X-ray crystallographic studies on Mlc and Aes, two transcriptional modulators from Escherichia coli

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X-ray crystallographic studies on Mlc and Aes, two transcriptional modulators from

Escherichia coli

Dissertation

zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften

Dr. rer. nat

Universität Konstanz

Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie

vorgelegt von Dipl. biol. Kinga Gerber

Konstanz 2005

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Dissertation der Universität Konstanz Tag der mündlichen Prüfung: 14.06.2005

Referent: Prof. Dr. Winfried Boos Referent: Prof. Dr. Wolfram Welte

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Abgrenzung der Arbeit

Die folgenden Experimente wurden von mir durchgeführt:

Klonierung, Reinigung, Kristallisation und Mutagenese von Mlc. Reinigung und Kristallisation von Aes. Sammlung von Diffraktionsdaten und Publikation mit Hilfe von André Schiefner. Mitwirkung bei der Publikation der Strukturdaten von Mlc.

Ich erkläre hiermit, dass ich mit der Darstellung von Frau Kinga Gerber über ihre Beteiligung an den oben aufgeführten Arbeiten einverstanden bin.

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Introduction

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Introduction 3

1.1. Regulation of gene expression in prokaryotes

Proteins need to be expressed according to the physiological conditions of the bacterial cell. This can be achieved by regulation at the level of transcription initiation, elongation and termination, as well as at the level of post-transcriptional mRNA processing and post-translational protein modifications (review: Adhya, 1996).

Transcriptional regulation can aim at controlling the rate of transcription or the time of transcription by turning specific genes off for a period of time. The presence of regulatory sequences in the bacterial genome is necessary for specific enzymes to regulate transcription of their target genes. Sequences recognized by RNA polymerases are called “promoters”, whereas regulatory proteins bind to “operator” sites. An operator is located immediately downstream from the promoter, just before the structural genes. Occupation of an operator site by a regulatory protein turns off the transcription of the genes immediately downstream from it since the RNA polymerase cannot proceed. Positive regulatory sites are located upstream of the promoter. The binding of a transcription factor to these sites enhances the activity of the RNA polymerase.

Prokaryotic genes to be regulated in the same manner are group together in “operons”

under the control of a single promoter and operator. There are two kinds of operons:

the inducible and the repressible one. An example for an inducible operon is the lac operon, composed of lacZ, coding for β-galactosidase to split lactose into glucose and galactose, lacY, a permease, increasing the permeability of the plasma membrane to lactose, and lacA, a transacetylase (Jacob and Monod, 1961; Beckwith, 1987; Adhya, 1996; Müller-Hill, 1996). The regulatory protein for the lac operon is called lac repressor, a protein encoded by the lacI gene. It can bind either to the operator

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Chapter 1 4

sequence or allolactose, the inducer molecule only available in the cell when lactose is present. Binding of allolactose causes an allosteric change in the repressor protein and prevents it from binding to its specific operator site. Lactose, therefore, is the inducer of the lac operon, enabling the synthesis of enzymes necessary for its metabolism.

Thus, in the absence of lactose the lac operon is occupied by the lac repressor.

However, for an effective repression of its operator site the lac repressor has to bind to the cAMP/CAP complex (catabolite activator protein with cyclic adenosine monophosphate). cAMP is produced by adenylatcyclase, an enzyme inhibited by glucose. Thereby the bacterial cell does not produce enzymes for the lactose metabolism when glucose, one of the products of lactose metabolism, is present (feedback inhibition).

An example for a repressible operon is the trp operon coding for 5 different enzymes (TrpA to Trp E) used in the biochemical pathway that synthetizes tryptophan (Yanofsky et al., 1999). The transcriptional regulator protein for this operon is the trp repressor and the signal molecule is tryptophan itself, a co-repressor. In the presence of low levels of tryptophan the trp repressor does not bind to its operator site.

However, in the presence of high amounts of tryptophan in the cell the trp repressor binds tryptophan, thereby undergoing a structural change that enables the protein to bind to the trp operon, blocking the transcription of the trp genes.

The arabinose (ara) operon is an example for positive transcriptional control (Englesberg and Wilcox, 1974). Similarly to the lac operon, the ara operon consists of 3 structural genes, araB, ara A and araD. AraBAD encode for enzymes required to convert arabinose into D-xylulose-5-phosphate, a molecule entering the pentose- phosphate pathway to generate energy for the cell. The regulator protein of the ara operon, AraC, can act both as a repressor and an activator of araBAD transcription and can regulate its own synthesis by repressing araC transcription. In the absence of L-arabinose, the inducer molecule, AraC binds to the regulatory sites I1 and O2,

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Introduction 5 thereby repressing the transcription of the araBAD operon (Schleif, 2000). In the presence of L-arabinose, AraC binds to the DNA sites I1 and I2 upstream of the araBAD promoter, thereby activating the transcription of the araBAD genes. At the same time, binding of the cAMP/CAP complex directly upstream of I1 is required for transcription to occur. In the absence of glucose, cAMP levels rise and the cAMP/CAP complex can bind to its specific site (catabolite repression).

Another remarkable example for transcription regulation in E. coli is the maltose system. It represents a member of the periplasmic binding protein-dependent ATP- Binding Cassette (ABC) high affinity transport systems of gram-negative bacteria (Boos and Lucht, 1996; Nikaido, 1994). The maltose/maltodextrin system is responsible for the uptake and efficient catabolism of α(1 ->4)-linked glucose polymers (maltodextrins) up to 7-8 glucose units (Boos and Shuman, 1998). The mal genes are organized in operons and are dispersed over the bacterial chromosome. They encode for several proteins localized in different compartements of the prokaryotic cell, the cytoplasm, the periplasm, the cytoplasmic membrane and the outer membrane (Boos et al., 1996). The transcriptional activator protein for all mal genes except for malI/X/Y is MalT, the product of the malT gene. MalT is a purely positive regulator of the mal genes (Figure 1A) (Danot et al., 1996) and is the prototype of a new family of transcription factors (Valdez et al., 1999). To become active, it has to bind to ATP and to maltotriose, the inducer molecule (Figure 1A and 1B) (Hofnung and Schwartz, 1971;

Debarbouille et al., 1978; Schreiber and Richet, 1999; Joly et al., 2002). Of the 5 known MalT-dependent operons in E. coli, expression of malK/lamb/malM and malE/malF/malG, is subject to catabolite repression and therefore requires the presence of the cAMP/CAP complex (Figure 1A) (Boos et al., 1996). The expression of malT itself is not autoregulated but requires cAMP/CAP as well (Debarbouille and Schwartz, 1979; Chapon, 1982).

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Chapter 1 6

A.

cAMP/CAP

mal genes +

mRNA except for malI/X/Y

+

MalT(a)/ATP/maltotriose complex

B.

ATP maltotriose cAMP/CAP

C.

Figure 1. Regulation of the maltose system in E. coli. Panel A. Regulation of mal gene expression by the MalT/ATP/maltotriose complex and cAMP/CAP. MalT(a) stays for the active, oligomeric conformation of the MalT protein. Panel B. Regulation of the maltose system by cAMP/CAP, Mlc, MalK, ATP and maltotriose. MalT(i)

malT mRNA MalT(i)

+ +

MalT(a)

- -

MalK, Mal Y or Aes Mlc

CAP Mlc malT +1

-70.5 +10

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Introduction 7 indicates the inactive conformation of MalT, MalT(a) stays for the active, oligomeric state of MalT. Positive regulation (activation) is indicated by a plus (+), negative regulation (repression) by a minus (-) sign. Panel C. Regulation of malT transcription by CAP and Mlc. CAP and Mlc binding sites are indicated. Sequences are numbered from the transcription start site (+1) of the malT gene shown in green.

MalT itself is subject to regulation by several proteins in E. coli and its activity is controlled by multiple regulatory signals. ATP and maltotriose are two effectors of the activator that positively control multimerization of MalT, a critical step in promoter binding (Figure 1B) (Boos and Shuman, 1998). Mlc, a transcriptional regulator of the phosphotransferase system of E. coli (Chapter 1.4), represses the expression of malT by binding to its operon (Figure 1C) (Hosono et al., 1995; Decker et al., 1998). MalK, the transport ATPase of the maltose system, is also able to inactivate MalT (Boos et al., 1996; Schreiber and Richet, 1999). It interacts directly with MalT in the absence of maltotriose and inhibits maltotriose binding to MalT (Panagiotidis et al., 1998; Joly et al., 2004). Another mal gene product, MalY, a cystathionase of E. coli, and Aes, an esterase from E. coli with homology to lipases (Chapter 1.6), both inhibit MalT activity when overproduced in the cell (Miyamoto et al., 1991 ; Reidl and Boos, 1991; Peist et al., 1997). MalY and Aes seem to inactivate MalT via the same mechanism (Figure 1B) (Boos and Shuman, 1998; Joly et al., 2002). In both cases, the inhibitory protein is a negative effector of MalT that competes with the inducer (maltotriose) for MalT binding and whose binding site is located in the N-terminal domain of MalT, which also contains the ATP binding site. However, the binding sites of Aes and MalY do not coincide. Recent studies indicate that the MalT/ADP complex might be the target of MalY and Aes in vivo (Joly et al., 2002).

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Chapter 1 8

1.2. Sugar transport through bacterial membranes

Bacterial cells are surrounded by a cell wall. Depending on whether they are gram- negative or gram-positive, the composition of their cell walls can differ considerably.

The cell wall of gram-negative bacteria (Figure 2A) is composed of an outer membrane of phospholipids, lipopolysaccharides and lipoproteins, followed by a periplasmic space with a thin layer of peptidoglycan. The cell wall of gram-positive bacteria (Figure 2B), on the other hand, lacks the outer membrane of gram-negative bacteria. A dense layer of peptidoglycan with teichoic acids prevents osmotic lysis of the cell, followed by a periplasmic space that separates the peptidoglycan layer from the cytoplasmic membrane.

The outer membrane of gram-negative bacteria, like Escherichia coli, serves as a selective permeator barrier for small, hydrophobic nutrient molecules (Nikaido, 2003). This selectivity is due to protein pores called porins (e.g. OmpF and OmpC) located in the outer membrane. Because of its semi-permeable nature, the outer membrane helps retain certain enzymes and prevents some toxic substances (e.g.

lysozyme) from entering. The surface proteins in the outer membrane, depending on the strain and species, carry out a variety of activities, including: functioning as enzymes; serving as adhesins and allowing the bacterium to adhere intimately to host cells and to other surfaces in order to colonize and to resist flushing, functioning as invasins, thereby allowing some bacteria to penetrate host cells and/or aiding certain bacteria in resisting phagocytic destruction. The periplasm is the gelatinous material between the outer membrane, the peptidoglycan, and the cytoplasmic membrane. It contains enzymes for nutrient breakdown as well as binding proteins to facilitate the transfer of nutrients across the cytoplasmic membrane harbouring many proteins that

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Introduction 9 A.

B.

Figure 2. Basic architecture of the cell wall of gram-negative (Panel A) and gram-positive bacteria (Panel B). For a detailed description see Chapter 1.2.

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Chapter 1 10

actively translocate solutes. Its transport systems are generally quite specific for one or a few substrates. Although many different types of transporters exist, all bacterial transport systems can be subdivided into only three categories on the basis of their energetics:

1) Primary transport systems convert chemical or light energy into electrochemical energy or solute gradients. Examples are the linear electron transfer chains (e.g. cytochrome C oxidase), the light driven ion-pumps (e.g.

bacteriorhodopsin), and the ATP-driven transporters (Poolman et al., 1992;

Higgins, 1992.)

2) Secondary transport systems use the free energy that is stored in the electrochemical gradients of protons, sodium ions or other solutes across the cytoplasmic membrane. Generally these systems are composed of a single polypeptide that binds and translocates the solutes, but it has also been shown that some secondary transport systems employ substrate binding proteins similar to that of binding protein dependent uptake systems that belong to the ABC superfamily (Jacobs et al., 1996). Secondary transport systems can be subdivided into three categories. The transport is called uniport when it is solely driven by the concentration gradient of the substrate. An example is the glucose transporter of Zymomonas mobilis (Weisser et al., 1995). The process is called symport when more than one substrate moves in the same direction. In this case the electrochemical gradient of one solute (usually proton or sodium ion) is used to drive the uphill transport of another solute (e.g. lactose permease, Kaback, 1997).

Finally, when substrates move in opposing directions transport is referred to as antiport (e. g. AcrAB-TolC system, Zgurskaya and Nikaido, 1999).

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Introduction 11 3) Group translocation systems couple the translocation to a chemical

modification of the substrate. The only group translocation systems found in bacteria are the phosphoenolpyruvate-dependent sugar phosphotransferase systems (PEP-PTS) (see below). The energy-rich phosphate bond of phosphoenolpyruvate is transferred to the membrane embedded translocator via cytoplasmic and/or membrane bound phosphoryl carrier proteins (for review see Postma et al., 1993). The phosphorylation state of the phosphoryl carrier proteins also plays an important role in regulatory processes such as catabolite repression.

At the energetic level the different transport reactions can be linked to each other as postulated by Michell in the chemiosmotic theory (Michell, 1961, 1963).

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Chapter 1 12

1.3. Phosphotransferase system (PTS)

The phosphoenolpyruvate (PEP) phosphotransferase system of Escherichia coli was discovered 40 years ago (Kundig et al., 1964). Today this system is known to be involved in the transport and phosphorylation of a large number of carbohydrates, as well as in the chemo-tactic movement toward these carbon sources and in the regulation of a number of other metabolic pathways in both gram-negative and gram- positive bacteria (Potsma et al., 1993). Regardless of the organism or carbohydrate, all PTSs that have been caracterized catalyse the following overall process:

PTS

Phosphoenolpyruvate⁽ⁱⁿ⁾ + carbohydrate^(out) → pyruvate⁽ⁱⁿ⁾ + carbohydrate-P⁽ⁱⁿ⁾

The phosphorylation and the translocation of a carbohydrate across the bacterial membrane are coupled and the energy needed for these processes is provided by the phosphoenolpyruvate. As an “energy currency”, PEP is equivalent to ATP since, during glycolysis, one ATP molecule is derived from one PEP molecule in the pyruvate kinase reaction. For carbohydrates that are actively accumulated by non-phosphotransferase systems, more than one ATP equivalent must be expended per monosaccharide unit for both transport and subsequent ATP-dependent phosphorylation (Potsma et al., 1993). Therefore phosphotransferase systems represent an energy conserving mechanism of carbohydrate uptake.

In all organisms studied, the following reactions compose the PTS-mediated translocation and phosphorylation of a given carbohydrate (Potsma et al., 1993):

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Introduction 13 (1) Phosphoenolpyruvate + enzyme I (EI) ‹−› P-EI + pyruvate

(2) P-EI + HPr ‹−› P-HPr + EI (3) P-HPr + EIIA ‹−› P-EIIA + HPr (4) P-EIIA + EIIB ‹−› P-EIIB + EIIA

(5) P-EIIB + carbohydrate(out) ‹−› EIIB + carbohydrate-P(in)

In most cases, enzyme I (EI) and HPr are soluble, cytoplasmic proteins that participate in the phosphorylation of all PTS carbohydrates in a given organism and thus have been called the general PTS proteins. Enzyme II (EII), on the other hand, is carbohydrate specific and may consist of a single membrane-bound protein composed of three domains, such as that for mannitol in E. coli, or of two or more proteins, such as the IICB^Glc-IIA^Glc pair for glucose in E. coli, with only EIIA^Glcbeing soluble, or the EIIA^Man-EIIB^Man pair for mannose in E. coli, fused together and soluble in the cytoplasm, while EIIC^Man and an additional domain EIID^Man are located in the membrane. In case of the cellobiose PTS in E. coli, EIIA^Cel and EIIB^Cel are separate proteins soluble in the cytoplasm, while only EIIC^Cel is membrane-bound (see Figure 3).

The PTS in E. coli constitutes an essential sensory system through which the cell monitors its environment. The information gathered through all PTS proteins is integrated at the level of the catalytic subunit kinase EI and (through HPr) of the signaller EIIA^Glc, the essential regulatory molecule of the PTS (Saier and Roseman, 1976). EIIA^Glc controls the activity of a large variety of membrane-bound transport systems as well as the synthesis of cAMP. Phosphorylated EIIA^Glc activates the adenylate cyclase (Potsma et al., 1993) and hence, positively regulates the cAMP concentration in the cell, thereby indirectly regulating transcription of several cAMP/CAP dependent genes (Kolb et al., 1993). Nonphosphorylated EIIA^Glc, on the other hand, is able to bind to and thereby inhibit several proteins essential in the

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Chapter 1 14

metabolism of non-PTS carbohydrates, e. g. lactose permease, glycerol kinase and MalK of E. coli (Lengeler, 1996). The phosphorylation state of EIICB^Glc is also involved in the regulation of PTS-dependent sugar uptake by being the target of Mlc, a transcriptional repressor of E. coli (Seitz et al., 2003). Its regulatory role will be discussed in more detail in Chapter 1.4.

Figure 3. The phosphotransferase system of Escherichia coli for cellobiose, glucose, mannitol and mannose. A schematic view of the reactions.

For a detailed description see Chapter 1.3.

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Introduction 15

1.4. Mlc from Escherichia coli

Mlc (makes large colonies) is a soluble 44.3 kDa protein consisting of 406 amino acid residues. It has been discovered as a regulator protein curbing the utilization of glucose (Hosono et al., 1995, for a recent review see Plumbridge, 2002). Mlc controls the expression of two operons encoding PTS-dependent transporters for glucose (Kimata et al., 1998; Plumbridge, 1998b) and mannose (Plumbridge, 1998a) as well as the genes encoding the general components of the PTS (Kim et al., 1999; Plumbridge, 1999; Tanaka et al., 1999) by repressing their operon sites. In addition, Mlc controls the expression of malT, encoding the central transcriptional activator of the maltose system (Decker et al., 1998). Thereby Mlc indirectly represses the malEFG, malK- lamB and malPQ operons, which encode genes for the uptake and catabolism of maltose. (Plumbridge, 1998a, 1999 and 2002; Kim et al., 1999; Tanaka et al., 1999).

The expression of mlc is auto-regulated (Decker et al., 1998) (Figure 3), furthermore rapidly decays when glucose is present, implying that mlc is also subject to post- transcriptional control (Shin et al., 2001; Plumbridge, 2002). The glucose PTS (ptsG) and Mlc are part of the heat shock regulon. Expression of both mlc and ptsG increases during heat shock, showing that the uptake of sugars is a stress-regulated process (Shin et al., 2001) (Figure 4).

In contrast to the classical mode of repressor inactivation by the cognate inducer, Mlc is inactivated by the sequestrating interaction with the actively transporting glucose transporter, the EIICB^Glc protein of the PTS (Figure 5B) (Lee et al., 2000; Tanaka et al., 2000; Nam et al., 2001). The interaction occurs at the EIIB^Glc domain of the transporter encompassing the phosphorylated cystein residue, the phosphate donor in glucose phosphorylation during its PTS-mediated transport (Seitz et al., 2003). The

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Chapter 1 16

membrane bound state of EIIB^Glc is essential for Mlc inactivation. Soluble EIIB^Glc, even though able to interact with Mlc (Nam et al., 2001; Seitz et al., 2003), does not prevent Mlc from binding to its operator regions and from its repressing activity. However, EIIB^Glc attached to the membrane by any lipophilic anchor, even unrelated to EIICB^Glc, binds Mlc in a fashion that prevents binding to the operator regions (Seitz et al., 2003). Also membrane attachment of Mlc itself prevents it from the interaction with its cognate operators (Tanaka et al., 2004). This indicates that Mlc alters its conformation when in close contact with the membrane as to suppress operator binding.

As judged by its amino acid sequence, Mlc belongs to the ROK family (Chapter 1.5. and Figure 6) (Repressors, Open reading frames and Kinases) (Titgemeyer et al., 1994;

Hansen et al., 2002) of transcriptional regulators encompassing xylose repressors, sugar kinases and transcriptional regulators with the widely conserved CXCGXXGCXE motif (Hosono et al., 1995; Hansen et al., 2002; Mesak et al., 2004). Mlc shares 40.5

% sequence identity with NagC, a transcriptional repressor for the nag regulon with N- acetylglucosamine-6-P as inducer. NagC is member of the ROK family and contains the conserved CXCGXXGCXE motif. The high similarity of their respective operator sites reflects the close relationship between Mlc and NagC (Plumbridge, 2001a, 2001b). Mlc recognizes the operator site of NagC at least in vitro but is unable to repress the nag operons in vivo. The DNA-binding motif of Mlc consists of a typical helix-turn-helix motif at its N-terminus and the protein behaves in dilute buffer solution as tetramer of a polypeptide of 44.3 kDa molecular weight (Nam et al., 2001;

Seitz et al., 2003). At its very C-terminus Mlc forms a putative amphipathic helix of 18 amino acid residues in length. The removal of this 18 amino acid long C-terminus leads to dimer formation, to the loss of EIICB^Glc binding as well as to the loss of operator interaction. Removing only 9 amino acid residues from the extreme C- terminus still allows EIICB^Glc binding, tetramerization as well as operator binding and

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Introduction 17 gene repression (Seitz et al., 2003). Thus, surprisingly, the C-terminus far removed in the primary sequence from the helix-turn-helix motif must be involved, directly or indirectly, possibly via a large conformational change, in EIICB^Glc binding as well as in operator recognition and subsequent repression. Regarding its unusual mode of inactivation, it was of interest to elucidate the crystal structure of this novel transcriptional regulator.

Figure 4. Organization of the Mlc promoter (Plumbridge, 2002). Mlc from Escherichia coli is expressed from two promoters, p1 and p2, which are recognised by RNA polymerase with σ⁷⁰. P2 is also recognised by RNA polymerase in complex with the heat shock sigma factor σ³². cAMP/CAP serves to regulate both promoters by repressing the upstream site (located at -58.5 relative to p1) and activating the downstream site (situated at -71.5 relative to p2). cAMP/CAP represses σ⁷⁰-directed transcription from p1 and activates transcription from p2. An Mlc site, centered at +15, overlaps p2 and represses E σ⁷⁰ expression from p2. Sequences are numbered from the p1 promoter (+1). A temperature upshift increases p2 expression from E σ³², especially in the absence of glucose (Shin et al., 2001).

CAP mlc

-58.5 Mlc +40 Eσ⁷⁰ Eσ³²

p1 p2 +1 +13

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Chapter 1 18

A.

.

igure 5. Model of Mlc activity. Panel A. In the absence of glucose. Panel B. In the EIIC^Glc

B

F

presence of glucose.

EIIB^Glc

ptsG Mlc

P

periplasm inner

cytoplasm membrane

EIIC^Glc

EIIB^Glc cytoplasm

periplasm inner membrane

Mlc glucose-6-P

ptsG

transcription translation glucose

EIICB^Glc

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Introduction 19

1.5. ROK family

he characteristics of the ROK family (Repressors, Open reading frames and Kinases)

ROK T

have been determined by amino acid sequence comparison (Titgemeyer et al., 1994, Hansen et al., 2002). The ROK family contains bacterial transcriptional repressors, bacterial fructokinases (SCRKs), bacterial glucokinases (GLKs), glucokinases from Archaea and mammalian N-acetylmannosamine kinases (Figure 6). The ROK repressors contain an N-terminal extension of about 80 amino acid residues, which includes a conserved DNA binding (helix-turn-helix) motif (Weickert and Adhya, 1992; Titgemeyer et al., 1994), whereas an ATP-binding site is conserved in the N- terminal region of the ROK kinases (Hansen et al., 2002). The C-terminal domain of the ROK kinases, on the other hand, harbors the sugar binding site (Titgemeyer et al., 1994). The domain common to all proteins of the ROK family comprises about 180 amino acid residues (Bateman et al., 2000). Due to the divers functions of the ROK family members only a few residues are conserved, forming the CXCGXXGCXE consensus motif (Hosono et al., 1995; Hansen et al., 2002; Mesak et al., 2004).

The structure of Mlc, published in this work, represents the first structure of a family protein described to date.

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Chapter 1 20

Figure 6. Phylogenetic relationships among ROK proteins (Hansen et al.

2002). Putative sequences are marked with an asterisk. Group I abbreviations: XYLRs, xylose repressors; Cs, Caldicellulosiruptor sp. (P40981); At, Anaerocellum

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Introduction 21 thermophilum (Q44406);Bs, Bacillus subtilis (P16557); Sx, Staphylococcus xylosus (P27159); Lp, Lactobacillus pentosus (P21940); NAGC-Ec, N-acetylglucosamine repressor from E. coli (P15301); and MLC-Ec, product of E. coli mlc (P50456); Group II abbreviations: SRCKs, fructokinases; Sm, Streptococcus mutans (Q07211); Pp, Pediococcus pentosaceus (P43468); Zm, Zymomonas mobilis (Q03417); ALSK-Ec, allokinase from E. coli (P32718). Group III abbreviations: Sc, Streptomyces coedicolor (P40184); Rs, Renibacterium salmoninarum (Q53165); Sg, Streptomyces griseus (Q9F1W1); Cg, Corynebacterium glutamicum (Q9KKE7); Bm, Bacillus megaterium (O31392); Bs, Bacillus subtilis (P54495); Sx, Staphylococcus xylosus (Q56198); Tm, putative Thermotoga maritime (Q9X1I0); Group IV abbreviations: YHCI, gene product of yhcI; Hi, Haemophylus influenzae (P44541); Ec, E. coli (P45425); Tv, putative Thermoplasma volcanicum (Q97AS0); Ta, putative Thermoplasma acidophilum (Q9HJY6); Pa, putative Pyrobactum aerophilum (PAE3437); H, GLK-H, putative Halobacterium sp. Strain NRC1 (Q9HMA7). Group V abbreviations; NAMK- h, N-acetylmannosamine kinase domain of the bifunctional enzyme UDP-N- acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (residues 413 to 722); h, human (Q9Y223); r, rat (O35826); m, mouse (Q9Z0P6).

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Chapter 1 22

1.6. Aes from Escherichia coli

Aes, a soluble 37 kDa protein consisting of 319 amino acid residues, is an acetyl esterase from E. coli (Figure 7). It belongs to the family of hormone-sensitive lipases (HSL) (Hemila et al., 1994, Peist et al., 1997, Kanaya et al., 1998). The catalytic domain of the members of the HSL family is the GXSXG motif with the active site serine residue (Yeaman, 1990, Hemila et al., 1994, Osterlund, 2001) (Chapter 1.7). In case of Aes, Gly¹⁶³, Asp¹⁶⁴, Ser¹⁶⁵, and Gly¹⁶⁷ are the components of the GXSXG motif with Ser¹⁶⁵, Asp²⁶², and His²⁹² as the catalytic triad (Kanaya et al., 1998) and Asp¹⁶⁴ as yet another critical residue for enzymatic activity (Haruki et al., 1999). Aes is able to hydrolyze various p-nitrophenyl esters of fatty acids (Kanaya et al., 1998, Figure 7). It prefers substrates with an acyl chain length of less than eight, with C4 and C5 being the best substrates.

Apart from its function as an esterase it acts as an inhibitor of MalT, the central transcriptional activator of the E. coli maltose regulon (Boos and Shuman, 1998, Joly et al., 2002). Joly et al. (2002) demonstrated that Aes inactivates MalT through direct protein-protein interaction in vivo (Chapter 1.1, Figure 1B).

O C H₃

O

NO₂ CH₃ C

O

O O NO₂

H₂O 2H⁺

Aes p-nitrophenyl acetate

+

acetate p-nitrophenolate

Figure 7. An example for the catalytic activity of Aes from Escherichia coli (Peist et al., 1997; Kanaya et al., 1998).

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Introduction 23

1.7. Hormone sensitive lipase family

Hormone-sensitive lipase (HSL) is an intracellular neutral lipase that is capable of hydrolyzing triacetylglycerols, diacylglycerols, monoacylglycerols, and cholesteryl esters, as well as other lipid and water soluble substrates (Kraemer and Shen, 2002).

HSL activity was first identified as an epinephrine-sensitive lipolytic activity in adipose tissue. Hormones such as catecholamines, adrenocorticotropic hormone (ACTH), and glucagons stimulate the activity of this lipase (Vaughan et al., 1964). Hormonal activation of HSL occurs via cyclic AMP dependent protein kinase (PKA), which phosphorylates HSL (Yeaman, 1990). As the enzyme responsible for the release of free fatty acids from adipose tissue, HSL plays an important role in providing the major source of energy for most tissues. Although its expression is highest in adipose tissue, HSL is also expressed in adrenal, ovary, testis, in skeletal and cardiac muscle and macrophages (Holm et al., 1988; Kraemer et al., 1993). The major isoform of HSL is a single polypeptide with a molecular mass of 84 kDa and comprises three major domains: a catalytic domain, a regulatory domain encoding several phosphorylation sites and an N-terminal domain involved in protein-protein and protein-lipid interactions (Yeaman, 2004). Members of the HSL family share the characteristic GXSXG motif with the active site serine residue (Holm et al., 1994). In human HSL the catalytic triad consists of Ser-424, Asp-693 and His723 (Holm et al., 1994, Contreras et al., 1996, Osterlund et al., 1997).

The structures of three members of the HSL family have been solved to date, revealing an α/β-hydrolase fold (PDB codes: 1JKM, 1EVQ and 1JJI). 1JKM, called BFAE, is a brefeldin esterase from Bacillus subtilis (Wei et al., 1999, Figure 8, Panel A), 1EVQ, called EST2, is a thermophilic carboxylesterase from Alicyclobacillus acidocaldarius

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Chapter 1 24

A. B.

C.

Figure 8. The crystal structure of three bacterial carboxylesterases of the HSL family. Panel A. The crystal structure of the brefeldin A esterase from Bacillus subtilis (PDB code: 1JKM, chain a) (Wei et al., 1999). Panel B. The crystal structure of the thermophilic carboxylesterase EST2 from Alicyclobacillus acidocaldarius (PDB code: 1EVQ) (De Simone et al., 2000). Panel C. The crystal structure of a hyper- thermophilic carboxylesterase, called AFEST, from the archaeon Archaeoglobus fulgidus (PDB code: 1JJI, chain a) (De Simone et al., 2001). In all three Panels the N- terminal helix is colored dark blue and the C-terminal helix is shown in dark red. The location of the catalytic triad is indicated with the help of black spheres (in Panel C:

Ser-160, Asp-255 and His-285; in Panel A: Ser-202, Asp-308 and His-338; and in Panel B: Ser-155, Asp-252 and His-282).

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Introduction 25 (De Simone et al., 2000, Figure 8, Panel B), and 1JJI, called AFEST, is a hyper- thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus (De Simone et al., 2001, Figure 8, Panel C). All of these proteins are bacterial carboxylesterases with a significant amino acid sequence similarity to Aes from Escherichia coli. BFAE shares an amino acid sequence identity of 25.3 % to Aes in 245 amino acid overlap, EST2 shows an amino acid sequence identity of 29.3 % in 232 amino acid overlap and sequence comparison with AFEST revealed an amino acid sequence identity of 31.3 % with Aes in 252 amino acid overlap (LALIGN server).

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Chapter 2

Purification and crystallization

of Mlc from Escherichia coli

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This work was published in the following journal:

Gerber, K., Boos, W., Welte, W., Schiefner, A. (2005) Crystallization and preliminary X-ray analysis of Mlc from Escherichia coli. Acta Cryst., F61, 183-185.

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Chapter 2 29

Abstract

Mlc is a prokaryotic transcriptional repressor controlling the expression of a number of genes encoding enzymes of the Escherichia coli phospho-transferase system (PTS), as well as manXYZ and malT, the gene of the global activator of the mal regulon.

The mlc gene containing the point mutation R52H has been cloned into a pQE vector and the recombinant protein was expressed and purified as the selenomethionine- labeled derivative. Crystallization of the SeMet-Mlc was carried out using the vapour- diffusion method. The crystals obtained belong to the monoclinic space group C2 with the unit cell parameters a=235.95 Å, b = 74.71 Å, c = 154.95 Å, β = 129.15° and diffract to 2.9 Å resolution.

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Crystallization of Mlc from E. coli 30

Introduction

Mlc was originally cloned onto a multicopy plasmid which rendered Escherichia coli cells more versatile to the use of glucose in complex media so that larger colonies could form „makes large colonies“, Mlc (Hosono et al., 1995). It was found that the mlc gene product slows down glucose uptake so that acetate produced in glycolysis can be more efficiently dissipated before it would inhibit cell growth (Hosono et al. 1995). Mlc is a member of the ROK family (Repressors, ORFs and Kinases) (Titgemeyer et al., 1994, Hansen et al., 2002) and acts as a transcriptional repressor for a variety of sugar- metabolizing enzymes and transport systems (Plumbridge, 1998a, 1998b, 1999, 2001b, 2002, Decker et al. 1998, Böhm and Boos, 2004). The major target for Mlc regulation is the phosphotransferase (PTS)-dependent transport of glucose (Potsma et al., 1993).

Mlc is known to control the expression of the genes for the general components of PTS I (enzyme I, and HPr) and PTS II (EIICB^Glc = PtsG for glucose transport), as well as the genes for the mannose transporter (EIIABCD^Man = PtsM, ManXYZ) (Plumbridge, 2002). In addition to these proteins, also MalT, the central transcriptional activator of the maltose regulon, is subject to transcriptional control by Mlc. The maltose regulon consists of ten genes encoding enzymes involved in the uptake and the metabolism of maltose and maltodextrins (Boos and Shuman, 1998). The particular feature that makes Mlc an attractive subject of study is its mode of regulation. In the absence of external glucose EIIB^Glcis phosphorylated and Mlc does not interact with it (Tanaka et al., 2000). Under this condition Mlc acts as a transcriptional repressor by binding to its target promoter sites. As soon as glucose is present in the external medium it is transported into the cell by the PTS. During this step EIIB^Glctransfers its phosphate to glucose and therebybecomes dephosphorylated. The dephosphorylated state of EIIB^Glc

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Chapter 2 31 is recognised by Mlc. By binding to EIIB^Glc, Mlc releases its specific promoter sites, thereby enabling the production of PTS proteins in the cell (Lee et al., 2000, Nam et al., 2001). Seitz et al. (2003) could show that some mutations within EIIB^Glc eliminate the binding of Mlc to EIIB^Glc. Studies with Mlc mutants suggest that both its N- terminal and C-terminal regions are involved in the interaction with EIIB^Glc (Seitz et al., 2003, Tanaka et al., 2004). Recently it could be demonstrated that membrane association of Mlc alone causes derepression of Mlc specific promoters (Seitz et al., 2003, Tanaka et al., 2004).

Here we describe the expression, purification and crystallization of SeMet-Mlc from Escherichia coli.

Materials and Methods

Expression and purification

For routine work with recombinant DNA, established protocols were used (Sambrook et al., 1989). For the construction of the Mlc expression plasmid, the mlc gene was amplified from the plasmid DNA pSL104 (Lee et al., 2000) using the oligonucleotides A (5'-cat gcc atg gtt gct gaa aac cag cct ggg -3') with an NcoI restriction site and B (5'- ggg aag ctt tta acc ctg caa cag acg-3') with a Hind III restriction site. The PCR mixture contained 50 ng plasmid template DNA, 1 µM of each primer, 10 mM deoxynucleoside triphosphates, 1× buffer for Pfu turbo DNA polymerase and 2.5 U Pfu turbo DNA polymerase. After an initial denaturation step (3 min at 95 °C), 30 cycles consisting of 30 sec at 95°C, 1 min at 44°C and 3 min at 72°C were carried out. The PCR mix was separated on a 1 % agarose gel and the expected 1.22 kbp fragment was isolated with a small glasswool column and purified with 2 volumes of phenol and chloroform (Sambrook et al., 1989). After restriction with NcoI and HindIII, the PCR fragment was again purified as before and ligated into pQE60 (Qiagen), resulting in pQE60mlc.

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The inserted mlc gene was sequenced commercially and one point-mutation was found: R52H. E. coli BL21(DE3) cells (Novagen) were then transformed with pQE60mlc. The Mlc protein was expressed with the selenomethionine-substitution method (Doublie, 1997). A seed culture of 20 ml LB/amp¹⁰⁰was grown over-night at 37°C and was then used to inoculate 8 x 0.25 liters of minimal medium M63 with supplements by adding 1 ml of the seed culture into each flask. One liter of this medium contained 3 g KH²PO⁴, 9.12 g K²HPO⁴, 2 g (NH⁴)²SO⁴, 1.25 mg FeSO⁴, 40 mg of all amino acids except methionine, 50 mg L-selenomethionine, 40 mg hypoxanthine, 40 mg uracil, 20 mg biotin, 20 mg nicotinamide, 2 mg riboflavin, 20 mg thiamine, 0.25 g MgSO4, 2 g glucose and 0.1 g ampicillin. The cells were grown until OD600=0.6 at 37°C and 180 rev/min and the culture was again supplemented with the following amino acids (numbers are given for one liter of culture): 100 mg of L-lysine, L-phenylalanine and L-threonine, 50 mg of L-isoleucine, L-leucine and L- valine and 60 mg of L-selenomethionine. 15 Minutes later the expression of SeMet- Mlc was induced with 0.3 mM IPTG (isopropyl-β-D-thiogalactopyranoside). Over- expression of SeMet-Mlc was continued for 20 hours after induction. Cells were then harvested at 10000 rpm at 4°C. The cell pellet was resuspended in 25 ml buffer A (10 mM glycine pH 9.5, 50 mM NaCl) and the cells were broken with a French press in two cycles at 20 000 psi. The lysate was spun down for 20 min at 11 000 rpm, then the supernatant was spun down again for 30 min at 35 000 rpm. The supernatant was loaded onto a 15 ml Q-sepharose column (Amersham Biosciences) pre-equilibrated with buffer A and washed with buffer A. SeMet-Mlc was eluted from the column with a linear gradient of buffer B (10 mM glycine pH 9.5, 500 mM NaCl) within 5 column volumes. SeMet-Mlc eluted at about 40% buffer B. The purity of the elution fractions was tested on a 12.5 % SDS PAGE and estimated to be about 95%. Samples containing SeMet-Mlc were pooled, dialyzed in 2 liters of buffer A over night at 4°C in dialysis tubing with 12 kDa cut-off (Serva). Afterwards the protein solution was concentrated

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Chapter 2 33 to about 45 mg/ml through a 30 kDa membrane (Vivaspin). The final yield was about 25 mg SeMet-Mlc per liter culture.

Crystallization and data collection

Crystal screening was performed using Screen I (Jancarik and Kim, 1991), Screen II (Hampton Research), Wizard Screens I and II (Emerald Biostructures), Stura Footprint Screens I and II (Molecular Dimensions), and JB screen Nr. 10 (Jena Biosciences) at 18°C in 96 well sitting drop plates (Douglas Instruments) with 100 µl reservoir solution and a drop size of 1.5 µl protein (at 45 mg/ml) plus 1.5 µl reservoir solution. Crystals of the size 150 µm x 100 µm x 50 µm grew within 2 weeks in JB screen Nr. 10/14 (1.6 M MgSO4, 100 mM MES pH 6.5) out of the precipitate (Fig. 1).

Crystals were then reproduced with the hanging drop vapour diffusion method in 24- well plates (Hampton Research) using 1 ml reservoir solution and a protein drop consisting of 2 µl protein and 2 µl reservoir solution. Before data collection single crystals were soaked in three drops of cryo-solution for one minute each and were then quickly transferred into liquid nitrogen. The cryo-solutions consisted of a 1:1 mixture of the reservoir buffer and the protein buffer A with increasing amounts of glycerol (5

%, 15 % and 25 %), respectively. The protein content of single crystals washed three times in cryo-solution was analyzed by 15% SDS-PAGE and Western blot using anti- Mlc antibodies (data not shown).

To solve the structure of Mlc by multiple anomalous dispersion, three data sets were collected from a single crystal of SeMet-Mlc in the order peak, inflection and remote high at the beamline X06SA at the Swiss Light Source (SLS) in Villigen, Switzerland to a resolution of 2.9 Å (Fig. 2). Due to the radiation sensitivity of the SeMet-Mlc crystal we have collected each data set at a different piece of the crystal. Raw data were processed using XDS (Kabsch, 1993).

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Results and discussion

In this report we describe the crystallization and preliminary X-ray analysis of Mlc from Escherichia coli. A search of the Protein Data Bank (PDB) (Berman et al., 2000) did not result in sequences sharing a high similarity with Mlc from E. coli, molecular replacement was therefore not possible. We have expressed Mlc in E. coli BL21 (DE3) strain with the accidental point mutation R52H as a seleno-methionine derivative.

SeMet-Mlc was purified to near homogeneity (data not shown) and crystallized at a concentration of 45 mg/ml with the vapour diffusion method at 18°C. Crystals grew within a few days to a size of 150 µm x 100 µm x 50 µm (Fig. 1) and at first did not diffract at our in-house X-ray source. One year later single crystals from the same crystallization plate diffracted to 4 Å at our in-house X-ray source and to 2.9 Å at the Swiss Light Source in Villigen/ Switzerland (Fig. 2). The reason for this significant improvement of crystal quality is not known. The space group of a single SeMet-Mlc crystal was determined to be C2 (Table 1.) with most likely four molecules per asymmetric unit as deduced from the Matthews coefficient VM=3.0 Å³/Da and a solvent content of 58.5 %.

Until now there are five structures published in the Protein Data Bank of ROK family members, all of them being glucokinases with only a very low sequence similarity to Mlc from E. coli as indicated by sequence alignment (Lalign server, data not shown).

Currently Mlc is the only repressor of the ROK family to be crystallized successfully.

Due to its diverse regulatory function and its special mechanism to “shuttle” between several operon sites and the membrane bound PtsG without any low molecular inducer, Mlc represents an especially interesting candidate for structural analysis.

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Chapter 2 35

Figure 1. A single SeMet-Mlc crystal. The size of the crystal is about 150 x 100 x 50 µm. For the growth conditions see Materials and Methods.

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Figure 2. X-ray diffraction pattern of a single SeMet-Mlc crystal measured at the Swiss Light Source in Villigen, Switzerland. Image edge: 2.7 Å.

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Chapter 2 37 Table 1. Crystal data and X-ray data-collection statistics for a single SeMet Mlc

peak inflection remote high

Wavelength (Å) 0.97637 0.97866 0.97780

egrees)

9 (3.0-2.9) 0 (3.1-3.0) 3 (3.4-3.3)

nit cell parameters (Å, degrees) a = 235.95, b = 74.71, c = 154.95, β = 129.15

.6) 9.3)

)

crystal. Numbers in parentheses refer to the highest resolution shell. Rmeas, the redundancy independent Rmerge, was calculated according to Diederichs and Karplus, 1997.

Rotation range (d 180 180 180

Resolution (Å) ∞- 2. ∞- 3. ∞- 3.

Space group C2

U

Observed reflections 174722 157372 118860

Unique reflections 89905 80926 61032

Completeness (%) 98.5 (99.7) 98.1 (99 98.3 (9

I/σ(I) 9.9 (2.3) 9.5 (2.1) 9.3 (2.2)

Rmerge (% 5.4 (36.6) 5.8 (41.3) 6.3 (38.8)

Rmeas (%) 7.5 (50.4) 8.0 (56.7) 8.8 (53.6)

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Chapter 3

The crystal structure of Mlc, a global regulator of sugar metabolism

in Escherichia coli

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This work is going to be published in the following journal:

Schiefner, A., Gerber, K., Seitz, S., Welte, W., Diederichs, K., and Boos, W. The crystal structure of Mlc, a global regulator of sugar metabolism in Escherichia coli.

(Journal of Biological Chemistry, paper in press)

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Crystal structure of Mlc from E. coli 41

Abstract

Mlc from Escherichia coli is a transcriptional repressor controlling the expression of a number of genes encoding enzymes of the phospho-transferase system (PTS), including ptsG and manXYZ, the specific enzyme II for glucose and mannose PTS transporters. In addition, Mlc controls the transcription of malT, the gene of the global activator of the mal regulon. The inactivation of Mlc as a repressor is mediated by binding to an actively transporting PtsG (EIICB^Glc). In this article we report the crystal structure of Mlc at 2.7 Å resolution representing the first described structure of a ROK (Repressors, Open reading frames and Kinases) family protein. Mlc forms stable dimers explaining its binding affinity to palindromic operator sites. The N-terminal helix-turn-helix domain of Mlc is stabilized by the amphipathic C-terminal helix implicated in EIICB^Glc binding earlier. Furthermore, the structure revealed a metal binding site within the cysteine-rich ROK consensus motif which coordinates a structurally important zinc ion. A strongly reduced repressor activity was observed when two of the zinc- coordinating cysteine residues were exchanged against serine or alanine, demonstrating the role of zinc in Mlc-mediated repressor function. The structures of a putative fructokinase from Bacillus subtilis, the glucokinase from Escherichia coli, and a glucomannokinase from Arthrobacter sp. showed high structural homology to the ROK family part of Mlc.

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Introduction

Mlc (makes large colonies) has been discovered as a regulator protein curbing the utilization of glucose in Escherichia coli (Hosono et al., 1995; Plumbridge, 2002).

Mlc, acting as a transcriptional repressor, controls the expression of malT, encoding the central transcriptional activator of the maltose system (Decker et al., 1998). In addition, Mlc controls the expression of two operons encoding PTS- dependent transporters for glucose ptsG (Kimata et al., 1998; Plumbridge, 1998b) and mannose manXYZ (Plumbridge, 1998a) as well as the genes encoding the general components of the PTS (Kim et al., 1999; Plumbridge, 1999; Tanaka et al., 1999). In contrast to the classical mode of repressor inactivation by a cognate inducer, Mlc is inactivated by the sequestrating interaction with the actively transporting glucose transporter, the EIICB^Glc protein of the PTS (Lee et al., 2000;

Tanaka et al., 2000; Nam et al., 2001). The interaction occurs at the EIIB^Glc domain of the transporter encompassing a critical cysteine residue (Cys421). This cysteine residue is phosphorylated in the resting transporter and becomes readily dephosphorylated during glucose transport by the transfer of the phosphoryl group onto the incoming glucose. Mlc binds only to the dephosphorylated form of EIIB^Glc (Seitz et al., 2003). The membrane bound state of EIIB^Glc is essential for Mlc inactivation. Soluble EIIB^Glc, even though able to interact with Mlc (Nam et al., 2001; Seitz et al., 2003), does not prevent Mlc from binding to its operator regions and from its repressing activity. However, EIIB^Glc attached to the membrane by any lipophilic anchor, even unrelated to EIICB^Glc, binds Mlc in a fashion that prevents binding to the operator regions (Seitz et al., 2003). This indicates that Mlc, when it is in close contact with the membrane, alters its conformation to suppress operator binding.

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Crystal structure of Mlc from E. coli 43 As judged by its amino acid sequence, Mlc belongs to the ROK family (Repressors, Open reading frames and Kinases) (Titgemeyer et al., 1994; Hansen et al., 2002) of transcriptional regulators encompassing xylose repressors, sugar kinases and transcriptional regulators with the widely conserved CXCGXXGCXE motif (consensus sequence 2). They also harbor another consensus motif (consensus sequence 1) consisting of 28 amino acid residues, located 9 residues upstream from consensus sequence 2 (Hansen et al., 2002). The DNA-binding motif of Mlc consists of a typical helix-turn-helix motif at its N-terminus and the protein behaves in dilute buffer solution as tetramer of a polypeptide of 44.3 kDa molecular weight (Nam et al., 2001; Seitz et al., 2003). The removal of the 18 C- terminal residues leads to dimer formation, to the loss of EIICB^Glc binding as well as to the loss of operator interaction (Seitz et al., 2003). Thus, surprisingly, the C- terminus which is far from the helix-turn-helix motif in the primary sequence must be involved, directly or indirectly, possibly via a large conformational change, in EIICB^Glc binding as well as in operator recognition and subsequent repression.

Regarding its unusual mechanism of derepression, it was of interest to elucidate the crystal structure of this novel transcriptional regulator. Here we report the 3- dimensional structure of dimeric Mlc R52H at 2.7 Å resolution.

Results

Structure of the Mlc monomer

The structure of Mlc represents the first described structure of a ROK family member. An Mlc molecule shown in Figure 1 consists of three domains: a) a helix- turn-helix (HTH) domain (Harrison and Aggarwal, 1990; Wintjens and Rooman, 1996) from amino acid residue 1 to 81 + 395 to 406 (domain 1, green), b) a smaller α/β−domain from residue 82 to 194 + 381 to 394 (domain 2, yellow) and c)

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a larger α/β-domain from residue 195 to 380 (domain 3, blue). The final Mlc model contains 382 out of 406 residues. Two segments in domain 1, residues 1 - 11 and 64 – 76, were structurally disordered in all four molecules within the asymmetric unit and were therefore not included into the final model. The structurally disordered region 64 – 76 of the HTH domain is known to be very flexible from previously described HTH motifs. It adopts its destined conformation, the so called hinge helix, only upon binding to the operator DNA (Lewis et al., 1996; Spronk et al., 1996; Kalodimos et al., 2002). Domains 2 and 3 are common to all ROK family members (Titgemeyer et al., 1994; Hansen et al., 2002) (see Figure 1, yellow and blue). Both α/β-domains (2 and 3) consist of a central β-sheet flanked by a pair of α-helices on one side and a single α-helix on the other side. Between domains 2 and 3 the polypeptide chain switches twice, so that domain 3 is formed by a continuous polypeptide while the fold of domain 2 is completed by the returning C-terminus from domain 3, packing as a C-terminal helix against the β-sheet of domain 2 (bright-yellow in Figure 1). The interface between domains 2 and 3 is mainly formed by the two single α-helices flanking the β-sheet in each domain. However, the packing of both domains toward each other is not very tight, allowing the domains to adopt different conformations with respect to each other. In addition to being part of domain 2, the C-terminal helix bends and is also part of domain 1 (bright-green in Figure 1), thereby stabilizing the orientation of the HTH domain (domain 1) with respect to domain 2. This stabilization might be the reason why the HTH part of domain 1 is structurally ordered while the connecting segment, including the hinge helix in domain 1, is not.

The three domains of an Mlc monomer behave as rigid groups but the pair wise arrangement of the domains is different in the four molecules within the asymmetric unit. The differences in the domain orientations were analyzed with

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Crystal structure of Mlc from E. coli 45 the program DYNDOM (Hayward and Berendsen, 1998) using domain 3 of the Mlc molecule A as the reference. Several rotational axes do not coincide in all possible domain pairs of the four Mlc molecules (A-D). Domains 1 and 2 are rotated as single units in molecules B and C by 18 and 5 degrees, respectively. In molecule D, however, domain 2 is rotated by 22 degrees and domain 1, with respect to domain 2, is rotated by 12 degrees around another axis at the same time. In addition, the comparison of molecule A with molecule B shows that residues 244 – 270 of domain 3, harboring parts of the two ROK motifs, are able to rotate separately by 14 degrees with respect to the rest of domain 3.

The ROK signature forms a zinc binding site

Domain 3 contains consensus motifs 1 and 2 that characterize the ROK family members (Titgemeyer et al., 1994; Hansen et al., 2002). Both motifs are highlighted in Figure 1 as red and orange ribbons. Consensus motif 1 (red) forms part of the central β-sheet in domain 3, leading into a loop followed by a short 3 - helix that ends with the invariant histidine His247. Nine residues downstream, consensus motif 1 is followed by consensus motif 2 (orange), starting with the conserved cysteine residues Cys257 and Cys259, followed by the conserved cysteine residue Cys264. The structural explanation for the conservation of these residues is the tetrahedral coordination of a zinc ion by the four residues His247, Cys257, Cys259, and Cys264 (see Figure 1, highlighted as a gray sphere). The presence of the zinc ion (0.9 +/- 0.1 zinc per protein) was confirmed by atom absorption spectroscopy (AAS), electron paramagnetic resonance (EPR) and UV-Vis spectroscopy.

10

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The Mlc dimer and its binding to DNA

The four molecules within the asymmetric unit are arranged as two homodimers AB (chains A and B) and CD (chains C and D). Figure 2A shows AB perpendicular to the 2-fold axis and Figure 2B shows AB along the 2-fold axis of the dimer. The dimerization occurs via domain 3 (blue) of each monomer, burying a surface area of 1378 Å²(in case of AB) and 1393 Å²(in case of CD). In the CD dimer there is an additional contact of 303 Å²between the HTH domains resulting from the above mentioned Mlc flexibility and the crystal packing but it is apparently not relevant for the dimer formation. Both dimers found in the asymmetric unit show different conformations. Whereas the superposition of chains A and C shows almost identical molecules, that of chains B and D reveals two conformationally distinguishable dimers (see superposition in Figure 3A). The conformational flexibility results in different distances between the two recognition helices in each dimer. Figures 3B-3D show the isolated HTH domains of both dimers (AB in yellow and CD in blue). In dimer AB the distance between the recognition helices is with ~31.7 Å very close to the helical pitch of B-DNA, which is 34 Å (Figures 3B and 3C). On the other hand, the CD dimer is much narrower with a distance of ~26.7 Å between the two recognition helices (Figures 3D and 3E) excluding its participation in DNA binding in this conformation. In addition, Figures 3C and 3E show that the recognition helices are not completely parallel, as one would assume from Figures 3B and 3D. The DNA would have to bend upon Mlc binding in order to accommodate both recognition helices of the AB dimer.

A tetramer of Mlc

Earlier studies using size exclusion chromatography indicated that Mlc forms tetramers in vitro (Nam et al., 2001; Seitz et al., 2003). In order to determine whether the biochemically described tetramer is present in the Mlc crystals as well,

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Crystal structure of Mlc from E. coli 47 we investigated all intermolecular contacts of the two Mlc dimers (AB and CD) within possible asymmetric units. The most symmetric arrangement relates both Mlc dimers by a pseudo 2-fold axis via domains 3. The single contacts are relatively weak with only 600 Å² between chains A and D and 636 Å² between chains B and C. Both contacts taken alone are not significant for a stable multimer formation (Janin, 1997). On the other hand, the sum of both contacts in a dimer of dimers with 1236 Å² could be relevant for a tetrameric structure. Nevertheless, we do not consider the crystallographic tetramer to be physiologically relevant.

Comparison of Mlc with related structures

A similarity search using the DALI server (Holm and Sander, 1993) revealed three bacterial kinases having the same structure as the ROK part of Mlc. In Table 3 the alignment lengths and the r.m.s. deviations of the identical Cα-positions are listed.

The highest structural homology to Mlc shows a putative fructokinase from Bacillus subtilis (Bs-FrcK), PDB code 1XC3, which has not been published so far.

Bs-FrcK belongs to the ROK family as well, a fact explaining the structural homology. Its zinc ion is coordinated by two histidines and two cysteines spaced by a different number of amino acids as compared to Mlc. The second and third most similar structures are the glucokinase from E. coli (Ec-GlcK), PDB codes 1Q18 and 1SZ2 (Lunin et al., 2004), and a bacterial inorganic polyphosphate/ATP glucomannokinase from Arthrobacter sp. (As-GMK), PDB code 1WOQ (Mukai et al., 2004). Both proteins, Ec-GlcK and As-GMK, are not regarded as ROK family proteins but are in fact very similar to the ROK part of Mlc consisting of domains 2 and 3.

Surprisingly, four of the five residues directly involved in glucose binding in Ec- GlcK and As-GMK are identical in Mlc and Bs-FrcK (Asp195, Glu244, His247, Glu266, Mlc numbering). The fifth residue, an asparagine in Ec-GlcK and in As-

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GMK is a histidine (His194) in Mlc and a threonine in Bs-FrcK. However, despite the high structural similarity of the corresponding region in Mlc to the binding site for glucose in Ec-GlcK and As-GMK, Mlc does not bind glucose or glucose-6-P as measured by the ammonium sulfate precipitation technique (Richarme and Kepes, 1983). The same technique readily revealed glucose binding of Ec-GlcK (data not shown). This suggested that the fifth position (His194 in Mlc) either discriminates between different sugars or between sugar binding and non-binding. However, altering His-194 to Asn did not result in glucose binding or glucokinase activity (Marc Erhardt, unpublished results).

The structural homology between the monomeric forms of Mlc, Bs-FrcK, Ec-GlcK and As-GMK indicated similar quaternary structures. Our Mlc structure clearly shows two dimers within the asymmetric unit. In Table 3 the contact interfaces within the crystal packings are listed, showing the same 2-fold symmetry as the Mlc dimer. According to their buried surfaces, Bs-FrcK, Ec-GlcK and As-GMK could be able to form stable homodimers of similar architecture as well (see Table 3).

Structural localization of Mlc mutants

All mutations in Mlc characterized so far are shown in Figure 4, highlighted by different colors. The mutant R52H has been accidentally selected on plates containing Luria Bertani (LB) medium during the cloning step for the structural analysis (Gerber et al., 2005). Although the mutation is located in the recognition helix of the HTH domain, the protein still showed full repression of a ptsG-lacZ fusion (Figure 5, gray histograms). In the presence of glucose, both the wild type Mlc and the R52H mutant show derepression of Mlc regulated genes (Figure 5, black histograms) demonstrating that the R52H mutation neither affects repression nor induction. The latter is equivalent to the ability of Mlc to be bound by EIICB^Glc.

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Crystal structure of Mlc from E. coli 49 To study the role of the bound zinc ion in more detail, we constructed two double mutants by changing C257 and C259 into alanine or serine, respectively, resulting in Mlc C257A/C259A and Mlc C257S/C259S. Both mutant proteins showed only residual ability to repress the ptsG-lacZ fusion, pointing to a structural role of the zinc ion necessary for DNA binding (Figure 5, gray histograms).

Seitz et al. (2003) found that C-terminal deletions of Mlc influence its ability to tetramerize in vitro as well as its activity as a transcriptional repressor and its capacity to bind EIICB^Glc in vitro. While the deletion of the last nine residues (Mlc∆C9) does not influence the activity of Mlc, the deletion of the last 18 residues (Mlc∆C18) produces a protein no longer able to tetramerize in vitro and being unable to bind its operator sites or EIICB^Glc. Figure 4 shows the parts deleted in Mlc∆C9 and Mlc∆C18, as yellow and yellow + orange ribbons, respectively.

Furthermore, four point mutations of Mlc have been characterized by Tanaka et al.

(2004). Two of these mutants, H86R and I34V, are impaired in EIICB^Glc binding (see Figure 4). This observation indicates that Mlc might bind to unphosphorylated EIICB^Glc with both the HTH domain and domain 2. On the other hand, Mlc mutants G211R and P294S show raised expression levels but they neither influence repression nor binding to EIICB^Glc (Tanaka et al., 2004).

Discussion

The structure of Mlc

Overall, the Mlc molecule consists of three domains. Only domain 3, which contains both ROK consensus motifs, is composed of one continuous polypeptide, whereas domains 1 and 2 are completed by the back-folding of the C-terminus of the molecule. This structural arrangement results in a defined orientation of domain 1 with respect to domain 2. The asymmetric unit of the Mlc crystals

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contains four molecules which are clearly arranged as two dimers with a buried surface of ~1400 Å²in each dimer. The dimer formation between two Mlc monomers occurs only via domain 3. Both domains 3 seem to form a stable scaffold with domains 1 and 2 flexibly attached to them. The hinge between domains 2 and 3 allows the movement of both domains 1 in an Mlc dimer with respect to each other. In this way the Mlc dimer is able to adopt different conformations. We conclude that the dimer contact is of biological relevance for three reasons: 1. the buried surface within the dimer is much larger than expected for an artificial crystal contact, 2. Mlc needs to be a dimer with the recognition helices being in proximity to bind to palindromic DNA, and 3. the structurally very similar molecules Bs-FrcK and Ec-GlcK apparently form dimers of similar architecture.

Furthermore, the structural similarity to bacterial sugar kinases suggests that Mlc represents a former kinase reused as a transcriptional repressor by the fusion of an HTH domain at its N-terminus.

What determines the ROK family proteins?

Mlc represents the first structure of a ROK family protein described to date. Two non-overlapping consensus motifs characteristic for ROK family proteins have been described by sequence comparison (Hansen et al., 2002). Based on the Mlc structure these two consensus motifs can be merged into a single one forming a zinc binding site. From structural and sequence data two similar zinc binding motifs GHX9-11CXCGX2GC/HXE and GHX11-17CX2HX2CXE, can be distinguished in ROK family proteins (metal binding residues highlighted in bold).

Mlc and most of the published protein sequences of ROK family proteins contain the first zinc binding motif, whereas the second one is found only in a minority of the investigated sequences. The underlined residues are in the same position in the ROK proteins Mlc, Bs-FrcK and in the non-ROK proteins Ec-GlcK and As-GMK.

X-ray crystallographic studies on Mlc and Aes, two transcriptional modulators from Escherichia coli