, dimers or tetramers The tetrameric enzymes exhibit 222 point-group symmetry Conventionally, the three mutually perpendicular two-fold axes are named P, Q and R [4] One of the most variable parts of the different SDR enzymes is a loop consisting of two a-helices that protrudes out of the compact tetramer This so-called substrate-binding loop, which is located between b-strand F and a-helix G, is involved in recognition of the structurally different substrates The substrate-binding loop differs significantly in length and sequence and also adopts different conformations when comparing open or closed forms of different SDR enzymes It has also been shown in some crystal structures that the loop undergoes a conformational change upon substrate binding [4–6] Substrate-induced conformational changes from an open to a closed conformation, or from a disordered (conformationally flexible) to an ordered structure, have also been observed Nakamura et al recently demonstrated by X-ray crystallography and CD spectroscopy that coenzyme binding to 3a-hydroxysteroid dehydrogenase alone induces a transition of the loop from a disordered structure to a conformation consisting of two a helices [7] Spectroscopic studies on 17-b-hydroxysteroid dehydrogenase by fluorescence energy transfer also indicated that a conformational change might occur upon coenzyme binding [8] To the best of our knowledge, a coenzyme-induced conformational change to a closed conformation of the substrate-binding loop has, to date, not been analyzed by crystallographic means D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida (PpHBDH) (EC 1.1.1.30, GenBank accession number AJ310211.2) catalyzes the reversible and stereospecific oxidation of D-3-hydroxybutyrate to acetoacetate using NAD+ as a coenzyme One subunit contains 256 amino acids with a calculated molecular weight of  26.6 kDa [9] X-ray structures of the homologous enzyme from Pseudomonas fragi (PfHBDH) in the presence of NAD+ and inhibitor (Protein Data Bank accession code: 1X1T) and without cosubstrate (Protein Data Bank accession code: 1WMB) have been recently determined [10] Interestingly, in these structures the substrate-binding loop was ordered in the absence of NAD+ and disordered in the complex structure with bound NAD+ In addition, a crystal structure of a human cytosolic HBDH 5768 (DHRS6) with bound NAD+ is available [11] Based on homology modelling, substrate and inhibitor docking studies, and site-directed mutagenesis, residues Gln91, His141, Lys149, Tyr152 and Gln193 were found to be involved in substrate binding in PpHBDH [9] With the exception of Gln193, these residues are located in the deep active-site cleft The exact boundaries of the flexible loop differ among enzymes Based on the structure of PpHBDH, the loop runs from residue 187 to residue 210 We will refer to the rest of the one-domain protein as the catalytic subdomain In this study we determined the structure of PpHBDH using three different crystal forms The X-ray structures showed a completely ordered conformation of the substrate-binding loop in at least one subunit in the open and closed forms A comparison of these conformers, and an analysis of the mobility of the loop in the crystals, allowed a detailed description of the enzyme dynamic properties and conformational change during HBDH catalysis Results and Discussion Monomer and tetramer structure Three different crystal forms of PpHBDH were obtained in the presence or absence of NAD+ (Table 1) In crystal form I the asymmetric unit was found to contain one tetramer and two dimers (the tetrameric structure is generated by a crystallographic two-fold axis) In crystal form II the asymmetric unit was found to contain two dimers, and in crystal form III one tetramer was present in the asymmetric unit In crystal form I, two of the eight subunits (designated as chains A and B) contained no bound NAD+, as shown by the electron density maps In crystal form II, only subunit A was NAD+ free Therefore, crystal forms I and II contain tetramers with all four binding sites occupied and tetramers with two bound NAD+ molecules In crystal form III the enzyme was completely devoid of NAD+ cosubstrates The comparison of several independent subunits, with and without bound NAD+, allowed an analysis to be made of cosubstrate-induced movements within the enzyme, in particular of the substrate-binding loop Each subunit of HBDH has an a ⁄ b doubly wound structure with the characteristic dinucleotide-binding motif known as the Rossmann fold (Fig 1) The subunit structure was made of a core b-sheet composed of seven parallel b-strands (bA, bB, bC, bD, bE, bF and bG) buried between three a-helices (aB, aC and aG, FEBS Journal 274 (2007) 5767–5779 ª 2007 The Authors Journal compilation ª 2007 FEBS K S Paithankar et al D-3-Hydroxybutyrate dehydrogenase Table Crystal and refinement data of crystal forms I, II and III au, Asymmetric unit I Space group Unit-cell ˚ dimensions (A) b (°) ˚ Resolution (A)a Completeness (%)a Rsym (%)a I ⁄ dIa Redundancya Mosaicity (°) ˚ Wilson B factor (A2) Monomers ⁄ a.u Solvent content (%) R ⁄ Rfree (%) ˚ rmsd bonds (A) rmsd angles (°) No of water molecules ˚ < Bprotein > (A2) ˚ < Bwaters > (A2) ˚ < BNAD > (A2) II III C2 261.5 59.9 116.5 113.7 30–2.0 (2.09–2.02) 96.6 (74.4) 8.2 (59.2) 7.8 (1.0) 7.5 (4.8) 0.69 36.1 36.9 20.2 ⁄ 27.4 0.028 2.28 473 C2 115.3 58.2 119.4 92.3 30–1.9 (1.97–1.9) 99.5 (98.3) 4.8 (15.1) 13.1 (4.9) 11.3 (11.0) 0.71 18.6 34.1 16.8 ⁄ 21.8 0.016 1.66 447 C2 117.6 58.8 119.4 93.7 30–2.1 (2.2-2.12) 99.1 (94.4) 6.7 (36.9) 8.3 (2.1) 9.9 (7.8) 1.4 34.4 36.1 18.1 ⁄ 24.6 0.028 2.04 320 42.7 43.5 45.7 18.6 20.7 17.6 A 33.0 33.8 – B a The values given in parentheses refer to the highest resolution shell or aD, aE and aF) located on both sides of the b-sheet The substrate-binding loop consisted of two helices, designated aFG1 and aFG2 With the exception of the substrate-binding loop, the catalytic subdomains formed a compact, flat tetra˚ meric structure of dimensions 70 · 80 · 40 A along the Q, P and R axes, respectively Only the substratebinding loop protruded from the main body of the tetramer along the R axis (Fig 1) This loop was partially disordered in most subunits (Table 2) However, the loop was completely defined in subunit A of crystal form II and in subunit B of crystal form III, both in the absence of NAD+, as well as in subunit D of crystal form I in the presence of NAD+ C Fig Crystal structure of P putida D-3-hydroxybutyrate dehydrogenase (PpHBDH) (A) Fold of one subunit in the closed conformation The substrate-binding loop is colored red and the bound NAD+ is colored yellow (B) View of the tetramer structure along the R axis (blue) The P and Q axes are marked in red and green, respectively Shown are subunits A, B, C and D of crystal form I Only in subunits C and D (green) is an NAD+ molecule (yellow) bound In subunits A and B (blue) the coenzyme-binding site is not occupied The substrate-binding loops are depicted in red (C) View along the P axis FEBS Journal 274 (2007) 5767–5779 ª 2007 The Authors Journal compilation ª 2007 FEBS 5769 5770 ˚ The number given as ‘crystal contacts’ describes all interatomic distances smaller than 4.5 A between an atom of the substrate-binding loop and an atom of a symmetry related molecule a – 39.1 – 35.8 – 39.5 – 46.5 19.7 19.5 20.5 17.7 – 29 – 57.3 – 50.9 63.2 45.2 36.2 41.2 39.6 42.1 51.0 48.5 42.9 43.1 42.2 42.1 12.8 15.8 32.8 33.0 32.3 32.8 18.5 19.5 18.4 43.2 42.8 42.6 42.7 42.3 42.3 B (substrate-binding ˚ loop 186–212) (A2) B (catalytic subdomain residues 2–185 ˚ 213–256) (A2) ˚ B (NAD+) (A2) B (residues ˚ 88–89) (A2) 43.2 42.8 43.8 E + 2–198 205–256 41 D + 12 2–256 C + 2–194 207–256 47.2 B – 31 2–198 202–256 42.4 A – 38 2–197 203–256 42.8 Chain NAD+ Crystal contactsa Residues I Crystal form Table Overview of the subunit structures of the three crystal forms F + 2–189 207–256 43.2 G + 11 2–199 204–256 41.6 II 17.6 18.4 33.4 A – 70 2–197 200–256 34 K S Paithankar et al H + 2–188 204–256 44.5 A – 89 2–256 B + 21 2–199 202–256 18.8 C + 14 2–197 205–256 19.2 D + 21 2–197 206–256 19.3 III B – 196 2–256 C – 150 2–196 201–256 34.3 D – 110 2–195 203–256 32.13 D-3-Hydroxybutyrate dehydrogenase Comparison of the subunit structures Crystal form I Figure 2A shows a superposition of the eight subunits in crystal form I It demonstrates that subunits with and without NAD+ adopted different conformations for regions in the substrate-binding loop, helix aC and the residues Ala88 and Gly89 after b-strand D Those regions that showed conformational variability in the superposition also displayed significantly higher B factors (Fig 2B) The average B factor for the atoms of ˚ helix C was 47.1 A2 (average over all molecules) and ˚ its residues superimposed with an rmsd of 0.6 A, indicating an increased mobility of this helix Whereas the subunits without NAD+ had their substrate-binding loop in an open conformation, all subunits with NAD+ exhibited a closed conformation Interestingly, in subunit H (with bound NAD+), residues 186–189 corresponded to the open conformation of the substrate-binding loop, whereas residues 204–208 (at the end of the loop) were in a position that is similar to the position of this region in the closed conformation Residues 190–203 were disordered This finding indicated that the loop can also change to the open conformation in the presence of NAD+ Crystal form II A superposition of the subunits from crystal form II showed that the substrate-binding loop of subunit A (NAD+ free) is in an open conformation and completely ordered whereas the corresponding loops in the NAD+-complexed subunits B to D are in a closed conformation and partially disordered (Fig 2C) Besides this, the largest variability was seen again in helix aC and in the region after b-strand D Compared ˚ with the average B factor of 19.7 A2 for all four protein molecules, helix aC had a somewhat higher B fac˚ tor, of about 29 A2, also in this crystal form In the NAD+-free subunit A, residues 88 and 89 of the central b-strand had a conformation different from the NAD+-containing subunits, similar to the situation in ˚ subunit IB Both residues shifted up to A upon NAD+ binding Crystal form III In crystal form III (all subunits are NAD+ free), subunit B possessed a completely ordered substrate-binding loop, whereas this loop was partially disordered in the other subunits All four subunits in crystal form III were in an open conformation with respect to the substrate-binding loop (Fig 2D) In contrast to crystal FEBS Journal 274 (2007) 5767–5779 ª 2007 The Authors Journal compilation ª 2007 FEBS K S Paithankar et al forms I and II, residues 88 and 89 adopted a conformation similar to that observed in the subunit structures with bound NAD+ As in the other crystal forms, helix C showed a higher variability, with an ˚ ˚ rmsd of 0.4 A, and a higher B factor, of 46 A2, com˚ pared with the average B factor of 40.4 A D-3-Hydroxybutyrate dehydrogenase A Crystal packing interactions As in other crystal structures of HBDH enzymes, the exposed substrate-binding loop, which forms two faces on opposite sides of the HBDH tetramer (Fig 1), was involved in crystal contacts in almost all subunits (Table 2) Interestingly, only in the NAD+-bound subunits IIC and IID, where the loop is in a closed conformation, it was not involved in crystal contacts Furthermore, the loop was in a closed conformation in all subunits with bound NAD+ and in an open conformation in all cosubstrate-free subunits These findings suggest that the closed conformation of the loop is a result of cosubstrate binding B Comparison of PpHBDH with PfHBDH and DHRS6 The major differences between the PfHBDH and PpHBDH structures are in the substrate-binding loop, in residues of the central b-strand D and in helix aC (Fig 3) The substrate-binding loop in the cosubstratefree PfHBDH structure has an intermediate position between the open and closed forms of this loop in PpHBDH The loop is completely disordered in the structure of PfHBDH in complex with NAD+ The conformational change of residues 88 and 89 in the central b-strand of PpHBDH upon NAD+ binding was not observed in PfHBDH In both structures of the latter enzyme, the b strand was in exactly the same conformation as in the NAD+-bound form of PpHBDH In addition to these conformational differences, helix aC is four residues shorter in PpHBDH because of a deletion of residues Moreover, the substrate-binding loop of PpHBDH is one residue shorter that that of PfHBDH, because of a deletion at the end of helix aFG1 In human DHRS6, a cytosolic type human HBDH enzyme [11], the substrate-binding loop is present in a C D Fig Superposition of subunit structures (A) Crystal form I: A, green; B, black; C, yellow; D, red; E, magenta; F, blue; G, cyan; H, brown (B) Ca traces of the eight monomers of crystal form I col˚ ˚ ored by the B factor (from blue at B < 30 A2 to red at B > 60 A2) (C) Crystal form II: A, green; B, red; C, blue; D, black (D) Crystal form III: A, black; B, red; C, blue; D, green FEBS Journal 274 (2007) 5767–5779 ª 2007 The Authors Journal compilation ª 2007 FEBS 5771 D-3-Hydroxybutyrate dehydrogenase K S Paithankar et al Asn87 Ala88 Fig Comparison of P putida D-3-hydroxybutyrate dehydrogenase (PpHBDH) (open form green, closed form red), Pseudomonas fragi D-3-hydroxybutyrate dehydrogenase (PfHBDH) (open form blue) and DHRS6 (black) The structure of the NAD+-bound form of PfHBDH (not shown) with the disordered substrate-binding loop superimposes closely with the open form closed conformation (Fig 3) A sulfate molecule from the crystallization solution is bound to the active site at the presumed binding site for the substrate The loop closure may be caused by the sulfate ion, as discussed by the authors, and also by interactions with NAD+, as described below The substrate-binding loop of DHRS6 is six residues shorter, as in the Pseudomonas HBDH enzymes Consequently, there is a shortening of helix aFG1 and a slight relocation of helix aFG2 NAD+ binding The conformation of NAD+ and the structure of the cosubstrate-binding pocket of PpHBDH are very similar to those of PfHBDH, with a distance of about ˚ 14 A between the C2 of nicotinamide and the C6 of the adenine ring As indicated in the superpositions of Figs and 3, the main difference is the displacement ˚ of Ala88 and Gly89 by about A in the NAD+-free subunits IB (chain B of crystal form I) and IIA The B ˚ value for these residues is around 10 A2 higher than the average B factor of the subunit In the subunits with bound NAD+, the B factor of the two residues is comparable with the average B value of the subunit Nevertheless, the conformation of residues 88 and 89 is clearly defined in all electron density maps The main chain torsion angles of residues 87–90 all correspond to allowed regions of the Ramachandran plot In crystal form III, residues 88 and 89 adopted a conformation similar to that of the NAD+-bound subunits in crystal forms I and II In this crystal form, the ˚ B factor of residues 88 and 89 was 5–10 A2 higher than the average B value of the corresponding subunit 5772 Gly89 Gln91 lle90 Fig Conformational change of residues 87–91 upon NAD+ binding The carbon atoms are colored grey in the conformation in the presence of NAD+ (yellow) and green in the NAD+-free subunit A of crystal form I The two residues are thus more mobile than in the presence of NAD+, but less than in the alternate conformations observed in subunits IB and IIA NAD+ interacts, via a hydrogen bond of one of its ribose hydroxyl oxygens, with the peptide carbonyl group of Asn87 (Fig 4) The loss of this interaction may be an important factor for the change of main chain conformation of this residue in the absence of NAD+ The present data thus indicate that residues 87–90 have a higher mobility in the absence of bound cosubstrate (as indicated by higher B factors) and they can adopt alternative conformations Translation, libration and screw (TLS) refinement At the end of the refinement procedure, anisotropic displacement parameters were determined by a TLS refinement [12,13] Translation (T) and libration (L) tensors describe the anisotropic motion of groups in the crystal Besides improving the fit of the model to the observed data, the TLS tensors may allow a description of correlated motions in the crystal It must be stressed, however, that a fit of the TLS model to the observed structure factor amplitudes implies nothing about the relative phases of the atomic displacements within the group [13] FEBS Journal 274 (2007) 5767–5779 ª 2007 The Authors Journal compilation ª 2007 FEBS K S Paithankar et al All three crystal forms were subject to TLS refinement with the substrate-binding loop region (186–212) and the catalytic domain (2–185, 213–256) in each subunit as two distinct TLS groups By TLS refinement, the R factors (Rfree factors) improved by 2.2% (3.3%), 0.7% (1.0%) and 1.7% (2.9%) for crystal forms I, II and III, respectively The total B factor (Btotal) of each atom is the sum of the TLS contribution (BTLS, from the rigid body motion) and the residual B factor (Bresidual, the individual mobility independent of the rigid bodies) The residual B factors of the substrate-binding loop were relatively constant and had values similar to those of other regions (data shown in the supplementary Fig S1) The significantly higher B factors of the substrate-binding loop are predominantly caused by a rigid body motion of the loop For all crystal forms, a significant librational movement is present for the substrate-binding loop (data shown in the supplementary Table S1) Furthermore, the libration is quite anisotropic in nature (Fig 5) How does the anisotropic librational motion of the substrate-binding loop compare with the closing motion of this loop? A superposition of the main axis Fig Principal axis (green) of the libration tensor of the substratebinding loop of subunit IIIA The anisotropic movement of the Ca atoms, as derived from the TLS tensors, is depicted by thermal ellipsoids for the loop (red) and for the catalytic subdomain (blue) For orientation, a superimposed NAD+ molecule is shown in yellow, although it is not bound in this crystal form D-3-Hydroxybutyrate dehydrogenase of the L tensors for all subunits showed that all L tensors roughly describe a similar libration movement in which the main rotation component is an axis approximately parallel to the two helices of the loop (Fig 5, superposition not shown) The second largest rotational component describes the closure motion of the loop Movement of the substrate-binding loop In order to characterize the nature of the movement of the substrate-binding loop, the structures of open and closed forms were compared using program dyndom to determine dynamic regions that move as pseudorigid structures, termed ‘dynamic domains’ (Fig 6) For stretches of five amino acid residues, the rotational movement between two enzyme conformations was analyzed Residues that belong to one rigid body show a similar rotation in the superposition and thus form a cluster, as shown in Fig 6B The analysis revealed two dynamic domains: residues 4–183 and 212–254 form one domain; and residues 187–210 of the substratebinding loop form the other domain However, within the substrate-binding loop, three subclusters were defined: residues 187–199 of helix aFG1; residues 204– 210 of helix aFG2; and residues 200–203 of the loop connecting the two helices Thus, the substrate-binding loop moves largely as a rigid body; however, the internal structure of the loop changes slightly by a re-orientation of the two helices Residues 184–186 and 211– 213 are the bending residues that connect the two dynamic domains (Fig 6) The substrate-binding loops in the open and closed ˚ conformations superimposed with an rmsd of 1.4 A The movement corresponded to a rotation by 46° with ˚ a small translational component of 0.06 A It is typical for hinge-bending movements that the rotation axis passes near the bending residues, which thus act as a mechanical hinge (Fig 6) [14] An analysis of the main chain conformations in the different loop structures showed that several small changes of main-chain torsion angles of the bending residues allowed the movement of the substrate-binding loop, but no large changes of the main chain conformation were observed (data not shown) A movement of the substrate-binding loop upon cosubstrate binding has not been observed before for other SDR enzymes Only for human estrogenic 17b-hydroxysteroid dehydrogenase [5,15], Drosophila lebanonensis alcohol dehydrogenase [16], Datura stramonium tropinone reductase [17] and Escherichia coli b-keto acyl carrier protein reductase [18,19] have structures of the apoenzyme and of the binary complex FEBS Journal 274 (2007) 5767–5779 ª 2007 The Authors Journal compilation ª 2007 FEBS 5773 D-3-Hydroxybutyrate dehydrogenase K S Paithankar et al 200 200 A 203 203 213 211 184 B 211 213 186 186 184 201 200 202 203 205 206 207 208 209 210 204 211 189 194 195 193 190 196 191 188 192 197 187 199 198 186 212 185 213 184 with cosubstrate been determined, without significant differences in the position of the substrate-binding loops In the crystal structure of 3a-hydroxysteroid dehydrogenase ⁄ carbonyl reductase from Comamonas testosteroni the substrate-binding loop is largely disordered in the absence and presence of bound NAD+[20] There are, however, crystal structures of SDR enzymes in the closed form available for binary complexes with cosubstrate, such as the structure of 5774 Fig Dynamic domains of P putida D-3hydroxybutyrate dehydrogenase (PpHBDH) on the basis of a comparison of conformers ID and IIA, which contain a fully defined substrate-binding loop (A) The two dynamic domains are colored blue and red for the fold of conformer ID (closed) and the bending residues are shown in green (B) Clustering of the rotational movements of stretches of five residues, on which the assignment of the domains moving as rigid bodies is based in program DYNDOM [31] Each sphere represents the rotation vector of a five-residue stretch For the substratebinding loop and the bending residues, the rotation vectors are labeled according to the residue in the center of the stretch human DHRS6 in complex with NAD [11] A structure of the DHRS6 apoenzyme is not yet available Residues inducing closure movement In an analysis of ligand-induced domain movements in other proteins, it was shown that a small number of residues from the closing domain interact with the ligand bound to the binding domain in the open FEBS Journal 274 (2007) 5767–5779 ª 2007 The Authors Journal compilation ª 2007 FEBS K S Paithankar et al D-3-Hydroxybutyrate dehydrogenase conformation to initiate and drive domain closure [21] In this model, specific interactions of the ligand with residues on the coenzyme-binding domain produce a torque about the hinge axis driving the domain closure Residues are assumed to induce closure movement if they satisfy specific conditions These residues are usually located in the bending regions or in the closing domain Additionally, thermal motions may also contribute to the closure movement A large number of interactions are involved in the binding of NAD+ to the catalytic subdomain of PpHBDH However, there are only a few interactions of the substrate-binding loop with NAD+ that might cause the conformational change A crucial residue for the coenzyme-induced conformational change might be Thr187, which forms hydrogen bonds to the nicotinamide NH2 and to a phosphate oxygen of NAD+ via its hydroxyl group (Fig 7) In the absence of NAD+, ˚ the alcoholic oxygen is displaced by about 1.5 A, such ˚ distant from the position of its putative that it is  A hydrogen-bonding partners This interaction might trigger the closure motion of the substrate-binding loop in the presence of NAD+ because Thr187 is located just behind the bending residues 184–186 and the torque produced by the interaction of Thr187 with NAD+ drives or supports the loop movement about the hinge residues A further, nonpolar interaction of the substrate-binding loop with the coenzyme is mediated by the side chain of Leu189, which makes hydrophobic contacts with the ribose group and nicotinamide ring of NAD+ in the closed conformation but is faced towards the solvent in the open conformation (Fig 7) Both residues are conserved in the bacterial HBDHs as part of a ‘TPLV’ motif Also, the interaction of the main chain NH and CO groups of Val185 might contribute to the conformational change by binding to the NAD+ nicotinamide group There are no further interactions that might explain the conformational change of the substrate-binding loop upon NAD+ binding The rest of the substrate-binding loop is also quite diverged, with the exception of Gln193 In the human enzyme DHRS6, Thr187 is conserved and makes a polar contact to NAD+, as in the bacterial enzyme Leu189 is replaced by a serine, which is in hydrogen-bonding distance to a phosphate oxygen atom of NAD+ and may thus also be involved in the induction of closure movement via cosubstrate binding The interaction of Val186 (Val185 in PpHBDH) and Thr188 (Thr187) of 3a-hydroxysteroid dehydrogenase with the cofactor has also been discussed to stabilize the substrate-binding loop in the loop–helix transition observed in this enzyme [7] Substrate binding A model for the binding mode of the substrate D-3-hydroxybutyrate to HBDH has been suggested based on a homology model of PpHBDH and molecular modelling techniques [9] In this model, the side chain of Gln193 belonging to the substrate-binding loop forms hydrogen bonds to the carboxylate group of the substrate Figure shows the modelled substrate in the Lys149 Gln193 Gln91 His141 Tyr152 Leu189 Leu189 Thr187 Val185 Fig Interactions of NAD+ with P putida D-3-hydroxybutyrate dehydrogenase (PpHBDH), which might drive a cosubstrate-induced conformational change of the substrate-binding loop The closed conformation is depicted in green and the open conformation in grey Also shown is the rotation axis in blue Fig A model for the binding mode of substrate D-3-hydroxybutyrate (cyan) to P putida D-3-hydroxybutyrate dehydrogenase (PpHBDH) Shown are the enzyme conformations in the open (green) and closed (red) forms, and selected residues, as discussed in the main text The cosubstrate NAD+ is shown with yellow carbon atoms Hydrogen bonds are shown as dashed lines, and the red dashed line between the nicotinamide C4 atom and the substrate carbon atom bound to the substrate alcohol group marks the distance between the reactive centers FEBS Journal 274 (2007) 5767–5779 ª 2007 The Authors Journal compilation ª 2007 FEBS 5775 D-3-Hydroxybutyrate dehydrogenase K S Paithankar et al crystal structure of PpHBDH in the closed form The side chain of Gln193 was shown to contribute significantly to substrate binding because the Km value increased from 0.6 mm for the wild-type enzyme to about 70 mm in a Gln193Ala mutant, whereas the kcat value decreased from 432Ỉs)1 in the wild-type enzyme to 215Ỉs)1 in the same mutant Gln193 was in hydrogen-bonding distance to the carboxylate group of the substrate in the closed conformation of the substratebinding loop In all subunits where this residue was defined in subunits with bound NAD+ (D, E and G of crystal form II as well as B and C of crystal form II), this residue was positioned to interact with the substrate This finding indicates that the loop is indeed in a position competent for catalysis upon NAD+ binding, even in the absence of the bound substrate Gln193 is the only residue of the substrate-binding loop that forms polar interactions with the substrate In addition, the side chain of Leu189 has hydrophobic contacts to the methyl group of the substrate model Further polar interactions to the substrate are formed by Lys149, Gln91, His141 and Tyr152 Of these residues, the important function for substrate binding has been demonstrated for a Gln91Ala mutant with Km 51 mm and kcat 411Ỉs)1 and for a Lys149Ala mutant that was essentially inactive [9] His141 appears to be also important for efficient catalysis because in a His141Ala mutant the Km increased to only mm, whereas the kcat decreased significantly to 13Ỉs)1 Tyr152 is known to be a core catalytic residue It is assumed to be present as a tyrosinate and to accept a proton from the alcohol group in order to facilitate H– transfer to NAD+ Our crystallographic study confirmed the repositioning of the substrate-binding loop in the closed conformation, as obtained from a molecular dynamics simulation of a PpHBDH model [9] A superposition of the active-site structures of PpHBDH and DHRS6 showed that the human enzyme developed a different environment for substrate binding: His141 was replaced by an alanine, Lys149 by an arginine, Gln91 by a valine and Gln193 (from the substrate-binding loop) by an arginine (data not shown) These replacements might account for the significant differences in the kinetic data of both enzymes Figure shows the molecular surface of HBDH in the open conformation, together with the model for the substrate binding mode The NAD+ coenzyme taken from the structures of PpHBDH in the closed form was included in the calculation of the surface Thus, the surface represented the protein in a state where NAD+ has just bound, but the loop is still in the open conformation The substrate was bound in a deep groove formed between the substrate-binding 5776 Fig Molecular surface of P putida D-3-hydroxybutyrate dehydrogenase (PpHBDH) in the open conformation (conformer IIA) colored by the electrostatic potential Positive potential is depicted in blue and negative potential in red Also shown are the model for substrate binding in green and the substrate-binding loop conformation in the closed form in yellow loop and the catalytic subdomain The carboxylate group of the substrate was located at a region with positive potential, which is mainly caused by Lys149 Also shown is the substrate-binding loop in the closed conformation In particular, residues Leu189 and Gln193 would block the entrance to the substratebinding pocket in the closed conformation A molecular surface drawn for the enzyme in the closed form revealed no access to the buried substrate-binding pocket (data not shown) Kinetic studies demonstrated that HBDH, similarly to other NAD+-dependent dehydrogenases, has an ordered sequential binding mechanism of cosubstrate binding followed by substrate binding (M Grunow, unpublished results) Therefore, although the binding of the coenzyme obviously induces a change of the enzyme to the closed form, the loop must exist in an equilibrium with the open form to enable binding of the substrate and release of the products This flexibility is demonstrated by a partial disorder of the substrate-binding loop in some subunits of NAD+-bound subunits of PpHBDH and also by the strong disorder of the loop in the NAD+– PfHBDH complex [10] In conclusion, the crystallographic analysis of PpHBDH in different crystal forms and in the presence and absence of bound NAD+ showed that the presence of the cosubstrate alone is able to induce a conformation of the substrate-binding loop that is competent for catalysis Such a conformational change of the substrate-binding loop has not been observed in previous crystallographic studies on other SDR enzymes Our results are in agreement with the FEBS Journal 274 (2007) 5767–5779 ª 2007 The Authors Journal compilation ª 2007 FEBS K S Paithankar et al role of the cosubstrate in promoting a loop–helix transition in 3a-hydroxysteroid dehydrogenase [7] A cosubstrate-induced conformational change is also in agreement with spectroscopic studies [8] These studies demonstrate an important contribution of cosubstrate binding in shifting the equilibrium between disordered and ordered, as well as open and closed, conformations of the substrate-binding loop Thus, the equilibrium is progressively shifted towards the closed conformation – initially upon cosubstrate binding and then by substrate binding Cosubstrate and substrate have interactions with the substrate-binding loop in the closed conformation It is possible that the shift towards the closed conformation induced by cosubstrate binding is necessary for substrate binding and ensures the sequential binding mechanism (i.e that substrate binding does not occur before cosubstrate binding) As outlined above, the current data strongly indicate that the conformational change is a result of cosubstrate binding and that it is not a result of crystal packing interactions The nature of the conformational change could be characterized in detail by a comparison of the different loop conformers and by an analysis of the loop mobility in the crystal In first approximation, the loop moves as a rigid body, with minor re-arrangements of the relative orientation of the two helices (aFG1 and aFG2) Thus, NAD+ binding increases the flexibility of the substrate-binding loop and shifts the equilibrium between the open and closed forms towards the closed form In all subunits the loop is in either the open or the closed conformation; no intermediate conformations have been observed Information on substrate binding is currently based on modeling of the substrate binding mode; however, it may be possible that the interactions between the enzyme and the substrate or analogues inhibitors can be studied by cocrystal structures in further studies D-3-Hydroxybutyrate dehydrogenase 10 mm acetoacetate Acetoacetate was added in order to study substrate binding However, under the specified conditions it did not bind to the enzyme The same crystals were also obtained in the absence of acetoacetate under identical conditions The presence of the substrate in the crystallization buffer had no influence on the refined structure, in particular concerning the position of the substrate-binding loop We present the structure refined from crystals obtained in the presence of acetoacetate because the best data were obtained from these crystals Many crystals of PpHBDH suffered from a significant orientational disorder of the PpHBDH molecules in the crystals, which resulted in high Wilson B factors and poor density This phenomenon was independent of the presence of acetoacetate For crystal forms I and II, mm NAD+ was added Crystals appeared within a couple of days The data sets of crystal forms I and II, used for refinement of the structures presented here, were in fact obtained from two fragments of the same crystal that broke apart upon transfer into the cryobuffer The smaller fragment belonged to crystal form I and the larger fragment to crystal form II Crystals for which data were collected at room temperature (22 °C) belonged to crystal form II Thus, crystal form I is probably the result of a phase change upon crystal cooling Data collection Crystals of size 400 · 150 · 150 lm were cryoprotected in the crystallization buffer, which included 15% (v ⁄ v) glycerol, and flash-frozen in a N2 stream at 100 K in a nylon loop The intensity data were collected using MAR345 image plate detectors (Mar Research Inc., Norderstedt, Germany) mounted to an Rigaku RU-H3R rotating anode generator (Rigaku Corp., Tokyo, Japan) or a Bruker FR591 Microstar generator (Bruker AXS, Delft, the Netherlands) Data processing and reduction was carried out using the hkl software, version 1.97.9 (HKL Research Inc., Charlottesville, VA, USA) [22] All crystals belonged to monoclinic space group C2 Details of data collection and refinement are listed in Table Experimental procedures Enzyme purification and crystallization Recombinant PpHBDH was expressed in E coli XL1Blue strain and purified by charge-controlled hydrophobic chromatography, as described previously [9] Before crystallization, PpHBDH was further purified by Sephadex G-100 size-exclusion chromatography (GE Healthcare Bio-Sciences, Uppsala, Sweden) For crystallization using the hanging drop vapor diffusion method, lL of PpHBDH (10 mgỈmL)1) was mixed with an equal volume of crystallization buffer containing 17–20% polyethylene glycol 1500, 0.1 m Tris-HCl, pH 7.1, 0.2 mm CaCl2 and Structure determination and refinement The phase problem was solved by molecular replacement with molrep [23] using the PfHBDH structure (Protein Data Bank accession code: 1WMB) as a search model Model building was performed using o [24] and crystallographic refinement with refmac [25] Before refinement of ˚ the TLS parameters, the B factors were set to 20 A2 The program tlsanl was used to calculate the principal axes of the translation and libration tensors The quality of the models was assessed with Ramachandran plots using the program procheck [26] FEBS Journal 274 (2007) 5767–5779 ª 2007 The Authors Journal compilation ª 2007 FEBS 5777 D-3-Hydroxybutyrate dehydrogenase K S Paithankar et al Structure analysis and generation of figures Electrostatic potential maps were calculated by solution of the Poisson–Boltzmann equation in a continuum electrostatic model, as implemented in the program delphi [27] A ˚ probe radius of 1.4 A was used, and the dielectric constant was set to for the protein region and to 80 for the solvent Full charges of Asp, Glu, Lys and Arg were used, in addition to the charges of the terminal amino acids The molecular surface was generated with the program msms [28] The molecular figures were generated with programs molscript [29] and raster3d [30] Protein Data Bank accession codes The crystallographic models are available from the RCSB Protein Data Bank under the accession codes 2Q2Q (crystal form I), 2Q2V (crystal form II) and 2Q2W (crystal form III) Acknowledgements The Deutsche Forschungsgemeinschaft is acknowledged for funding to MG and NS The BESSY synchrotron in Berlin, Germany, is acknowledged for beamtime at the PSF beamlines References Tanaka N, Nonaka T, Nakamura KT & Hara A (2001) SDR: structure, mechanism of action, and substrate recognition Curr Org Chem 5, 89–111 Jornvall H, Persson B, Krook M, Atrian S, GonzalezDuarte R, Jeffery J & Ghosh D (1995) Short-chain dehydrogenases ⁄ reductases (SDR) Biochemistry 34, 6003–6013 Oppermann U, Filling C, Hult M, Shafqat N, Wu X, Lindh M, Shafqat J, Nordling E, Kallberg Y, Persson B et al (2003) Short-chain dehydrogenases ⁄ reductases (SDR): the 2002 update Chem Biol Interact 143–144, 247–253 Ghosh D, Weeks CM, Grochulski P, Duax WL, Erman M, Rimsay RL & Orr JC (1991) Three-dimensional structure of holo alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family Proc Natl Acad Sci USA 88, 10064–10068 Ghosh D, Pletnev VZ, Zhu DW, Wawrzak Z, Duax WL, Pangborn W, Labrie F & Lin SX 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Maser E, Mobus E, Klebe G, Reuter K & Ficner R (2000) The crystal structure of 3alphahydroxysteroid dehydrogenase ⁄ carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase ⁄ reductase family J Biol Chem 275, 41333–41339 21 Hayward S (2004) Identification of specific interactions that drive ligand-induced closure in five enzymes with classic domain movements J Mol Biol 339, 1001– 1021 22 Otwinowski Z & Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode Methods Enzymol 276, 307–326 23 Vagin A & Teplyakov A (2000) An approach to multicopy search in molecular replacement Acta Crystallogr D Biol Crystallogr 56, 1622–1624 24 Jones TA, Zou JY, Cowan SW & Kjeldgaard M (1991) Improved methods for building protein models in electron density maps and the location of errors in these models Acta Crystallogr A 47, 110–119 25 Murshudov GN, Vagin AA & Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method Acta Crystallogr D Biol Crystallogr 53, 240–255 26 Laskowski RA, MacArthur MW & Thornton JM (1998) Validation of protein models derived from experiment Curr Opin Struct Biol 8, 631–639 27 Gilson MK, Sharp KA & Honig BH (1987) Calculating the electrostatic potential of molecules in solution: method and error assessment J Comput Chem 9, 327– 335 28 Sanner MF, Olson AJ & Spehner JC (1996) Reduced surface: an efficient way to compute molecular surfaces Biopolymers 38, 305–320 D-3-Hydroxybutyrate dehydrogenase 29 Kraulis PJ (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures J Appl Crystallogr 24, 946–950 30 Merritt EA & Murphy ME (1994) Raster3d Version 2.0 A program for photorealistic molecular graphics Acta Crystallogr D Biol Crystallogr 50, 869–873 31 Hayward S & Lee RA (2002) Improvements in the analysis of domain motions in proteins from conformational change: DynDom version 1.50 J Mol Graph Model 21, 181–183 Supplementary material The following supplementary material is available online: Fig S1 B factor plot for the residues of subunit IIIA Shown is the total B factor and its contribution from the rigid body movements (BTLS, obtained from a refinement of translation, libration and screw tensors) and from the individual atom or residue movement (Bresidual) Table S1 Magnitudes of the translation and libration tensors for the catalytic domain and the substratebinding loop of the different subunits in crystal forms I to III For the libration tensor of the substrate-binding loop the eigenvalues of the tensor are listed and the mean value shown in brackets This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 5767–5779 ª 2007 The Authors Journal compilation ª 2007 FEBS 5779 ... A of crystal form II and in subunit B of crystal form III, both in the absence of NAD+, as well as in subunit D of crystal form I in the presence of NAD+ C Fig Crystal structure of P putida D-3-hydroxybutyrate. .. dehydrogenase from Pseudomonas putida Chembiochem 7, 1410–1418 10 Ito K, Nakajima Y, Ichihara E, Ogawa K, Katayama N, Nakashima K & Yoshimoto T (2006) D-3-hydroxybutyrate dehydrogenase from Pseudomonas. .. FEBS 5771 D-3-Hydroxybutyrate dehydrogenase K S Paithankar et al Asn87 Ala88 Fig Comparison of P putida D-3-hydroxybutyrate dehydrogenase (PpHBDH) (open form green, closed form red), Pseudomonas