Structural Studies of Glutamine Synthetases – Towards ...172536/FULLTEXT01.pdf · Antitubercular Agents WOJCIECH W. KRAJEWSKI ISSN 1651-6214 ISBN 978-91-554-7283-2 urn:nbn:se:uu:diva-9286

ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2008

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 553

Structural Studies of GlutamineSynthetases – Towards theDevelopment of NovelAntitubercular Agents

WOJCIECH W. KRAJEWSKI

ISSN 1651-6214ISBN 978-91-554-7283-2urn:nbn:se:uu:diva-9286

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List of Papers

This thesis is a comprehensive summary of the results presented in the fol-lowing publications and manuscript, which will be referred to by their Ro-man numerals:

I Krajewski, W.W., Jones, T.A., and Mowbray, S.L. (2005) Struc-

ture of Mycobacterium tuberculosis glutamine synthetase in complex with a transition-state mimic provides functional in-sights. Proc. Natl. Acad. Sci. USA 102, 10499-10504.

II Krajewski, W.W., Collins, R., Holmberg-Schiavone, L., Jones,

T.A., Karlberg, T., and Mowbray, S.L. (2008) Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes, and provide opportunities for drug and herbicide design. J. Mol. Biol. 375, 217-228.

III Nordqvist A., Nilsson, M.T., Röttger, S., Odell, L.R., Krajewski,

W.W., Andersson, C.E., Larhed, M., Mowbray, S.L., and Karlen, A. (2008) Evaluation of the amino acid binding site of Mycobac-terium tuberculosis glutamine synthetase for drug discovery. Bioorg. Med. Chem. 16, 5501-5513.

IV Nilsson, M.T.*, Krajewski, W.W.*, Srinivasa, B.R., Yahiaoui, S.,

Larhed, M., Karlen, A., Jones, T.A., and Mowbray, S.L. (2008) Structural basis for the inhibition of Mycobacterium tuberculosis glutamine synthetase by novel ATP-competitive inhibitors. Manuscript.

* Joint first authorship Articles I, II, and III have been reproduced with permission from the respec-tive copyright holders.

Contents

Introduction.....................................................................................................9

Background...................................................................................................11 Glutamine synthetase and its role in nitrogen metabolism.......................11 Classification of GSs................................................................................11 Reaction mechanism of GS ......................................................................12 Other reactions catalyzed by GS ..............................................................12 Taut and relaxed forms of bacterial GS....................................................13 Regulation of bacterial GS activity ..........................................................13 GS inhibition by methionine sulfoximine and phosphinothricin .............14 Structures of bacterial and eukaryotic GSs ..............................................15 M. tuberculosis GS as a potential drug target...........................................18

General Methods...........................................................................................19 Protein expression and purification..........................................................19 Activity and inhibition assays ..................................................................19 X-ray crystallography...............................................................................20

Results and discussion ..................................................................................21 Structure of MtGS in complex with a transition-state mimic (Paper I)....21

Complex formation and crystallization................................................21 Overall structure ..................................................................................21 Active site ............................................................................................22 Implications for catalysis and inhibition..............................................24

Structures of mammalian GSs (Paper II) .................................................25 Thermal shift assay ..............................................................................25 Crystallization......................................................................................26 Overall structures.................................................................................27 Active site ............................................................................................27 Substrate-induced conformational changes .........................................29 Comparison of HsGS and MtGS structures – implications for inhibitor design...................................................................................................29

Targeting the amino acid-binding site (Paper III) ....................................31 Literature survey..................................................................................31 Virtual screening..................................................................................32

Targeting the nucleotide-binding site (Paper IV).....................................35

Structure of a relaxed MtGS in complex with PA ...............................35 MSO-P as a crystallization aid ............................................................35 Structure of a taut MtGS in complex with PA and MSO-P.................37 Prospects for selective inhibition of MtGS by PA...............................37

Future perspectives .......................................................................................39

Summary in Swedish ....................................................................................40

Acknowledgements.......................................................................................42

References.....................................................................................................44

Abbreviations

ATase Adenylyl transferase CfGS GS from Canis familiaris GS Glutamine synthetase HsGS GS from Homo sapiens HTS High-throughput screening MSO L- Methionine-S-sulfoximine MSO-P Methionine sulfoximine phosphate Mtb Mycobacterium tuberculosis MtGS GS from Mycobacterium tuberculosis NCS Noncrystallographic symmetry PEG Polyethylene glycol PDB Protein Data Bank RAPID Rational Approaches to Pathogen Inhibitor Discovery rms Root mean square StGS GS from Salmonella typhimurium TB Tuberculosis UTase Uridylyl transferase �-GCS �-glutamyl:cysteine synthetase ZmGS GS from Zea mays

9

Introduction

Tuberculosis (TB) is a contagious disease caused by a pathogenic bacillus, Mycobacterium tuberculosis (Mtb). It has plagued humans for thousands of years, and despite the existence of treatment and control programs, continues to claim 1.6 million lives each year (The World Health Organization, http://www.who.org). In 2005, there were an estimated 8.8 million new cases of TB, 90% of which occurred in Asia and sub-Saharan Africa. While it is estimated that one-third of the world’s population is currently infected with M. tuberculosis, only 5-10% will develop the active form of TB. Particularly at risk are people with a weakened immune system.

Although existing drugs can often cure TB, the treatment is lengthy, and requires taking a combination of drugs for at least 6 months. However, the therapy is not always successful and often results in patient relapsing. Fur-ther, poor patient compliance has led to the emergence of drug-resistant TB, which is even more difficult to treat. The situation is further exacerbated by the emergence of HIV/AIDS, which is a serious risk factor for TB (Corbett et al. 2003).

The above facts indicate an urgent need for the development of novel, more effective drugs that would shorten the current treatment, and act on persistent and drug-resistant TB.

Identification of novel antimicrobial compounds relies mainly on two ap-proaches, a so-called “empirical” process involving whole-cell screening, and a target-based approach, often referred to as a “mechanistic” path. Both approaches have advantages and disadvantages, but with the advances in genomics (Cole et al. 1998) and the availability of essentiality data, the latter seems to dominate the drug discovery landscape (Cole and Alzari 2007).

During the past 10 years there has been an increase in research activities aimed at developing novel drugs against TB. The RAPID (Rational Ap-proaches to Pathogen Inhibitor Discovery) center that started at Uppsala University in 2003 is one such example. Research groups with expertise in structural biology, medicinal chemistry, and computational chemistry have combined to form the center. Within RAPID we target essential enzymes from Mtb, determine their three-dimensional structures and develop assays for monitoring their activities. With this in hand, the search for inhibitors can begin, either by high-throughput screening (HTS) of large compound librar-ies or through structure-aided drug design. Projects are performed in collabo-ration with AstraZeneca India, which carries out HTS and organic synthesis

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of compounds. The medicinal chemistry group uses virtual screening and de novo approaches to identify potential inhibitory molecules, and then synthe-sizes them together with analogues for evaluation in the inhibition assays. These assays, as well as protein cloning and expression, structural studies and interpretation of the combined data are the responsibility of the struc-tural biology unit. Visualization of protein-ligand interactions is important for validation of hits, as well as subsequent lead optimization. The computa-tional chemistry group uses molecular dynamics for evaluation of inhibitors based on free energy calculations, and helps guide the optimization of com-pounds’ specificity.

Glutamine synthetase (GS) plays an important role in nitrogen metabo-lism by catalyzing an ATP-dependent condensation of ammonia and gluta-mate to form glutamine. Recent studies from the Horwitz laboratory (as de-scribed below) showed the importance of Mtb GS (MtGS) for growth and survival of Mtb, and demonstrated its potential as a drug target. Given its importance for the pathogen, we focused on MtGS in an effort to develop novel inhibitors against the enzyme, which could be of therapeutic interest.

The aim of this thesis is to present the results of our work on MtGS and mammalian GSs, which have led to the identification of novel inhibitors, with a main emphasis on the structural aspects of the work. The GS struc-tures presented in this thesis have been solved using X-ray crystallography.

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Background

Glutamine synthetase and its role in nitrogen metabolism Glutamine synthetase (GS; EC 6.3.1.2, also known as �-glutamyl:ammonia ligase) catalyzes an ATP-dependent condensation of glutamate and ammonia to yield glutamine, as shown in (1). Glutamine is one of the building blocks of proteins, and its amide nitrogen is essential for the biosynthesis of a num-ber of molecules such as AMP, CTP, tryptophan, histidine, carbamoyl phos-phate, and glucosamine-6-phosphate.

The roles of GS in the nitrogen metabolism of various species differ. For bacteria, ammonia constitutes a preferred nitrogen source, and GS together with glutamate synthase provide a major route for its assimilation. In plants, GS plays an important role in assimilation of ammonia, as well as in the refixation of photorespiratory ammonia. In mammals, the role of GS de-pends on tissue localization; in the brain, it regulates the levels of toxic am-monia and eliminates neurotoxic glutamate, while in the liver it is one of the enzymes responsible for the removal of ammonia.

Classification of GS GS enzymes, which are present in all organisms, exist in at least three dis-tinct forms, known as GS I, GS II, and GS III (Pesole et al. 1995). GS I has so far been found only in prokaryotes, while GS II is present in eukaryotes and some bacterial genera such as Rhizobium, Frankia and Streptomyces (Merrick and Edwards 1995). GS III was first identified in Bacteroides fragilis and subsequently found in some cyanobacteria. The three GS types share a relatively low sequence identity and have distinct quaternary struc-tures. GS I proteins are dodecamers of identical subunits (molecular weight,

12

~55 kDa), whereas GS II enzymes are decamers composed of smaller ~40 kDa polypeptide chains. GS IIIs are also dodecameric (van Rooyen et al. 2006), but these assemblies are larger than GS Is, as they are composed of ~80 kDa subunits.

Reaction mechanism of GS Synthesis of glutamine by all types of GS is believed to occur via a two-step mechanism, as summarized and reviewed earlier (Eisenberg et al. 2000). First, the �-phosphate of ATP is transferred to the �-carboxylate of gluta-mate, forming an activated �-glutamyl phosphate. The second step involves an attack of ammonia on the intermediate with subsequent release of gluta-mine and free phosphate. The structural basis for these catalytic steps was elucidated by Eisenberg and coworkers (Eisenberg et al. 2000), and will be discussed later in the text.

Other reactions catalyzed by GS The reaction shown in (1) is often referred to as the biosynthetic reaction, and is considered to be that most physiologically relevant. For assay work, a variation of the biosynthetic reaction is sometimes used, in which ammo-nium is replaced with hydroxylamine (NH2OH). The product of that reac-tion, �-glutamylhydroxamate can then be assayed colorimetrically.

GS is also known to catalyze a number of other reactions (Eisenberg et al. 2000). The most well known and commonly used for assaying GS activity is the transferase reaction. This reaction is very similar to the reverse of the biosynthetic reaction, and is shown below (2).

During the transferase reaction, arsenate is thought to bind at the phosphate site and react with glutamate to form �-glutamyl arsenate intermediate. Sub-sequently, hydroxylamine binds at the ammonium-binding site to attack the intermediate, forming �-glutamylhydroxamate through displacement of am-monia (Eisenberg et al. 2000).

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Taut and relaxed forms of bacterial GS For its catalytic activity, GS is completely dependent on the presence of divalent cations, Mg2+ or Mn2+. Besides their role in catalysis, the metal ions play an important role in stabilizing the enzyme’s native conformation. Based on biochemical studies, Stadtman and coworkers identified and char-acterized two forms of GS, metal ion-bound (“taut”), and metal ion-free (“relaxed”) form, as reviewed elsewhere (Shapiro and Stadtman 1970).

The taut form of GS is catalytically active and resists dissociation into subunits when exposed to alkaline pH, 1 M urea or even 1% sodium dodecyl sulfate (SDS). The relaxed form of GS can be obtained by treating the native GS with EDTA, which removes the divalent cations from the protein. In contrast to the taut form, the relaxed GS is inactive and the above-mentioned treatments cause it to dissociate into subunits in a time-dependent way. Upon relaxation, cysteine sulfhydryl groups become accessible to titrating re-agents, and an increase in tryptophan exposure to solvent is observed. An-other characteristic of the relaxed form is that it can bind AMP but not ATP (Ginsburg 1972), suggesting that there are changes in the nucleotide-binding site.

Interconversion of the taut and relaxed forms could be viewed as a means of regulating GS activity, but it remains unclear if this actually occurs in vivo.

Regulation of bacterial GS activity The importance of GS in nitrogen metabolism is underscored by a tight regu-lation of its activity. GS is thought to be subject to feedback inhibition by a wide variety of end products of glutamine metabolism: alanine, glycine, serine, AMP, CTP, glucosamine-6-phosphate, carbamoyl phosphate, his-tidine, and tryptophan (Woolfolk and Stadtman 1967). In addition to feed-back inhibition, GS activity can be modulated by covalent addition (adenyly-lation) or removal (deadenylylation) of an AMP group (Shapiro et al. 1967).

The control of GS activity by adenylylation is achieved through a bicyclic cascade (Figure 1), comprising two bifunctional enzymes, uridylyl trans-ferase (UTase) and adenylyl transferase (ATase), and the signal transduction protein, PII, as reviewed in (Stadtman 2001). In a nitrogen-rich environment, indicated by a high glutamine-to-�-ketoglutarate ratio, UTase removes a UMP group from PII-UMP, yielding unmodified PII. The PII protein then stimulates the adenylylation activity of ATase, which catalyzes the addition of an AMP molecule to GS, lowering its activity. Under nitrogen-limiting conditions, UTase converts PII to PII-UMP, which interacts with ATase to stimulate its deadenylylation activity. During the deadenylylation step, ATase removes an AMP group from GS, yielding an active enzyme.

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Figure 1. Control of GS activity by adenylylation, adapted from (Stadtman 2001). Unmodified PII stimulates the adenylylation reaction, while uridylylated PII (PII-UMP) stimulates the deadenylylation. The uridylylation/deuridylylation is catalyzed by uridylyl transferase (UTase). The adenylylation/deadenylylation is catalyzed by adenylyl transferase (ATase). The “+” indicates a stimulatory effect, while “-“ indi-cates an inhibitory effect.

GS inhibition by methionine sulfoximine and phosphinothricin Apart from the adenylylation and feedback control of GS activity, there has been considerable interest in studying the inhibition of GS by compounds of natural or synthetic origin. Such inhibitors have been used to probe the mechanistic and structural features of the enzyme, as well as to investigate the effects of inhibition on the growth of different organisms (Eisenberg et al. 2000). A comprehensive review of GS inhibitors has been presented in (Eisenberg et al. 2000), and shows that the vast majority of them target the glutamic acid-binding site. Among the best-known and most potent inhibi-tors of GS are structural analogues of glutamic acid, methionine sulfoximine (MSO) and phosphinothricin (PPT) (Figure 2).

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Figure 2. The GS inhibitors, methionine sulfoximine (MSO) and phosphinothricin (PPT) are structural analogues of glutamic acid.

MSO was first isolated from nitrogen trichloride treated maize protein, zein (Bentley et al. 1949), and identified as the toxic factor that causes epi-leptic fits in certain animals (Gershoff and Elvehjem 1951). The level of toxicity varies significantly between different species, with dogs and cats being highly sensitive, and humans relatively insensitive (Newell et al. 1949).

PPT (also known as glufosinate) is part of the tripeptide L-phosphinothricyl-L-alanyl-L-alanine produced by Streptomyces viridochro-mogenes (Bayer et al. 1972). Glufosinate is the active agent of several com-mercially available herbicides, which are often used in combination with resistant crop plants, such as the Aventis Liberty Link series.

The mechanism of action of MSO and PPT is similar. Both inhibitors compete with glutamate for binding to the GS active site (Eisenberg et al. 2000), and become phosphorylated in the presence of ATP and Mg2+ (or Mn2+) ions, yielding methionine sulfoximine phosphate (MSO-P) and phosphinothricin phosphate (PPT-P), respectively (Ronzio et al. 1969; Logusch et al. 1989). As a result, a tight complex consisting of GS, ADP, MSO-P (or PPT-P) and metal ions is formed. MSO-P (or PPT-P) is thought to mimic the phosphorylated tetrahedral adduct at the transition state (Gass and Meister 1970; Weisbrod and Meister 1973). MSO and PPT have a simi-lar inhibitory effect on MtGS with Ki values reported to be 1.1 and 0.6 �M, respectively (Harth and Horwitz 1999).

Structures of bacterial and eukaryotic GSs The first insights into the architecture of bacterial GSs came from electron microscopy studies of the Escherichia coli enzyme (Valentine et al. 1968). EM data revealed a dodecamer with 622 symmetry, where identical subunits are arranged in two hexameric rings stacked onto each other. More detailed structural information was obtained from X-ray crystallographic studies on the Salmonella typhimurium GS (StGS) performed in David Eisenberg’s

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laboratory. Initially, a substrate-free, Mn2+-bound StGS structure was solved (Almassy et al. 1986; Yamashita et al. 1989), followed by structures in com-plexes with substrates, feedback inhibitors, and PPT (Gill and Eisenberg 2001). Overall structure of StGS is shown in Figure 3A and B. More recently a structure of a relaxed MtGS has been solved (Gill et al. 2002). The bacte-rial GS structures present in the Protein Data Bank (PDB), including those described here, are listed in Table 1.

GS dodecamers have 12 active sites, each located at the interface of two adjacent subunits in the hexameric ring. The active site has a shape of a “bi-funnel”, which ATP and glutamate enter through opposite ends (Eisenberg et al. 2000). Divalent metal ions (Mg2+ or Mn2+) bind at the center of the bifun-nel, and, as mentioned earlier, are important for catalysis and structural sta-bility. The ammonium ion binds to a negatively charged pocket, which was identified based on a structure of StGS in complex with thallium (Liaw et al. 1995). Binding of substrates to GS is associated with local conformational changes affecting several loops (Eisenberg et al. 2000), which are discussed later in this thesis.

Figure 3. Overall structures of bacterial and plant GSs. (A) StGS hexamer with one subunit colored in black. (B) StGS dodecamer shown from a view 90� away from that in (A). (C) ZmGS pentamer with one subunit colored in black. (D) ZmGS de-camer shown from a view 90� away from that in (C).

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Table 1. Structures of bacterial GSs available in the PDB.

Structure PDB code

Resolution (Å) Reference

StGS/Mn2+ 2GLS 3.50 (Yamashita et al. 1989) StGS/Mn2+/glutamate 2LGS 2.80 (Liaw et al. 1993) StGS/MnAMP 1LGR 2.80 (Liaw et al. 1994) StGS/MnADP/Tl 1F1H 2.67 (Gill and Eisenberg 2001) StGS/MnADP 1F52 2.49 (Gill and Eisenberg 2001) StGS/PPT/MnADP 1FPY 2.89 (Gill and Eisenberg 2001) MtGS/citrate/MnAMP 1HTO 2.40 (Gill et al. 2002) MtGS/citrate/MnAMP 1HTQ* 2.40 (Gill et al. 2002) MtGS/MSO-P/MgADP 2BVC 2.10 (Krajewski et al. 2005)

* Multi-copy model Until very recently, structural information concerning eukaryotic GSs was

lacking. During the course of our work on mammalian GSs (canine GS, CfGS, and human GS, HsGS), the structures of the maize (Zea mays) en-zyme (ZmGS) in complexes with inhibitors were solved (Unno et al. 2006). The currently available structures of eukaryotic GSs, including those that will be described here, are listed in Table 2.

Plant GS is a decamer of identical subunits that are assembled into two pentameric rings stacked onto each other (Figure 3C and D). The decameric molecule has 10 active sites, which, as is the case for bacterial GSs, are lo-cated between two neighboring subunits within a ring. Binding of substrates to the plant enzyme is essentially the same as that seen in bacterial GS struc-tures. Structural features of eukaryotic GSs are discussed in more detail later in this thesis and in Paper II. Table 2. Structures of eukaryotic GSs available in the PDB.

Structure PDB code

Resolution (Å) Reference

ZmGS/MnADP/MSO-P 2D3A 2.63 (Unno et al. 2006) ZmGS/MnAMP-PNP/MSO 2D3B 3.50 (Unno et al. 2006) ZmGS/MnADP/PPT-P 2D3C 3.81 (Unno et al. 2006) CfGS/Mg2+ 2UU7 3.00 (Krajewski et al. 2008) HsGS/MnADP/Pi 2OJW 2.05 (Krajewski et al. 2008) HsGS/MnADP/MSO-P 2QC8 2.60 (Krajewski et al. 2008)

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M. tuberculosis GS as a potential drug target The potential of MtGS as a drug target was first suggested by studies from the Horwitz laboratory (Harth et al. 1994; Harth and Horwitz 1999). Besides its well-established role in nitrogen metabolism, MtGS is believed to be in-volved in the synthesis of a cell wall component, poly-L-glutamate/glutamine, which is found only in pathogenic mycobacteria (Hirschfield et al. 1990; Harth et al. 1994).

MtGS encoded by the glnA1 (or rv2220) gene has been classified as es-sential for optimal growth based on a transposon mutagenesis study in H37Rv strain (Sassetti et al. 2003). The null glnA1 mutant is auxotrophic for glutamine and has no detectable GS activity (Tullius et al. 2003). Further-more, the mutant was shown to be attenuated for intracellular growth in hu-man THP-1 macrophages, and was avirulent in the highly susceptible guinea pig model of pulmonary TB (Tullius et al. 2003). The essentiality of the glnA1 gene for growth has also been demonstrated in the mouse model of TB (Lee et al. 2006).

Apart from the glnA1 gene, the Mtb genome has three additional GS genes, glnA2 (rv2222c), glnA3 (rv1878), and glnA4 (rv2860c) (Cole et al. 1998). GlnA2 catalyzes the synthesis of D-glutamine and D-isoglutamine, whereas the GlnA3 and GlnA4 enzymes catalyze the synthesis of L-glutamine (Harth et al. 2005). It has been shown that GlnA2, GlnA3, and GlnA4 are not essential for growth in vitro (Harth et al. 2005) and in vivo (Lee et al. 2006), and cannot be detected in Mtb (Harth et al. 2005).

MSO was shown to inhibit growth of Mtb in vitro in both broth culture and in human THP-1 macrophages (Harth and Horwitz 1999). The inhibitory action of MSO is accompanied by a marked reduction in the amount of the poly-L-glutamate/glutamine in the cell wall. Moreover, MSO blocks the growth of pathogenic mycobacteria in culture and in THP-1 cells, but non-pathogenic mycobacteria or nonmycobacterial species, such as E. coli are unaffected (Harth and Horwitz 1999). Inhibition of growth and reduction of the levels of poly-L-glutamate/glutamine were also observed when Mtb were treated with antisense oligonucleotides directed at MtGS mRNA (Harth et al. 2000).

Despite its impact on growth of Mtb in vitro and in vivo, MSO cannot be used as a therapeutic agent. Epileptogenic properties of MSO observed in various animal species are associated with inhibition of brain GS (Griffith and Meister 1978). In addition, MSO inhibits �-glutamyl:cysteine synthetase (�-GCS), an enzyme involved in glutathione synthesis (Richman et al. 1973). Therefore, there is a need for the development of novel inhibitors of MtGS, which would not interfere with the activities of human GS and �-GCS en-zymes.

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General Methods

Protein expression and purification MtGS and mammalian GSs were overexpressed heterologously in E. coli. In all three cases, the proteins were fused with His6-tags for purification using nickel-immobilized metal affinity chromatography. The purified proteins were subjected to size exclusion chromatography and concentrated for stor-age and crystallization trials.

Expression of MtGS using E. coli raised the possibility that the host’s ATase (GlnE) could modify the mycobacterial enzyme. This would result in decreased biosynthetic activity and sample heterogeneity, a potentially det-rimental factor for protein crystallization. However, analysis of the structure of the taut MtGS described later (Paper I), indicated that the protein is un-modified at the relevant Tyr406, with the residue being buried in the struc-ture. Following a report that MtGS becomes partially adenylylated when expressed in E. coli (Mehta et al. 2004), characterization of the original preparation by mass spectrometry revealed that approximately 10-20% of the enzyme had been modified (unpublished). The low levels of modification may reflect the inability of the natural levels of E. coli ATase to “keep up” with the high levels expressed for GS. This indicated that the crystal was able to “select” for the unmodified molecules. To avoid the unwanted modi-fication, for subsequent structural studies and assay work, MtGS was ex-pressed in a GlnE-deficient E. coli strain (a generous gift from AstraZeneca India, Ltd.), similar to that reported earlier (Gill et al. 2002).

Activity and inhibition assays As outlined above, the enzymatic activity of GS can be assayed using the biosynthetic or the transferase reaction. Since the biosynthetic reaction is considered to be the most physiologically relevant (Eisenberg et al. 2000), inhibitors were evaluated using this assay system. The biosynthetic activity can be measured either by quantifying the release of Pi or the formation of ADP in a coupled assay system.

To avoid possible complications associated with the coupled assay (two additional enzymes that could be themselves affected), the release of Pi was measured. Pi was quantified using a commercially available PiColor-

20

Lock�Gold kit (Innova Biosciences). In this system, a phosphomolybdate complex in the presence of malachite green produces a green color with an absorption maximum at ~ 635 nm. The assay has a stable end-point and can be used with relatively high concentrations of acid-labile substrates such as ATP. A linear response was seen from 1.6 �M to 50 �M Pi.

The assay conditions chosen for testing inhibitors depended on whether the compounds were intended to compete with glutamate or with ATP. The ATP-competitive inhibitors were assayed using a 100 �L reaction containing 50 mM Hepes (pH 7.5), 25 mM MgCl2, 1 mM ATP, 30 mM NH4Cl, 30 mM glutamate, and 7 nM of GS per subunit. For testing the inhibitors competing with glutamate, lower concentrations (10 mM) of that substrate were used. The incubation was done for 1 h at room temperature. Inhibitors were typi-cally evaluated at 1 mM.

X-ray crystallography Structural models of GSs presented in this thesis were obtained using X-ray crystallography. Below is a short description of the particular considerations and methods used.

Crystallographic data for all the structures were collected at the European Synchrotron Radiation Facility beamlines in Grenoble, France. Because of relatively large unit cell dimensions, it was necessary to collect data using oscillation ranges as small as 0.1º to minimize overlap of reflections. At the same time, it was important to find an optimal crystal-to-detector distance, which often resulted in a compromise between data completeness and the maximum diffraction resolution that could be obtained from a crystal. Data reduction and scaling was performed using the software suites HKL (Otwinowski and Minor 1997) and XDS/XSCALE (Kabsch 1988) or with a combination of MOSFLM (Leslie 1999) and SCALA (Evans 1993).

The structures discussed in this thesis were solved by molecular replace-ment in Phaser (McCoy et al. 2007), AMoRe (Navaza and Saludjian 1997), and MOLREP (Vagin and Teplyakov 1997). The solutions were inspected for good crystal packing using the graphics program O (Jones et al. 1991) or Coot (Emsley and Cowtan 2004).

For refinement, REFMAC5 (Murshudov et al. 1997) and CNS (Brunger et al. 1998) were used. The presence of noncrystallographic symmetry (NCS) was taken advantage of during refinement, as it improved the data-to-parameter ratio, resulting in better phases and maps.

Where appropriate, ARP/wARP was used for automated model building (Lamzin et al. 2001). Manual model rebuilding was done in O or Coot. NCS averaging of the electron density maps in O was helpful in rebuilding, espe-cially in the poorly defined map regions. Water molecules were added to the models using ARP/wARP or water-picking options in O.

21

Results and discussion

Structure of MtGS in complex with a transition-state mimic (Paper I) The aim of this study was to determine a crystal structure of MtGS in com-plex with its known inhibitor MSO. The understanding of how GS binds the inhibitor was expected to facilitate future efforts to identify novel inhibitors of the enzyme and provide additional functional insights. In addition, it was believed that a higher resolution structure was required to provide a firm basis for such work.

Complex formation and crystallization As mentioned earlier, inactivation of GS by MSO is associated with the in-hibitor’s phosphorylation in the active site. We therefore attempted to form the inactivation complex prior to crystallization screening. The reaction mix-ture containing 25 mg/ml enzyme, 10 mM ATP, and 11 mM MSO (pure S-enantiomer) was incubated for 30 min at room temperature and then sub-jected to crystallization trials. Well-defined crystals grew from 30% poly-ethylene glycol (PEG) 400, 100 mM Mes (pH 6.8), and 200 mM MgCl2 within 48 h. A round of streak-seeding under the same conditions resulted in improved crystal quality.

Overall structure The structure of MtGS in complex with MSO-P and MgADP was solved by molecular replacement using a subunit of MtGS/citrate/MnAMP (Gill et al. 2002) as search model and refined at a resolution of 2.1 Å. Statistics for data collection and refinement are shown in Table 1, Paper I. The asymmetric unit includes one hexameric ring (Figure 4A), which is part of a dodecamer by virtue of a 2-fold crystallographic axis.

The structure of a single subunit is composed of two compact domains. The N-terminal domain (residues 1–107) is referred to as the �-grasp do-main, whereas the C-terminal one (residues 108–478) is known as the cata-lytic domain. Superposition of the A subunit of the current model on that of the MtGS/citrate/MnAMP structure gives an rms difference of 1.0 Å for 452 C� atoms (out of 478 in the complete sequence). The most notable differ-

22

ence between the two structures is a three-residue register shift of the �-strand starting at Glu214, altering the local environment in the nucleotide and amino acid-binding sites (Figure 4B). The shift was attributed to a loss of metal at the n1 site, creating a relaxed form of the enzyme (Gill et al. 2002). The current metal-bound structure represents a taut form of MtGS, similar to that observed for the Salmonella enzyme.

Figure 4. Overall structure of taut MtGS. (A) The MtGS hexamer with bound ADP and MSO-P shown as black sticks. (B) The �-strand register shift that distinguishes the taut MtGS structure (light grey carbons) from the relaxed one (dark grey car-bons). Residue names of the relaxed structure are underlined.

Active site Inspection of the electron density map revealed the presence of ADP and MSO-P molecules in each active site, which indicates that the �-phosphate of ATP has been transferred to MSO (Figure 5A).

MSO-P was placed assuming that the phosphorylation takes place at the sulfoximido nitrogen (Rowe et al. 1969) and the stereochemistry is S. The binding mode of MSO-P differs from that proposed for PPT in the StGS/PPT/MnADP structure (Gill and Eisenberg 2001). While there is a good overlap for the amino acid part of the inhibitors, the orientation of the sulfoximine and phosphinyl groups is different. The methyl group of PPT points �180� away from that of MSO-P model, while the two oxygen atoms are placed unfavorably for interaction with protein and for the phosphate transfer.

The binding of ADP in the taut MtGS structure is well supported by elec-tron density. Comparison with previous structures shows that the nucleotide base is now rotated by 180� around the glycosidic bond, placing it more favorably for interactions with protein and water. In the current binding mode, N1 makes a hydrogen bond with Ser280, while N6 interacts with

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Lys361-O and an active-site water (Figure 5B). Additionally, we find Lys215 interacting with the �-phosphate of the nucleotide, which helps sta-bilize it and possibly assists in the phosphoryl transfer.

In addition to the two known metal binding sites (n1 and n2) seen in the taut StGS structures, we observe a third magnesium ion at position n3, a feature that has not to been reported for GSs so far. This third metal ion is involved in bridging the �- and �-phosphates of ADP and the phosphate of MSO-P, as well as in interactions with Glu133 and Glu227 (Figure 5B).

Figure 5. MtGS active site. (A) Electron density (2Fo-Fc) of ADP, MSO-P, and magnesium ions contoured at 1�blue;= 0.29 e-/Å3) and 3�magenta). (B) Polar interactions with ligands in the MtGS/MSO-P/MgADP structure. (C) Ammonium-binding site and its surroundings. Thallium from the StGS/MnADP/Tl structure marks the ammonium site, which is blocked by the methyl group of MSO-P.

Superposition of the MtGS/MSO-P/MgADP and StGS/MnADP/Tl struc-tures indicates that the methyl group of MSO-P blocks the ammonium-

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binding site identified earlier by binding thallium and cesium ions (Liaw et al. 1995). The negatively charged ammonium pocket consists of residues Tyr186, Glu219, Glu335, as well as Asp54 and Ser57 of the adjacent subunit (denoted Asp54’ and Ser57’) (Liaw et al. 1995), all of which are clearly visible in the present structure (Figure 5C). Interestingly, Asp54’ forms a previously unreported strong (2.6 Å) hydrogen bond with Glu335.

Implications for catalysis and inhibition The structure of MtGS/MSO-P/MgADP complex offers several insights into catalysis and inhibition. Binding of the nucleotide base as seen here explains data concerning the enzyme’s nucleotide specificity (Harth et al. 1994). While ATP is preferred, GS can also make use of GTP, although with lower specific activity. As both nucleotides would be expected to interact with Ser280, the lower activity with GTP could be explained by a loss of interac-tion with Lys361-O.

Comparison of the MtGS/MSO-P/MgADP and �-GCS [PDB:1V4G] (Hibi et al. 2004) active sites revealed remarkable similarities in the way they bind their ligands (see Figure 3B, Paper I). The �-GCS structure was also solved in complex with MgADP and a transition-state analogue. Despite only 10% structure-based sequence identity between the two structures, the placement of the nucleotide, phosphorylated inhibitor and the arrangement of the three magnesium ions is essentially the same. The bridging coordination by the n2 and n3 metals has been seen in other ADP-forming ligases, and was sug-gested to be a common catalytic motif for these enzymes, increasing the reactivity of the �-phosphate of ATP and stabilizing the �-glutamyl phos-phate intermediate (Hibi et al. 2004). The methyl group of MSO-P in the MtGS complex overlaps with the cysteine mimic in the �-GCS structure, suggesting that both enzymatic mechanism and the mode of inhibition for the two proteins will be similar.

During catalysis, the �-glutamyl phosphate intermediate needs to be pro-tected from non-productive hydrolysis. Based on the current structure, we suggest that Glu335 fulfills such a role by forming a tight hydrogen bond with Asp54’ (Figure 5C). Glu335 is part of a loop, referred to as the “flap”, while Asp54’ is part of the so-called “latch” (Eisenberg et al. 2000). Such interaction between the flap and latch residues is also expected to strengthen the subunit-subunit interactions. Both residues have been shown to be impor-tant for ammonium binding, and Asp54’ is believed to be involved in depro-tonation of the ammonium ion during catalysis (Alibhai and Villafranca 1994). Additional stabilization of this region is provided by Tyr406 and Tyr186, whose side chains interact with main chain atoms of Ala336 and Asp54’, respectively. The stabilizing role of Tyr186 correlates well with kinetic studies of a GS mutant from A. variabilis (Healy et al. 2003). Substi-tution with cysteine at this position led to a 825-fold increase in Km for am-

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monium. It is interesting to note that the latch is also involved in additional interactions with the residues from the neighboring catalytic domain, one of them being Asp68’ hydrogen-bonding to Arg352, which in turn coordinates the �-phosphate group of ADP.

The �-strand shift, representing a structural transition between the relaxed and taut forms of MtGS, can be viewed as a potential regulatory mechanism of GS activity. Regardless of whether such regulation occurs in vivo, the existence of structurally distinct forms of the enzyme may have implications for inhibitor design. For example, using the available structural information, one could design compounds that would stabilize the relaxed (inactive) con-formation of the protein.

Structures of mammalian GSs (Paper II) The taut MtGS structure described in the previous section provided new insights into substrate binding and catalysis, and constitutes a solid frame-work for the design of novel inhibitors against the enzyme. A major question that remained open at this stage was whether it would be possible to design or discover inhibitors that would only bind to the bacterial enzyme, i.e. not inhibit the human GS. To help answer that question, the availability of the three-dimensional structure of the human enzyme would be beneficial.

For our structural studies, we chose to work with canine GS (CfGS) (Shin and Park 2004), which is a close homologue (97% sequence identity) of the human enzyme. The choice of the canine GS rather than human one was based on the availability of a well-characterized recombinant expression system. In parallel to our efforts to solve the structure of CfGS, the Struc-tural Genomics Consortium in Stockholm began work on the structure de-termination of HsGS. The structural studies of the mammalian GSs pre-sented in this chapter (and Paper II) are the result of collaboration between Uppsala and Stockholm groups. The high sequence identity between the two proteins (97%), and the conservative nature and location of amino acid sub-stitutions, allow us to consider them as essentially identical in terms of struc-ture and function.

Thermal shift assay The mammalian GSs described in this thesis appear to be less stable than the bacterial enzymes. CfGS tended to precipitate when stored on ice and was fairly unstable at 4 °C. Similarly, HsGS was difficult to handle, tending to precipitate during the concentration step. Since protein solubility, homoge-neity, and stability correlate well with crystallizability (Ericsson et al. 2006), HsGS was subjected to a fluorescence-based thermal shift (ThermoFluor®)

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assays in order to identify ligands that would promote its stabilization (Pantoliano et al. 2001).

The thermofluor assay measures ligand-dependent changes in the thermal stability of a target protein. The assay is based on the signal changes of an environmentally sensitive fluoroprobe; the probe is quenched in aqueous solutions, whereas binding to an exposed hydrophobic core of the protein results in an increase of the fluorescence signal as a function of temperature. The melting temperature (Tm) is defined as the midpoint temperature of the transition between the folded and unfolded states (Ericsson et al. 2006). In the presence of ligands, a positive shift in Tm reflects a gain in stability as the conformational flexibility of the protein is reduced (Geerlof et al. 2006).

The effects of various concentrations of glutamate, glutamine, MgCl2, MnCl2, nucleotides and their analogues on the Tm of HsGS were investi-gated by the Stockholm group. The Tm for HsGS in the absence of ligands was 56 °C. Interestingly, neither glutamate nor glutamine affected the Tm significantly. Magnesium had only a small effect on the enzyme’s stability (an increase of ~2 °C) even at concentrations as high as 500 mM. Upon the addition of 1 mM MnCl2 and 1 mM ATP, the Tm increased 4.1 °C and 11.5 °C, respectively. When both MnCl2 and ATP were added to the protein, the Tm of HsGS was raised by 15.5 °C. A similar stabilization effect was ob-served with MgATP for HsGS (Listrom et al. 1997). It was therefore decided to add the two ligands to all subsequent preparations of HsGS prior to con-centrating the protein to 40 mg/ml.

Crystallization Crystallization conditions for CfGS and HsGS were identified using sparse-matrix screens, JCSG+ Suite (Qiagen) and Index (Hampton Research), by the vapor diffusion method.

CfGS was crystallized in hanging drops at room temperature. The drops contained equal volumes of 9-mg/ml protein and mother liquor solution con-sisting of 15% (w/v) PEG 3350 and 200 mM potassium citrate (pH 8.3). To improve crystal quality, drops with reduced protein and mother liquor con-centrations were streak-seeded. Crystals were cryo-protected in 10% (w/v) PEG3350, 100 mM Tris-HCl (pH 8.5), 100 mM MgCl2, 30% (v/v) PEG400, and flash-cooled in liquid nitrogen.

Crystals of the HsGS/MnADP/Pi complex were grown in sitting drops at 4 ºC. A 20-mg/ml protein solution (containing 10 mM each of ATP and MnCl2) was mixed with an equal volume of reservoir solution containing 10% (v/v) isopropanol, 200 mM NaCl and 100 mM HEPES (pH 7.5). For cryo-protection, crystals were transferred to a mother liquor solution sup-plemented with 30% (v/v) glycerol.

The HsGS/MnADP/MSO-P complex was obtained in a similar way to that described for the taut MtGS structure. A mixture of 20 mg/ml protein

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solution (containing 10 mM ATP and 10 mM MnCl2) and 2 mM MSO was incubated at room temperature for 45 min and then mixed with an equal volume of well solution consisting of 1.1 M sodium malonate, 0.5% (w/v) Jeffamine ED-2001 and 0.1 M HEPES (pH 7.0). Crystals were cryo-protected in mother liquor solution supplemented with 30% (v/v) glycerol.

Overall structures Since the ZmGS structure (Unno et al. 2006) became available during the course of these investigations, both Uppsala and Stockholm groups were able to use it for molecular replacement. The structure of apo CfGS was solved by molecular replacement using a pentamer of ZmGS [PDB: 2D3A] as search model and refined to a resolution of 3 Å. The structure of the HsGS/MnADP/Pi complex was solved by molecular replacement using a single subunit of ZmGS and refined to a resolution of 2 Å. For the subse-quent determination of the HsGS/MnADP/MSO-P complex structure, a pen-tamer of HsGS/MnADP/Pi model was used as a probe; this structure was refined to a resolution of 2.6 Å. Statistics for data collection and refinement for all three models are shown in Table 1, Paper II.

Mammalian GSs like the plant enzymes are decamers of identical sub-units, arranged as two pentameric rings stacked on top of each other (see Figure 3C and D). The dimensions of the decamer are ~90 Å measured along the 5-fold axis and ~110 Å along the 2-fold axis. In each pentamer, there are five active sites located at the interface of two adjacent subunits. Interactions between pentamers are relatively weak and only involve loop residues 150–156 of each subunit, such that five loops from one ring interact with their 2-fold symmetry-related equivalents of the other ring. Each sub-unit is composed of two compact domains and an N-terminal meander (resi-dues 3–24). As is the case for the bacterial GSs, the N-terminal domain (residues 25–112) is referred to as the �-grasp domain, whereas the C-terminal one (residues 113–373) is known as the catalytic domain.

Active site Each active site is located in the funnel-shaped pocket formed by the N-terminal �-grasp domain of one subunit and the C-terminal catalytic domain of the adjoining subunit (Figure 6A). Both domains contribute to the forma-tion of a continuous, highly curved �-sheet. The �-strands from the C-terminal domain contribute most catalytic residues.

Protein-ligand interactions in the mammalian GSs are best illustrated by the HsGS/MnADP/MSO-P structure (Figure 6A and B). In each active site, an ADP molecule occupies the top part of the funnel, whereas the bottom part contains MSO-P together with three manganese ions, n1–n3. Residues Trp130, Pro208, Arg262, and Tyr336 form a hydrophobic cluster that sur-

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rounds the adenine ring of ADP. Interactions between Ser257 and the N1 and N6 atoms represent the only hydrogen bonds between the adenosine moiety and the protein. The phosphate groups of ADP, on the other hand, are engaged in numerous interactions with protein ligands and two manganese ions, n2 and n3. MSO-P, which occupies the glutamic acid-binding site, is positioned near the �-phosphate of ADP. The inhibitor is tightly bound by several protein side chains and by all three manganese ions.

Figure 6. HsGS active site. (A) HsGS active site with bound ADP, MSO-P, and manganese ions. The catalytic domain is in grey, while the �-grasp domain of the neighboring subunit is in black. Hydrophobic residues surrounding the adenine ring of ADP are shown as sticks. (B) Polar interactions with ligands in the HsGS/MnADP/MSO-P structure.

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Substrate-induced conformational changes Inspection of the superposed apo and liganded GS structures reveals that binding of substrates to the enzyme is associated with conformational changes occurring near the active site (Figure 7). The main movements af-fect residues 311-337 of the catalytic domain, as well as residues 63-77 of the �-grasp domain of the adjacent subunit. These changes are followed by a shift of the helix at 267-278 and the adjustment of the flap residues 302-307. As a result, more closed forms of the enzyme are generated when substrates are bound. The fact that no real differences are observed between the two HsGS complex structures indicates that binding of the nucleotide is respon-sible for the active site closure, and few conformational changes take place on glutamic acid substrate binding. The interactions of the latch Asp76’ with Arg324 and Arg319 are similar to those seen in the MtGS structure, underly-ing its importance for nucleotide binding.

The thermofluor experiments described earlier in the text correlate well with the structural observations, i.e. binding of nucleotide and Mn2+ ions has the most profound effect on the enzyme’s conformation, and, in turn, stabil-ity.

Comparison of HsGS and MtGS structures – implications for inhibitor design Despite the overall low sequence identity between MtGS and HsGS (~20%) and their different oligomeric states, the shape of active site cavities is very similar (Figure 8A). The respective �-grasp and catalytic domains forming each active site superimpose quite well on each other.

In Figure 8B, superposition of the active sites of HsGS and MtGS shows that the glutamic acid-binding site together with the three metal ions are completely conserved. On the other hand, most residues defining the nucleo-tide-binding site are different, and some of the �-strands around the nucleo-tide are shifted.

The differences in the nucleotide-binding site of MtGS and HsGS thus seem to provide the best opportunities for the design of specific inhibitors against the mycobacterial enzyme.

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Figure 7. Conformational changes near the active sites of mammalian GSs. Super-position of C� backbones of CfGS (blue) and HsGS/MnADP/MSO-P (red). The catalytic domain of molecule A and the �-grasp domain of the neighboring subunit are shown in stereo. MSO-P and ADP of HsGS are shown as sticks, and manganese ions as spheres.

Figure 8. Comparison of HsGS /MSO-P/MnADP and MtGS/MSO-P/MgADP struc-tures. (A) Superposition of catalytic and �-grasp domains of HsGS (red) and MtGS (blue). ADP of HsGS is shown as sticks. (B) The active sites of HsGS (gold car-bons) and MtGS (grey carbons) are compared. Metal ions of the two structures are shown in magenta and grey, respectively. Interacting residues of HsGS are labeled.

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Targeting the amino acid-binding site (Paper III) When this work began, only MSO and PPT had been reported in the litera-ture as MtGS inhibitors. This section describes our efforts to identify new inhibitors of MtGS that target the amino acid-binding site of the enzyme. The following work was done in collaboration with the medicinal chemistry group of Anders Karlén at Uppsala University as part of the RAPID center.

In this study, we combined a literature survey, structure-based virtual screening and the synthesis of a small library of compounds.

Literature survey Our initial attempts focused on the evaluation of inhibitors that were re-ported to be active against GSs from other species, either prokaryotic or eukaryotic. Most of the GS inhibitors found in the literature are analogues of the glutamic acid substrate, and display activities in the micromolar to mili-molar range. Given the highly conserved nature of the amino acid-binding site, we anticipated that such compounds could also inhibit the mycobacte-rial enzyme.

In total, 13 compounds (known GS inhibitors and their analogues) sug-gested by the literature search were purchased or synthesized, and evaluated for their inhibition of MtGS (Table I, Paper III). All the compounds were linear glutamic acid analogues, with different functional groups at the delta position of the scaffold. Enzyme inhibition was tested at 1 mM compound concentration using the biosynthetic assay. Apart from MSO and PPT, three compounds (4, 5, and 7) were found to weakly inhibit the enzyme (Figure 9).

Figure 9. Literature-based compounds found to inhibit MtGS.

The most potent of those, compound 4, reduced MtGS activity by 58% under the standard assay conditions (see the general methods section), while com-pounds 5 and 7 showed 26 and 33% inhibition, respectively. Compound 4, (2S,5R)-2,6-diamino-5-hydroxyhexanoic acid had an IC50 of 610 � 15 �M, a

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value which is significantly poorer than that obtained for MSO (IC50 of 51 � 6 �M) and PPT (IC50 of 1.9 � 0.4 �M) under these conditions. The docking of 4 in the amino acid-binding site is shown in Figure 1, Paper III. Com-pound 4 overlays well with MSO-P, retaining all the important interactions to Arg329, His276, Gly272 and Glu335, while the 5-aminomethyl group docks close to the ammonium-binding site. It is interesting to note that the 5S-isomer of 4 was inactive, suggesting that the stereochemistry of the hy-droxyl group is important for binding. The inactivity of the 5S-isomer could be explained by the loss of a hydrogen bond between the hydroxyl group and Arg368, as seen for 4.

Virtual screening The availability of the high-resolution structure of MtGS with the transition-state mimic MSO-P prompted us to carry out a structure-based virtual screening in order to identify novel inhibitors of MtGS based on the amino acid-binding site. The process of virtual screening used is outlined in Figure 10. First, a library of 2.1 million commercially available compounds from different vendors was assembled. The library was filtered to select for com-pounds of molecular weights below 300 g/mol containing carboxylic acid bioisosters or functional substitutes of the MSO sulfoximine group. The 46,400 compounds obtained in that way were then converted to structures and processed with Ligprep (Maestro, Schrödinger) to generate stereoisom-ers, two ring conformations, and tautomers, thus expanding the set to 99,251 structures. Docking of structures was done with FlexX (Tripos Inc.) with a protocol optimized to successfully reproduce the binding mode of MSO-P seen in the crystal structure. The highest-ranking pose of each compound was then subjected to a rigid MSO-based pharmacophore search. The phar-macophore model consisted of two hydrogen bond donors and one acceptor with a tolerance criterion of 2 Å. Compounds with at least two matching pharmacophoric points were visually inspected. Of those, 29 compounds were selected for purchase and evaluation in the activity assay.

Among the 29 compounds tested at 1 mM concentration, we identified three active hits, 14, 15, and 23, with 24%, 26% and 48% inhibition, respec-tively (Figure 11). The three compounds contain aromatic scaffolds and are not related to any previously known inhibitors of GS. The docking of these compounds in the amino acid-binding site is shown in Figure 4, Paper III. Compound 14 docks to the active site with the urea group close to Arg329 and Gly272, while the sulfonamide moiety sits near the sulfoximine group of MSO-P. Docking results for compounds 15 and 23, suggest that their car-boxyl groups interact with Arg329 and His276 in the same way as MSO-P. The sulfonic group of 23 overlays well with the phosphate of MSO-P, while the triazole group of 15 extends further than MSO-P into the active site.

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Figure 10. Virtual screening flowchart.

Compound 23 was selected as a starting point for a small synthetic li-brary, where the sulfonic moiety was replaced with different functional groups, including carboxylic acid, thioether, sulfoxide, sulfonamide, and phosphonate ester. An additional 10 compounds were synthesized by intro-ducing L- and D-serine into the aromatic scaffold instead of glycine. Screen-ing for activity revealed four weak inhibitors (Figure 12), among which a phosphonate ester, compound 28, was the most potent, showing 42% inhibi-tion. The level of inhibition seen here for compound 28 suggests that the phosphonate moiety is a reasonable replacement for the sulfonic group of 23.

It is also interesting to note that several compounds showed an apparent activation of the enzyme. Further structural and kinetic studies will be needed to provide an explanation for such activation, an effect that has been reported previously for plant GSs (Evstigneeva et al. 2003). At the moment, it is tempting to speculate that the activation may be associated with binding of the compounds at an allosteric site. Such binding could, for example, sta-

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bilize one of the flexible loops, which are known to be important for the catalytic activity.

Figure 11. Inhibitors identified in virtual screening.

Figure 12. Synthetic library compounds found to inhibit MtGS.

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Targeting the nucleotide-binding site (Paper IV) This section describes the structural basis for inhibition of MtGS by a com-pound that targets the nucleotide-binding site. The inhibitor, 1-[(3,4-dichlorophenyl)methyl]-3,7-dimethyl-8-morpholin-4-ylpurine-2,6-dione, will be referred to as the purine analogue (PA) (Figure 13A). PA is a com-mercially available analogue of a class of compounds found to inhibit MtGS in an HTS assay carried out by our collaborators at AstraZeneca, India. PA was found to inhibit MtGS with an IC50 value of 2.8 ± 0.4 �M.

Structure of a relaxed MtGS in complex with PA The structure of a relaxed MtGS in complex with PA was solved at 2.55 Å resolution by molecular replacement using a dodecamer of MtGS/citrate/MnAMP structure as search model. In the final model, residues 64-66 and 405-412 were not seen in the electron density maps, and were omitted.

In each active site, a molecule of compound PA is found occupying the upper part of the cavity (Figure 13). Binding of the inhibitor by the protein is achieved via non-polar interactions and two hydrogen bonds (Figure 13C). The diketopurine group is oriented roughly perpendicular to the beta-barrel wall, being sandwiched between the side chains of Phe232 and Arg364. A single hydrogen bond links the O2 atom of the diketopurine moiety and Ser280. The morpholino ring is stacked near the side chain of His278, with its oxygen atom involved in hydrogen bonding to the Asn229 side chain. The dichlorophenyl moiety extends toward solvent near Lys361, surrounded by a cluster of hydrophobic residues, Tyr129, Phe232, and Trp282.

Comparison of the relaxed MtGS/PA and MtGS/MSO-P/MgADP active sites shows a partial overlap of the inhibitor with the nucleotide (Figure 13D) The morpholino group occupies the position of the ADP-ribose, while the diketopurine moiety lies in the plane of the adenine base.

Since the MtGS/PA complex was obtained at low pH, in the absence of metals, it represents the relaxed (inactive) form of the protein. There was concern that PA could, in principle, bind differently bind to the taut (active) form. Indeed, the inhibitor had been identified using the assay, which by definition refers to the active form of the enzyme. Moreover, growing well-diffracting crystals of MtGS with this and other inhibitors was difficult. We therefore tried to obtain a complex of the taut form of MtGS with PA.

MSO-P as a crystallization aid Despite extensive crystallization screening, conditions that would yield a taut form of MtGS in the presence of PA could not be identified. One strat-egy that we pursued was to synthesize MSO-P in the hope that it would help

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stabilize the protein, thus influencing its crystallizability. MSO-P was syn-thesized by Samir Yahiaoui from the Department of Medicinal Chemistry at Uppsala University. Following the incubation of MtGS with synthetic MSO-P, the mixture was subjected to crystallization trials. MtGS in the presence of MSO-P was found to crystallize under the same conditions and in the same space group as the MtGS/MSO-P/MgADP complex.

Figure 13. Active site of the relaxed MtGS with a bound PA. (A) Chemical structure of PA. (B) Electron density (2Fo-Fc) for PA contoured at 1 = 0.23 e-/Å3. (C) Bind-ing of PA in the active site. (D) Binding of PA (gold carbons) compared with that of ADP (yellow carbons) of the MtGS/MSO-P/MgADP structure.

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Structure of a taut MtGS in complex with PA and MSO-P

The MtGS/MSO-P/Pi/PA complex structure was refined at 2.2 Å resolution using the ligand-free hexamer of the isomorphous MtGS/MSO-P/MgADP structure as the starting point. Inspection of the active sites revealed the presence of bound ligands, MSO-P, Pi, PA, and magnesium ions (Figure 14A). Binding of PA is essentially the same as that seen in the relaxed MtGS/PA structure (Figure 14B). The most notable difference is the loss of a hydrogen bond between the inhibitor and Asn229, as this residue is rotated away to interact with a neighboring water molecule and Glu227. The differ-ence is associated with the presence of metal, and an altered local environ-ment caused the �-strand shift. The water molecule interacting with Asn229 replaces the O1A atom of ADP, and takes over its role of coordinating the n3 magnesium ion.

Comparison of the MtGS/MSO-P/Pi/PA and MtGS/MSO-P/MgADP complexes shows that MSO-P and the three magnesium ions occupy the same positions in both structures (Figure 14C). The free phosphate (possibly from the hydrolysis of MSO-P) sits at the position of the �-phosphate of ADP.

Prospects for selective inhibition of MtGS by PA As discussed earlier in this thesis, the nucleotide-binding site of MtGS dif-fers from that of HsGS, providing opportunities for designing inhibitors di-rected at MtGS. Superposition of the MtGS/MSO-P/Pi/PA and HsGS/MnADP/Pi nucleotide-binding sites is shown in Figure 15. The dike-topurine and morpholino groups of PA overlay with the base and the ribose moieties of ADP, respectively. The dichlorophenyl group of the inhibitor, however, clashes with Trp130 and Arg262 side chains of HsGS. Since HsGS lacks the hydrophobic pocket that could accommodate the dichlorophenyl group, we expect that PA and similar compounds would not be able to bind to the human enzyme. The structural differences described above, as well as other architectural features of the MtGS nucleotide-binding site can be ex-ploited to design potent and selective inhibitors.

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Figure 14. Active site of taut MtGS with bound PA and MSO-P. (A) Electron den-sity (2Fo-Fc) for the ligands contoured at 1 = 0.30 e-/Å3. (B) Comparison of PA binding by the taut (gold carbons) and relaxed (turquoise carbons) MtGS. Hydrogen bonds are shown as dashed lines. (C) Superposition of the MtGS/MSO-P/Pi/PA (gold carbons) and MtGS/MSO-P/MgADP (turquoise carbons) active sites.

Figure 15. Comparison of the nucleotide-binding sites of MtGS/MSO-P/Pi/PA and HsGS/MnADP/Pi. MtGS residues (together with PA) are shown gold carbons, and HsGS (together with ADP) are shown with grey carbons. Residues of the HsGS structure clashing with PA are underlined.

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Future perspectives

The structural studies of GS enzymes presented in this thesis have provided many functional insights, as well as a structural basis for inhibition of MtGS by a novel class of compounds represented by PA.

In future enzymatic studies, PA will be tested for inhibition of human GS and �-GCS enzymes. Ideally, a good inhibitor would not interfere with the activities of these proteins, or display a favorable selectivity index.

PA will be tested for inhibition of growth of Mtb in vitro. If antimycobac-terial properties of PA can be demonstrated, further studies will be required to confirm that the compound acts specifically on MtGS. Following that, the inhibitor could be evaluated in vivo in an animal model. Structural studies are expected to play an important role in guiding the opti-mization process aimed at improving potency, selectivity, and ADME (ab-sorption, distribution, metabolism and elimination) properties of PA and its analogues.

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Summary in Swedish

Strukturstudier av glutaminsyntetaser –med syfte att fram-ställa nya antibakteriella läkemedel mot tuberkulos

Tuberkulos är en infektionssjukdom som orsakas av bakterien Mycobacteri-um tuberculosis (M. tuberculosis). Världshälsoorganisationen uppskattar att ca 2 miljoner människor dör varje år och att en tredjedel av jordens befolk-ning är bärare av tuberkulosbakterien. Av dessa smittade utvecklar omkring 10% aktiv tuberkulos under sin livstid. Särskilt lätt drabbas människor som har nedsatt immunförsvar. Situationen försämras av förekomsten av HIV som är en allvarlig riskfaktor för tuberkulos. Enligt Världshälsoorganisatio-nen rapporterades ca 9 millioner av nya fall 2005, varav 90 % av dessa före-kom i sydöstra Asien och Afrika. Ett flertal läkemedel mot tuberkulos an-vänds idag men behandlingen tar minst 6 månader och kan orsaka obehagli-ga biverkningar. Felaktig användning av dessa mediciner har lett till utveck-ling av resistenta bakteriestammar som är mycket svårare att behandla. För att förkorta den långa behandlingstiden och för att få kontroll över de resis-tenta stammarna behövs nya läkemedel mot tuberkulos utvecklas.

Glutaminsyntetas (GS) spelar en viktig roll vid metabolismen av kväve

genom att den katalyserar den ATP-beroende kondensationen av glutamat och ammoniak till glutamin. För bakterier är ammoniak en kvävekälla och GS tillsammans med glutamatsyntas är involverad i dess assimilering. Tidi-gare studier har visat att GS från M. tuberculosis (MtGS) är nödvändig för bakteriens tillväxt och överlevnad, vilket gör den till en lämplig måltavla för läkemedelsdesign.

I denna avhandling presenteras strukturella studier av MtGS och GS en-

zymer från däggdjur med syftet att identifiera och utveckla nya inhibitorer mot MtGS. De tredimensionella strukturerna har bestämts med hjälp av röntgenkristallografisk metodik. I röntgenkristallografi belyser man en prote-inkristall med röntgenstrålning för att få ett diffraktionsmönster. Denna in-formation analyseras med hjälp av olika datorprogram för att bygga upp en så kallad elektrondensitetskarta. En tredimensionell struktur av proteinet konstrueras genom att placera atomer i elektrondensitetskartan.

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Strukturen av MtGS i komplex med inhibitorn metionin sulfoximinfosfat, ADP och magnesiumjoner representerar den hittills mest detaljerade bilden av enzymets aktiva yta. Komplexet har erhållits genom att aktivera metionin sulfoximin i närvaro av ATP och MtGS före kristallisering. Två metalljoner har tidigare varit kända att vara viktiga för enzymaktivitet. Strukturen visar en tredje metalljon som stabiliserar den höga negativa laddningen i den akti-va ytan. Strukturen avslöjar dessutom hur metionin sulfoximinfosfat inhibe-rar MtGS, vilket utgör en viktig grund för strukturbaserad läkemedelsdesign.

Tre GS enzymer från däggdjur har strukturbestämts, ett GS apoenzym

från hund samt två humana GS enzymer med bundna ligander. Strukturjäm-förelse av dessa enzymer visar substratinducerade konformationsändringar i proteinet. Bindningen av ligander orsakar rörelse av looparna, vilket genere-rar mer slutna former av enzymerna. De största ändringarna är kopplade till bindningen av nukleotiden. Dessutom skiljer sig nukleotidbindningsfickan i humant GS från denna av MtGS, vilket ger en möjlighet att designa selektiva inhibitorer.

För att identifiera nya MtGS inhibitorer har vi fokuserat på såväl bindnings-fickan för en aminosyra som bindningsfickan för en nukleotid. Aminosyrans bindningsficka har undersökts genom en kombination av olika metoder, bl.a. strukturbaserad virtuell screening. Som resultat har några svaga inhibitorer hittats. För att kunna hitta fler inhibitorer som binder till nukleotidbindnings-fickan har en s.k. high-throughput screening använts och några starka inhibi-torer har hittats. I våra strukturella studier visar vi hur en av inhibitorerna binder till enzymet. Skillnader i nukleotidsbindningsfickorna mellan humant GS och MtGS antyder att det är möjligt for selektiv inhibering av MtGS.

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Acknowledgements

First of all, I would like to thank my supervisor Sherry for excellent support, long scientific discussions, and guidance. I have learnt a lot from you. Thank you Alwyn for accepting me as a PhD student in your group and for all the support throughout these years. Special thanks to my collaborators: Nisse for the assay and X-ray work on the GS project. The Medicinal Chemistry group: Anneli, Luke, Olle, Prasad, Samir, Svenja, Anders, and Mats for their contributions to the GS project. Srinivasa Bachally, Tanjore Balganesh, and their colleagues at Astra-Zeneca India Pvt. Ltd, for their contributions to the GS project. Tobias and Lovisa from the SGC in Stockholm, for the human GS story. Ulrika and Daniel for help with various TB projects. Many thanks to: The current and past RAPID group members: Adrian, Alina, Anna J, Anna L, Annette, Christofer, Daniel, Evalena, Henrik, Jimmy, Lena, Martin, Nina, Nisse, Patrik, Tex, and Torsten. Thank you for creating a good atmosphere in the lab, supportive environment, and … the “fikas”. I wish you guys the famous “genombrott” on a daily basis. Pavel and Magnus for all those laughs, sense of humor, and pumping iron. The fellow Poles, Agata and Urszula, for chats and good company. Gerard, Mark, Erling, Christer, and many others for all the technical help and advice. The current and former members of the Structural Biology labs: Al, Anatoly, Andrea, Anton, Emma, Fariborz, Fredrik, Glareh, Gerard, Gunnar, Göran, Hasse, Henke, Hugo, Jens, Jenny, Jerry, Johan, Jonas,

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Karin, Kaspars, Lars, Linda, Malin, Maria, Marian, Martin, Mats, Rosie, Saeid, Sanjee, Sara, Seved, Stefan, Wimal, Yang, and alla@xray for making the BMC a great place to work in. The Flogsta gang: Michiel, Pavel, Pavel K, and others for the movie ses-sions. Andrzej, Aneta, Yogesh, and Jennifer for good times spent together. Thanks to my family for support and encouragement. Finally, my greatest thanks go to Monia, my love and best friend. Thank you for always being there for me, for your understanding and patience.

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