Crystal structures of the Mycobacterium tuberculosis secretory antigen alanine dehydrogenase (Rv2780) in apo and ternary complex forms captures “open” and “closed” enzyme conformations

Structural work has demonstrated that the enzyme comprises of a NAD binding domain made up largely of the C-terminus residues and a catalytic domain which binds to pyruvate. Both domains are separated by a cleft. Bacterial Ald exhibit larger sequence divergence compared with other aminoacid dehydrogenases including phenylalanine dehydrogenase,11 leucine dehydrogenase12 and glutamate dehydrogenase.13 Earlier work involving kinetics has resulted in the understanding of several aspects of the enzyme mechanism. These studies have suggested that the reaction is ordered with NAD+ binding before alanine in the forward reaction involving oxidative deamination while NADH binds first in the reverse reaction involving reductive amination.14-16 However the order of release/binding of pyruvate and ammonia apparently varies between the enzymes from different species.14, 15 Work on the B. subtilis enzyme suggested that imino acid and carbinolamine intermediates are formed during the catalytic cycle14 (Scheme 1). The P. lapideum Ald structure in the apo and binary complexes with NAD and pyruvate revealed conformational changes in certain loops upon NAD binding but the comparative analysis between the structures led the authors to propose that either an alteration in intersubunit interactions or a domain rearrangement should take place to allow the pyruvate and NAD molecules to come close to initiate the hydride transfer step.17 The authors were however unable to structurally characterize a ternary complex in the enzyme. Reaction mechanism of L-alanine dehydrogenase adapted from Refs.14 and15. Alanine reacts as a monoanion and a hydride transfer takes place between Cα of alanine and C4 of the nicotinamide ring resulting in an iminopyruvate intermediate. The next step involves attack by a water molecule in the presence of the catalytic histidine (base) to form a carbinolamine intermediate. Proton removal from the hydroxyl group of the carbinolamine results in elimination of ammonia and formation of pyruvate. We report here the crystal structures of the apo enzyme as also a ternary complex with NAD and pyruvate. Each chain of the enzyme can be divided into "catalytic" and "NAD binding" domains, respectively. The individual subunits associate into a hexamer with the catalytic domains on the outside as observed in the apo and holo enzyme crystals. The structures have captured "open" and "closed" conformations of the enzyme and clarify that a domain rearrangement step must take place during the reaction as opposed to alterations in intersubunit interactions. The clear electron density maps for the bound pyruvate and NAD allows for the characterization of their interactions with the enzyme. The structural analysis has led to the identification of a water molecule which is hydrogen bonded to the active site His 96 and probably drives the conversion of the iminopyruvate intermediate to a carbinolamine. The reported work clarifies several aspects of the reaction and can serve as the blueprint for structure aided inhibitor design to exploit its potential as a novel anti-TB target. Ald gene (Rv2780) was amplified from Mycobacterium tuberculosis H37Rv genomic DNA by standard PCR techniques and cloned in pET23a between NheI and HindIII restriction sites. A detailed report on over expression, purification, and crystallization of the unliganded form of the enzyme has been submitted elsewhere (manuscript submitted). Briefly, IPTG-induced cells were harvested and resuspended in 50 mM Tris-HCl, pH 7.2, 300 mM NaCl, and 10 mM imidazole (Buffer A) and sonicated. A Ni++-IDA column was used in affinity chromatography to purify the protein as per the manufacturer's recommendation. Protein fractions were pooled, precipitated using ammonium sulfate (60%) and resuspended in 50 mM Tris-HCl pH 7.2, 50 mM NaCl, 5 mM EDTA, 2 mM β-mercaptoethanol (Buffer B). After gel filtration using Superdex S-200 (GE healthscience) column, protein was concentrated to 15 mg/mL as determined using the Bradford reagent18 with BSA as standard. Protein remained stable at 4°C without degradation up to 1 week. Purity of the protein was confirmed using 12% SDS-PAGE. Photometric determination of enzyme activity was accomplished by measuring the rate of the production of NADH at 340 nm that accompanies the conversion of L-alanine into pyruvate in the oxidative deamination. The hanging drop method was used for crystallizations and the plates were incubated at 22°C. MtbALD native crystals were obtained by mixing 2 μL of 10–12 mg/mL protein and 1 μL of reservoir solution containing ammonium sulfate ranging from 0.8 to 1.6M. Crystals of the complex were obtained under similar conditions by supplementing the crystallization drop with 10 mM pyruvate and 5 mM NAD. Diffraction data for native and ternary complex were collected using a Rigaku MICROMAX-007 X-ray generator coupled to MAR345-dtb image plate detector. The Diffraction images were indexed integrated and scaled using the HKL-2000 package.19 Amino acid sequence alignment showed 45% identity with the Phormadium lapideum L-alanine dehydrogenase17 (PDB; 1PJB). Therefore, a search model derived from this structure was used in the molecular replacement calculations using the program AMoRe20 implemented in the CCP4 package.21 The best solution gave a correlation coefficient of 56.2% and R-factor 47.8% using data between 15 and 3 Å. The transformed model was subjected to rigid body refinement using REFMAC5.22 The phases were improved and extended to 2.6 Å incrementally. The 2Fo − Fc and Fo − Fc electron density maps were visualized using the program TURBO-FRODO23 and COOT24 which was used for model building. The crystallographic R-factor and R-free were monitored at each stage to avoid any bias. Water molecules were added by the automatic water-picking algorithm of COOT24 and inspected manually. A few additional waters were manually identified on the basis of electron density maps. The loop regions between residues 234–245 in each subunit exhibit relatively weaker electron density in the maps. The refined model of MtbALD contains six chains and 224 water molecules. The final refinement statistics are given in Table I. The structure of the ternary complex was solved by using PHASER,25 searching for three copies of each domain based on the structure of the apo enzyme and was refined using similar procedures described above. The final model contains three chains in the asymmetric unit and 159 water molecules. Bound NAD and pyruvate are present in all the subunits. The geometry of the refined models was checked using PROCHECK26 and WHAT-IF.27 All the structural superimposition and r.m.s.d. calculations were carried out using the programs ALIGN28 and PROFIT (http://www.bioinf.org.uk/software/profit). The most favored and additionally allowed regions of the Ramachandran plot contained more than 90% of nonglycine and nonproline residues. B-factor analysis was carried out by using the BAVERAGE program from CCP4 package.21 Interactions were calculated using program CONTACTS module from CCP4 package. NACCESS29 was used for surface area calculations. The program DynDom30 was used to analyze the domain motions. The program identifies dynamic domains by clusters of rotation vectors corresponding to main-chain segments, which may be composed of several regions moving in a concerted fashion without necessarily forming a globular unit. This program was used to identify fixed and moving domains and hinge residues at the transition points between the two domains. Figures were prepared using the program CCP4MG,31 InsightII,32 and Pymol.33 Binding of NAD and NADH was monitored by following the change in tyrosine fluorescence upon ligand binding. Measurements were made on a Perkin–Elmer flourimeter (λexcitation = 276 nm, λemission = 309 nm). Titrations were performed at 25°C by addition of NAD (500 mM stock) or NADH (10 mM stock) to 0.6 mL of 50 mM Tris (7.2), 50 mM NaCl, 5 mM EDTA, and 2 mM β-mercaptoethanol containing 100 μg protein. To avoid dilution effects, volume change during titration was limited to only 3% of the total volume. The coordinates and structure factors of the apo enzyme and its ternary complex have been deposited in the Protein Data Bank (http://www.rcsb.org) with accession numbers 2VOE and 2VOJ, respectively. Each subunit of MtbALD consists of 371 residues and the purified enzyme ran as a 40 kD protein consistently in SDS-PAGE gels. Its oligomeric state from size exclusion chromatography was found to be hexameric as observed in many members of this family of enzymes as also reported earlier.34, 35 Binding of NAD and NADH to MtbALD was probed by fluorescence titration assays. Addition of NAD or NADH quenches the fluorescence emission signal of MtbALD. Titration of MtbALD with NAD and NADH shows that NADH has very strong binding in comparison to NAD with Kd value 12.81 ± 2.5 μM and 0.276 ± 0.017 mM for NADH and NAD, respectively at neutral pH. The lower Kd value for NADH at physiological pH assumes that MtbALD catalyses exclusively the formation of L-alanine in vivo. Each subunit of MtbALD folds into two compact domains that are separated by a cleft. Well defined electron density maps for the cofactor and substrate were observed in the ternary complex [Fig. 1(A)] allowing for a detailed analysis of their interactions which will be dealt with in later sections. The two domains share structural homology with r.m.s.d deviations between them of 4.47 Å for 181 Cα atoms. The overall fold of the subunit in this enzyme has been described in detail in related structures earlier.17 Briefly, both domains consist of mainly a parallel central β-sheet, flanked by helices. The NAD binding domain consists of residues 130–310 while the catalytic domain consists of residues 1–129 and 311–371. There are a total of 13 α-helices and 15 β-strands in the subunit [Fig. 1(B)]. The two domains are linked by two α-helices. Most of the active site residues are clustered around a cleft between the two domains. The accessible surface area of the subunit in the apo and holo enzyme forms is 15,785 and 15,056 Å2, respectively. Approximately, 40% of the accessible surface area is polar. Crystal structure of M. tuberculosis L-alanine dehydrogenase. (A) Omit electron density map contoured at 3.0 σ around NAD and pyruvate in the ternary complex. (B) Superposition of respective subunits from the apo and ternary complex structures showing the rotation of the catalytic domain by 16.2° due to binding of NAD and pyruvate (shown in stick representation). Residues in the hinge region are shown in red. The bound NAD and pyruvate are colored yellow. The cartoon representation of the apo and ternary complex structures are shown in cyan and blue, respectively. (C) Hexameric association in MtbALD. The hexamer is formed as a trimer of dimers. The individual subunits are colored distinctly. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] The purified protein was consistent with a hexameric association as observed in the size exclusion chromatography experiments. Each subunit associates to form a dimer which exhibits extensive interactions (Fig. 2). These interactions largely involve the respective NAD binding domains of the individual subunits while the respective catalytic domains are situated at either end of the dimer. The total buried surface area upon dimerization is 3631 and 3435 Å2, respectively in the apo and holo enzymes out of which about 30% is polar. Three such dimers associate into a hexamer which can also be described as a "trimer of dimers" [Fig. 1(C)]. The apo enzyme crystals contain a hexamer in the asymmetric unit while three subunits are found in the asymmetric unit of the holo enzyme crystals which associate through crystal symmetry to form the hexamer. Approximately 20,825 and 17,860 Å2 surface area in the apo and holo enzymes, respectively are buried upon hexamer formation out of which about 35% is polar in nature. The superimposed Cα backbone of the dimer from apo and ternary complex of MtbAld. The catalytic and NAD binding domains are marked. The dotted arrow indicates the movement of catalytic domain in ternary complex structure. Catalytic domains of the apo and ternary complex structures are colored red and pink, respectively, while the NAD binding domains are depicted in dark and light blue colors. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] The catalytic domains of the respective subunits rotate by ∼16.2° relative to the NAD binding domains in the ternary complex with NAD. This rotation is centered on the residues 127–132 and 309–315 which are part of two helical stretches linking the two domains. Mapping of the changes onto the oligomer makes it clear that the catalytic domain moves towards the NAD binding domains and therefore leads to the "closed" conformation [Figs. 1(B) and 2]. A comparison of the apo enzyme structure and the binding of NAD reveal a change in the conformation of loop 238–247 on NAD binding. This loop moves by about 2 Å accompanied by other subtle changes to presumably optimize the interactions of the NAD moiety with the enzyme. Similar changes were observed in its binary complex with the P. lapideum Ald17 although no relative rotation of the catalytic and NAD binding domains were observed. The ribose moiety adopts the C2′ endo conformation in the complex while the adenine ring is placed in a predominantly hydrophobic pocket. NAD interacts extensively with the enzyme and includes polar interactions with Met 301, Asn 300, Asp 270, Ile 267, Val239, Ser 134, Thr 178, Ala 179, Ala 240, Asp 198, Gly 177, and Lys 203 (Fig. 3) (see also Supplementary Table). Overall the mode of binding is similar to that seen earlier in other dehydrogenases.36, 37 The pyruvate moiety is stabilized by interactions of the carboxyl group with Arg 15. Unlike its binary complex with the P. lapideum Ald, the moiety comes close to the nicotinamide ring (Fig. 3). Other interactions include those with Lys 75 and Wat 53. The latter water molecule interacts with His 96 and Glu 76 also. The pyruvate is about 4.1 Å from the nicotinamide moiety and about 3.8 Å from Asp 270, an important active site residue. The si face of the moiety stacks against the side chain of Phe 94 on one side while the re face is adjacent to the nicotinamide moiety. The O3 oxygen of pyruvate is at 3.7 Å from the ND1 and NE2 atoms of the side chain of His 96. Protein-NAD and protein-pyruvate interactions in (Left) the ternary complex of MtbALD. The pyruvate and NAD moieties are shown in stick representation. Hydrogen bonds are depicted by black dotted lines while water oxygens are shown as red spheres. The protein chain is shown in cartoon representation. Selected residues are labeled for clarity. (Right) Superposition of the ternary complex of MtbALD (shown in dark blue) with the binary complexes of NAD (PDB code: 1PJC) and pyruvate (PDB code: 1SAY) with P. lapideum Ald (depicted in light blue). The cofactor and substrate molecules are depicted as sticks while the respective protein chains are in cartoon representation. Earlier work and kinetic characterization has given rise to a generally accepted mechanism14-17 (Scheme 1). Asp 270 and His 96 are presumably the active site catalytic residues in MtbALD as seen from the crystal structures. L-alanine reacts as a monoanion in the forward hydride transfer step involving Cα of alanine and C4 of nicotinamide with subsequent formation of the iminopyruvate intermediate. The subsequent step involves attack by a water molecule and is facilitated by the cationic acid (His 96 in case of MtbALD) and results in the formation of a carbinolamine. Ammonia and pyruvate are finally released in the last step. From the biochemical work on the B. subtilis enzyme14, 15 and the subsequent structures of the P. lapideum Ald17 it became clear that attack by ammonia in the reverse reaction or by water in the forward reaction will have to occur on opposite faces of pyruvate or iminopyruvate, respectively. The question arises as to whether the water is on the same side as the nicotinamide ring or is it trapped between the substrate and enzyme surface. The present ternary complex clearly identifies the water as being trapped between the substrate and the enzyme surface thereby fixing the position of attack by ammonia from the nicotinamide ring side during the reaction. The rotation of the two domains while going from the apo to the ternary complex brings the pyruvate moiety in the earlier binary structure with P. lapideum Ald17 (Fig. 3) close to the nicotinamide ring of NAD in the present complex. This is necessary for the initiation of the hydride transfer reaction step. From the P. lapideum structures17 the question remained as to whether the pyruvate can be brought close to the nicotinamide by changes in intersubunit-interactions or more simply by a domain rearrangement. The present structures clarify this aspect and the observed domain rotation brings the pyruvate within "striking" distance of the nicotinamide moiety to initiate the hydride transfer step. Other intermediate steps of the reaction can conceivably occur without major rearrangements to the active site. The authors thank Dr. Amit Sharma, ICGEB, New Delhi, India, for use of the X-ray generator. SMT is a UGC senior research fellow. This is communication number 7474 from C.D.R.I. The Supplementary Material referred to in this article can be found online at http://www.interscience.wiley.com/jpages/0887-3585/suppmat/ Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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