Structure and function of peptide deformylase

Crystallization, data collection, and structure determination of substrate- or inhibitor-bound XoPDF .... Crystallization, data collection, and structure determination of fragment chemic

Dissertation for Degree of Doctor

Supervisor: Prof Lin-Woo Kang

Structure and Function of Peptide

Deformylase

Submitted by

HO THIEN HOANG

February, 2018 Department of Biological Sciences

Graduate School of Konkuk University

Structure and Function of Peptide

Deformylase

A Dissertation submitted to the Department of Biological Sciences and the Graduate School of Konkuk University in partial fulfillment of the requirements for the degree of Doctor

of Philosophy

Submitted by

HO THIEN HOANG

December, 2017 This certifies that the Dissertation of

HO THIEN HOANG is approved

Approved by Examination Committee:

TABLE OF CONTENTS

List of Figures……… … ……… iv

Abstract……….……… v

Chapter 1 Introduction 1

1.1 Peptide deformylase 1

1.1.1 The important of peptide deformylase in cell 1

1.1.2 Structure and mechanism of peptide deformylase 5

1.1.3 Xanthomonas oryzae pv oryzae 8

1.1.4 Acinetobacter baumannii 10

1.1.5 Three-dimensional structure determination methods 12

Chapter 2 Methods 18

2.1 Cloning of peptide deformylase from Xanthomonas oryzae pv oryzae 18 2.2 Overexpression and purification of XoPDF 18

2.3 Crystallization, data collection, and structure determination of substrate- or inhibitor-bound XoPDF 21

2.4 Crystallization, data collection, and structure determination of fragment chemical compound-bound XoPDF 22

2.5 Structure determination and refinement of XoPDF 23

2.6 Cloning of AbPDF 24

2.7 Overexpression and purification of AbPDF 24

2.8 Crystallization and X-ray data collection of AbPDF 25

Chapter 3 Results 27

3.1 Overall apo-XoPDF structure 27

3.2 Substrate-complex structure 36

3.3 Inhibitor-complex structure 39

3.4 Fragment chemical-complex structures 41

3.5 Overall apo-AbPDF structure 42

3.6 Inhibitor-complex AbPDF structure 47

Chapter 4 Discussion 49

References 58

Abstract (in Korean) 66

List of Tables

Table 1 Data collection and refinement statistics 33 Table 2 Data-collection and processing statistics 44

List of Figures

Figure 1.1 Proposed catalytic mechanism of PDF 8

Figure 1.2.Workflow for solving the protein structure by X-ray crystallography 13

Figure 1.3 Schematic illustration of a protein crystallization phase diagram 16 Figure 1.4 Two methods of preparing A: Hanging drop B: Sitting drop 17

Figure 2.1 Purified XoPDF is shown on a 15% SDS-PAGE gel 20

Figure 3.1 Native structure of XoPDF (a) 28

Figure 3.2 Metal binding and coordination of XoPDF 29

Figure 3.3 Sequence alignment of PDFs 30

Figure 3.4 Superimposed apo-, MA-bound, and MAS-bound XoPDF structures 31

Figure 3.5 Structures of PDF CD-loops 32

Figure 3.6 Refined electron density map of bound substrates and inhibitor actinonin 37

Figure 3.7 Substrate methionine-binding site 39

Figure 3.8 Comparison of actinonin-bound and FCC-bound XoPDF structures 40

Figure 3.9 Chemical structure and refined electron density maps of bound FCCs 42

Figure 3.10 Purified AbPDF on a 12% SDS-PAGE gel Molecular-mass markers are shown in lane M and purified AbPDF is shown in lane P 43

Figure 3.11 Native structure of AbPDF 46

Figure 3.12 Structure of CD-loop AbPDF 47

Figure 3.13 Superimposed apo-, Actinonin-bound AbPDF structure 48

Figure 4.1 Time-resolved transcriptional gene expression of XoPDF (Xoo1 075, def) and Xoo0585 genes 50

Figure 4.2 Sequence alignment of XoPDF and eukaryotic PDFs 52

Figure 4.3 Structure comparison between XoPDF and AtPDFs 53

Figure 4.4 Bottom view of substrate methionine-binding site 56

Figure 4.5 Exposure of Phe134 residue in XoPDF with the flexible open conformation of CD loop 57

Figure 4.6 Actinonin structure in XoPDF 57

ABSTRACT

Structure and Function of Peptide Deformylase from

Acinetobacter baumannii and Xanthomonas oryzae pv oryzae

Ho, Thien Hoang Department of Biological Sciences Graduate School of Konkuk University

Peptide deformylase (PDF) is an enzyme responsible for catalyzing the removal of the N-formyl group from the N-terminus following translation in bacterial cells and is an important target to develop antibacterial agents I

determined crystal structures of two PDFs from Acinetobacter baumannii and Xanthomonas oryzae pv oryzae Acinetobacter baumannii is a Gram-

negative opportunistic pathogen and has a high risk infection among immune impairment patients, particularly those who have stayed in hospital long-

term Acinetobacter baumannii is one of the most common and serious multidrug-resistant pathogens Xanthomonas oryzae pv oryzae causes

bacterial blight on rice; this species is one of the most devastating disease of rice worldwide In addition to the apo form of PDF structures, substrate and fragment chemical bound PDF structures were also determined, which include two substrate-bound (methionine-alanine or methionine-alanine-

serine), an inhibitor-bound (actinonin), and six fragment chemical-bound structures Six fragment chemical compounds were bound in the substrate- binding pocket The fragment chemical-bound structures were compared to the natural PDF inhibitor actinonin-bound structure The structural studies will be useful to design new inhibitors specific to AbPDF and XoPDF and

potential antibiotics against Acinetobacter baumannii and Xanthomonas

oryzae pv oryzae

Key words: Acinetobacter baumannii, Xanthomonas oryzae pv oryzae,

peptide deformylase, Bacterial blight (BB) disease, opportunistic bacterial pathogen

1 Chapter 1 Introduction 1.1 Peptide deformylase

1.1.1 The important of peptide deformylase in cell

Peptide deformylase are metalloprotease in bacteria, plants (chloroplast), and

humans (mitochondrial) that removes the formyl moiety on the methionine of

N-formyl-methionine peptide in newly synthesized polypeptides which is essential process for them (Escobar-Alvarez et al., 2010) The protein synthesis usually stars

with an N-formyl-methionine residue In its initiation complex, the key components

in protein synthesis are the initiator fMet-tRNAf

Met

, the methionine-tRNAf

Met

molecule is N-formylated by a methionyl-tRNAf

Met

formyltransformylase, so that the newly synthesized proteins are formylated at the amino terminus (Miesel, Greene, & Black, 2003) The formylation of the methionine tRNAf

Met

molecule is a crucial role in recognition of IF2 factor, and finally the codon is adjusted in the P site, contributing to the accuracy of translation initiation (Petersen, Roll, Grunberg-Manago, & Clark, 1979; Sundari, Stringer, Schulman, & Maitra, 1976) After the process of initiation, elongation of protein translation lead to the displacement of IF2 factor by EF-Tu molecules and GTP, which protect Met-tRNAf

Met

against enzymatic hydrolysis and

recognize non-formylated tRNAf

Met

(Hansen, Wikman, Clark, Hershey, & Uffe Petersen, 1986) The interaction of complex component is necessary to process of translation to complete the polypeptide transcription (Kaczanowska & Ryden-Aulin, 2007)

In bacteria, a formyl-methionine residue is inherently incorporated at the terminus of all nascent polypeptides After that, the N-formyl is removed from the mature protein Peptide deformylase is required to remove the formyl group from a newly synthesized polypeptide chain Finally, methionine aminopeptidase (MAP) remove the N-terminal methionine residue from the nascent protein PDF is broadly conserved in more than 90 sequence bacterial genome (Miesel et al., 2003; Yuan, Trias, & White, 2001) The gene knockout experiments showed that it is essential

N-for the growth of Escherichia coli, Streptococcus pneumoniae and Staphylococcus aureus (Apfel et al., 2001; Margolis et al., 2000; A S Waller & Clements, 2002)

Methionine residue excision by MAP depends on the properties of the adjacent amino acids (Ben-Bassat et al., 1987) The preferences have been showed that MAP reaction is more efficient when the adjacent amino acid is either Val, Thr, Ala, Gly, Ser, Pro, or Cys, but not when the adjacent amino acid is Phe, Leu, Met, Glu, Arg,

or lysine (Ben-Bassat et al., 1987; Hirel, Schmitter, Dessen, Fayat, & Blanquet, 1989) However, activity of MAP is inhibited by the N-blocked polypeptides (Ben-Bassat et al., 1987; Hirel et al., 1989; Solbiati, Chapman-Smith, Miller, Miller, & Cronan, 1999) PDF is necessary to remove the N-formyl group for subsequent N-terminal processing by MAP cleavage of the N-terminal methionine residue So that,

PDF is an essential enzyme in bacteria and promising target for antibacterial (Ranjan, Mercier, Bhatt, & Wintermeyer, 2017)

So far, the functional of formylation in translation initiation is still poorly understood More than 50% of all bacterial protein is subject to N-terminal methionine removal, but it is a unique feature in the eukaryotic cytosol mitochondrial, plastid translation systems and archaea (Ranjan et al., 2017) Although protein synthesis with an N-formylmethionine in the organelles of all eukaryotes, deformylation is apparently absent from mitochondrial protein

translation in yeast Saccharomyces cerevisiae or in the nematode Caenorhadditis elegans (Giglione, Pierre, & Meinnel, 2000; Giglione, Serero, Pierre, Boisson, &

Meinnel, 2000; Guillon, Mechulam, Schmitter, Blanquet, & Fayat, 1992), honey (Polz & Kreil, 1970), and bovine (Steffens & Buse, 1976) where their N-formylmethionine moiety is retained The recently studies about whole genome

sequencing of higher plants such as Solanum lycopersicum, Arabidopsis thaliana, and Oryza sativa, which identified as deformylase-like genes in plant code for

functional eukaryotic PDFs (Giglione, Serero, et al., 2000) These enzymes occur to

be localized in the mitochondria and chloroplasts of plant (Bracchi-Ricard et al., 2001; Dirk, Williams, & Houtz, 2001; Giglione, Serero, et al., 2000; Hauska, Nitschke, & Herrmann, 1988; Schmidt, Herfurth, & Subramanian, 1992; Shanklin, DeWitt, & Flanagan, 1995) and these eukaryotic PDFs are active in vitro and in vivo (Bracchi-Ricard et al., 2001; Dirk et al., 2001; M D Lee et al., 2003; Nguyen

et al., 2003; Serero, Giglione, Sardini, Martinez-Sanz, & Meinnel, 2003) Another

PDF was identified and characterized in Plasmodium falciparum, the primary

parasite responsible for malaria in humans (Bracchi-Ricard et al., 2001) This catalytically active deformylase (PfPDF) is inhibited by actinonin and other know PDF inhibitors, which suggests PfPDF as a potential target for antimalarial therapies (Bracchi-Ricard et al., 2001; Dirk et al., 2001; Guilloteau et al., 2002; Kumar et al., 2002; M D Lee et al., 2003; Nguyen et al., 2003; Serero et al., 2003) Recently, the

human Pdf gene has been determined This gene contains 2 exons on chromosome

16, and the gene product is homologous to other characterized PDFs with some significant differences (Escobar-Alvarez et al., 2010; M D Lee et al., 2004) The human Pdf mRNA was expressed at the same level in all types of human tissues (Giglione, Serero, et al., 2000) More recently, the studies have shown that a recombinantly expressed human PDF is active in vitro (M D Lee et al., 2003; Nguyen et al., 2003; Serero et al., 2003) Therefore, the data suggest that N-terminal protein processing is evolutionary conserved and may be important in chloroplasts

or mitochondria of at least some eukaryotic organisms, although its role in mammals is controversial (Cynamon, Alvirez-Freites, & Yeo, 2004; Nguyen et al., 2003)

There are 3 classes of PDFs based on structural and sequence analyses (Giglione, Pierre, et al., 2000; Guilloteau et al., 2002) Type 1 is divided into 2 subclasses, whereas PDF1b includes enzymes found in Gram-negative bacteria, some Gram-Positive bacteria, and plants The eukaryotic PDF1b enzymes are targeted to both plastids and mitochondria (Giglione, Serero, et al., 2000) Type 2

and type 3 PDFs are found only in Gram-positive bacteria; however, type 3 PDFs are found in archaea and trypanosomatids and have not been studied experimentally yet (Bouzaidi-Tiali et al., 2007; M D Lee et al., 2004)

1.1.2 Structure and mechanism of peptide deformylase

The first PDF was identified and detailed characterization studies due to its extreme instability by Adam in 1978 (Adams, 1968) Another PDF was later cloned,

overexpressed from a plasmid carrying fms gene by Meinnel in 1993 (Meinnel &

Blanquet, 1993) These studies show the greatly facilitated our mechanistic

understanding of PDF The first PDF Overexpressed and purification of PDF from E coli have been characterized However, enzyme activity was extremely poor for formyl-Met-Ala-Ser substrate (kcat/KM of 80 M-1s-1), It has been shown that an

zinc ion inside a polypeptide (Meinnel & Blanquet, 1995) However, in 1997, Rajagopalan indicated that under oxygen-free conditions, the activity of pure PDF enzyme was highly active forward the formyl-Met-Ala-Ser substrate with a

kcat/KM of 2.9 x 104 M-1s-1 , and one ferrous ion was also observed in a monomeric polypeptide (Rajagopalan, Datta, & Pei, 1997)

Subsequently, the X-ray structure indicated that the Fe2+ was presented at the putative active site in PDF (Groche et al., 1998; Rajagopalan P T Ravi 1997) Extraordinary lability of the ferrous-containing PDF has been explained the initial results The results demonstrated that the Fe2+ ion could be oxidized in to the Fe3+ ion by atmospheric O2 (Meinnel & Blanquet, 1993; Rajagopalan & Pei, 1998), the

Fe2+ metal ion was also oxidized and replaced by Zn2+ ion during their purification

when exposed to air, which observed lower catalytic activity.(Rajagopalan P T Ravi 1997)

The structure of PDF have been researched and show that the containing PDF can be easy replaced by a divalent metal such as Ni2+ (Groche et al., 1998) or Co2+ (Rajagopalan, Grimme, & Pei, 2000) in purification to form a highly stable structure and function of enzyme Among the metals, nickel and cobalt are oxygen insensitive and activity of them as the ferrous-containing PDF Through structural and mechanistic studies recently have been confirmed that the important

ferrous-of oxygen in future biochemical studies ferrous-of peptide deformylase (Rajagopalan & Pei, 1998)

PDF is a novel class of iron metalloenzyme, and belong to the metalloproteinase superfamily (Frank et al., 2013) Protein from family share a common structure containing three highly conserved motifs that together form the entire active site: GΦGΦAAXQ, EGCXS, and HEXXH (where Φ is any hydrophobic amino acid and X is any amino acid) (Giglione, Boularot, & Meinnel, 2004) Several structure of Fe-PDF, Zn-PDF, and Ni-PDF(free) forms have been determined by X-ray and NMR method (Becker, Schlichting, Kabsch, Groche, et al., 1998; Becker, Schlichting, Kabsch, Schultz, & Wagner, 1998; Chan et al., 1997; Dardel, Ragusa, Lazennec, Blanquet, & Meinnel, 1998; Meinnel, Blanquet, & Dardel, 1996) In each case, the catalytic metal ion has been shown to coordinate two histidine residues of the conserved HEΦDH motif, one cysteine residue from EGCΦS motif, and a water molecule (H P Ngo et al., 2016) The co-crystallization

of PDF (Fe-PDF, Zn-PDF, Cd-PDF and Ni-PDF) with a reaction product

(Met-Ala-Ser), inhibitor actinonine, transition state analog, and caproyl-L-leucyl-p-nitroanilide (PCLNA) revealed that the S1‟ site has a

(S)-2-O-(H-phosphonoxy)-L-hydrophobic pocket and interact with the methionine side chain (Becker, Schlichting, Kabsch, Schultz, et al., 1998; Hao et al., 1999; H P Ngo et al., 2016) Moreover, the S1‟ and S2‟ are formed from hydrogen bonding between the carbonyl and amide protons of the peptide backbone

A proposed mechanism of catalysis is shown in (Figure 1.1) Step 1 represents the initial state, the metal-bound water molecule is hydrogen bonded to the side chain of Glu133 In the next step, the formylated peptide binds as the model

of the enzyme-substrate complex Consequently water W2 is replaced by the carbonyl oxygen of the formyl group Formyl group is stabilized by the hydrogen bonds to the amide of Leu91 and the side chain Gln50 This supports the nucleophilic attack of W1 on the carbonyl carbon of the formyl group The carbonyl oxygen is tetrahedrally ligated by the metal, carbonyl carbon, side chain amide of Glu50, and main chain amide of Leu91 (Becker, Schlichting, Kabsch, Groche, et al., 1998) The proton of W1 is transferred to the amide at the N-terminus of peptide with the help of Glu 133, the providing of positive charge make the nitrogen suitable as a leaving group Therefore the ternary enzyme-formate-peptide complex was form The five-coordinated metal and the free N-termimus are bound to the formate by the hydrogen bonds A proton is transferred from Glu133 which lead to release an activated enzyme-formate complex and is replaced by a water to

complete the catalytic cycle (Becker, Schlichting, Kabsch, Groche, et al., 1998)

Figure 1.1 Proposed catalytic mechanism of PDF (Becker, Schlichting,

Kabsch, Groche, et al., 1998)

1.1.3 Xanthomonas oryzae pv oryzae

Rice is the staple food for more than half of the human population, especially

in Asia Bacterial blight on rice, caused by the gram-negative bacterium belongs to

Gracillicutes, Xanthomonas oryzae pv oryzae (Xoo), is a serious problem in rice

cultivation and no effective pesticide currently exists against it Xoo infections are not only endemic to Asia and West Africa, but also have been reported from Australia and Latin America As the world population grows rapidly, the rice

production should also increase by at least 25% by 2030 (Li, Wang, & Zeigler, 2014) The complete genome sequence of Xoo was determined (B M Lee et al., 2005; Ochiai, Inoue, Takeya, Sasaki, & Kaku, 2005) and systematic efforts have been made to find pathogenicity-related genes from Xoo and resistance genes from rice (Zhang & Wang, 2013) In addition, several crystal structures of target proteins from Xoo were determined which are essential in bacterial cell wall synthesis, lipid synthesis, and peptide synthesis (Doan et al., 2014; J K Kim et al., 2013; Natarajan

et al., 2012)

In bacteria, Proteins are synthesized with N-formyl methionine, which is

formylated from methionyl-tRNAf

Met

by a formyl-transferase (Becker, Schlichting, Kabsch, Groche, et al., 1998; Lucchini & Bianchetti, 1980) The formyl group of methionine is subsequently removed by peptide deformylase (PDF; EC 3.5.1.31) and methionine aminopeptidase during translational elongation (Dohmen, 2015; J P

Waller, 1963) The N-formyl group at the N-terminus of proteins is removed by PDF Subsequently deformylated N-methionine is cleaved by methionine aminopeptidase

(MAP) (Adams & Capecchi, 1966; Fry & Lamborg, 1967) MAP can recognize

only deformylated N-methionine, and not N-formyl methionine The inhibition of

PDF in bacteria can disrupt the protein maturation process and eventually prohibit bacterial cellular processes and cause bacterial cell death (Chang, McGary, & Chang, 1989; Miller, Kukral, Miller, & Movva, 1989) PDF‟s essential role in protein maturation has been proven for various pathogenic bacteria such as

Escherichia coli, Streptococcus pneumonia, Staphylococcus aureus, and

Mycobacterium tuberculosis (Apfel et al., 2001; Margolis et al., 2000; Mazel, Coic,

Blanchard, Saurin, & Marliere, 1997; Teo et al., 2006)

PDFs usually work in monomeric form and require metal ions such as Fe2+,

Co2+, or Ni2+ for hydrolytic activity (Groche et al., 1998; Miller et al., 1989; Pei, 2001; Ragusa, Blanquet, & Meinnel, 1998; Rajagopalan et al., 2000; Vallee & Auld, 1990).Like other PDFs, XoPDF share a common structure containing three highly conserved motifs that together form the entire active site: GΦGΦAAXQ, EGCXS, and HEXXH (where Φ is any hydrophobic amino acid and X is any amino acid) (Meinnel, Lazennec, Villoing, & Blanquet, 1997), are positioned next to each other

in the three-dimensional structure and form the active site pocket with the metal ion (Mazel et al., 1997)

Thus far, no plant pathogen PDF structure has been determined based inhibitor/drug screening study has been limited mostly to human pathogen targets (Hoover et al., 2015; Kumari, Issar, & Kakkar, 2014; H Y Lee et al., 2016; Yang et al., 2014) The present study reports crystal structure of XoPDF as apo and

Structure-in complex with substrates (methionStructure-ine-alanStructure-ine, MA or methionStructure-ine-alanStructure-ine-serStructure-in

e, MAS), actinonin, and six fragment chemical compounds (FCCs) and systematic approaches to screen inhibitors against XoPDF using structural studies The FCCs will provide useful information for the development of XoPDF inhibitors

1.1.4 Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen whichhas a high incidence among immunocompromised individuals, particularly those who have

experienced a prolonged (> 90 d) hospital stay (Montefour et al., 2008).Commonly associated with aquatic environments (Turton et al., 2006),it has been shown to colonize the skin as well as being isolated in high numbers from the respiratory and oropharynx secretions of infected individuals (Sebeny, Riddle, & Petersen, 2008).In recent years, it has been designated as a “red alert” human pathogen, generating alarm among the medical fraternity, arising largely from its extensive antibiotic resistance spectrum.(Cerqueira & Peleg, 2011; Montefour et al., 2008) This phenomenon of multidrug-resistant (MDR) pathogens has increasingly become a cause for serious concern with regard to both nosocomial and community-acquired infections (Peleg, Seifert, & Paterson, 2008) Indeed, the World Health Organization (WHO) has recently identified antimicrobial resistance as one of the three most important problems facing human health (Bassetti, Ginocchio, & Mikulska, 2011).The most common and serious MDR pathogens have been encompassed within the acronym “ESKAPE,” standing for Enterococcus faecium,

Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp (Rice, 2008)

While in the 1970s A baumannii is thought to have been sensitive to most antibiotics, today the pathogen appears to exhibit extensive resistance to most first-line antibiotics (Fournier et al., 2006).More recently, A baumannii has become a major cause for concern in conflict zones, and has gained particular notoriety in the resent desert conflicts in Iraq, earning it the moniker “Iraqibacter.” In particular,

high incidences of MDR bacteremia (bloodstream infections) have been noted among US Army service members following Operation Iraqi Freedom (OIF) (Centers for Disease & Prevention, 2004)

1.1.5 Three-dimensional structure determination methods

Three-dimensional determination of molecules and molecular assemblies allows us to understand biological processes at the most basic level: which molecules interact, how they interact, how enzymes catalyze reactions, how drugs act (Berruyer et al., 2017; Raines et al., 2010) The information of 3D structure determination allow us understanding disease at an atomic level and become useful for designing and developing new drugs In the field of structural biology, some techniques as X-ray crystallography, 3D electron microscopy, dual polarization interferometry, and atomic force microscopy, nuclear magnetic resonance spectroscopy, etc are used to determine the structure of a protein (Pan et al., 2016; Stout, 1989)

1.1.5.1 X-ray crystallography

X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions (Bök, 2003; crystallography, 2017a) From the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal (Crystallography, 2017b) From this

electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder and various other information (Crystallography, 2017b)

X-ray crystallography method includes three basic steps:

The first step is considered as the most difficult step, which obtain an adequate crystal of the protein

The second step, the crystal is carried out in an intense beam of X-rays The third step, these data are combined computationally to produce and

crystal(Crystallography, 2017b) The final, refined model of the atomic arrangement-now called a crystal structure-is usually stored in a public database (Crystallography, 2017b)

Figure 1.2.Workflow for solving the protein structure by X-ray crystallography (Crystallography, 2017b)

1.1.5.1.1 Protein crystallization principle

In the X-ray structural analysis of a protein, obtaining suitable single crystals is also the least understood step Protein crystallization is mainly a trial-and-error procedure in which the protein is slowly precipitated from its solution (J., 1999) As

a general rule, the purer the protein is, the better the chances to grow crystals (J., 1999)

To obtain crystals suitable for crystallographic studies, the macromolecule must be purified to homogeneity, or as close as possible to homogeneity The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; Dessau & Modis, 2011; McPherson, 2004) Crystallization requires bringing the macromolecule to supersaturation The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of macromolecule (Dessau & Modis, 2011) Introducing the sample to precipitation agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution (Dessau & Modis, 2011) Crystallization proceeds in two phases: nucleation and growth To easily design crystallization experiments, a crystallization phase diagram was created to show which state liquid, crystalline or amorphous solid (precipitate) is stable under a variety of crystallization parameters (Sanderson & Skelly, 2007)

The phase diagram is obtained experimentally by varying two parameters at a time, thus representing a two-dimensional „slice‟ of the multidimensional space of

parameters relevant to crystallization (Chayen & Saridakis, 2008) In a typical crystallization phase diagram (Fig.13), it is distinguished between four areas: an area of very high supersaturation, where the protein will precipitate; an area of moderate supersaturate, where spontaneous nucleation will take place; an area of lower supersaturation just below the nucleation zone, where crystals are stable and may grow, but no further nucleation will take place (referred to as the metastable zone, this area is thought to contain the best conditions for growth of large, well-ordered crystals); and an undersaturated area, where the protein is fully dissolved and will never crystallize (Chayen, 2004; Sanderson & Skelly, 2007)

There are two main techniques to obtain crystals: Vapor diffusion and batch crystallization In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant Water vapor then diffuses out of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop (Dessau & Modis, 2011)

Figure 1.3 Schematic illustration of a protein crystallization phase diagram (Chayen & Saridakis, 2008)

1.1.5.2 Vapor diffusion methods for protein crystallization

There are at least seven practical methods used for macromolecule crystallization including micro-batch experiment, vapor diffusion, bulk crystallization, free interface diffusion, dialysis, temperature-induced, and seeding Among these methods, vapor diffusion method is one of the most widely used for crystallization (Crystallography, 2017b)

Vapor diffusion can be carried out in hanging drop or sitting drop format Hanging-drop apparatus involve a drop of protein solution placed on an inverted cover slip, which is then suspended above the reservoir(Crystallography, 2017b) Sitting-drop crystallization apparatus place the drop on a pedestal that is separated

from the reservoir Both of these methods require sealing of the environment so that equilibration between the drop and reservoir can occur (McRee, 1999; Rhodes, 2006)

Figure 1.4 Two methods of preparing A: Hanging drop B: Sitting drop (Crystallography, 2017b)

2 Chapter 2 Methods

oryzae pv oryzae

XoPDF protein from the Xoo bdf (Xoo1075; XoPDF) gene was produced and

crystallized as previously published (P T Ngo et al., 2008) The sequences of oligonucleotide primers used for gene cloning were base on the published genome

sequence (B M Lee et al., 2005) Forward (5‟- GGG GGG CAT ATG ATT CGC

GAC ATT ATC CGC ATG – 3‟) and reverse (5‟- GGG GGG GGA TCC CTA CAG

ATC GTA AGA CAA GAC – 3‟) primers were designed to introduce NdeI and BamHI digestion sites (bold) (P T Ngo et al., 2008) The PCR amplified DNA

fragments were purified using a PCR purification kit (Bioneer) and inserted into the same restriction enzyme-digested pET11a expression vector (Novagen)

E.coli BL21 (DE3) cells containing pET11-XoPDF were grown at 288 K in LB medium until the OD600mm reached 0.6 (P T Ngo et al., 2008) Protein expression was induced with 0.5 mM IPTG The cells were cultured at 288 K After 16 hours

growth, the cells were harvested by centrifugation at 9780× g for 30 min at 277 K

and resuspended in 25 mM Tris-HCl buffer (pH 7.5) containing 3 mM mercaptoethanol The cells were disrupted using a sonicator (Sonomasher); after

β-centrifugation (9780 × g at 4°C for 30 min), a crude protein sample was adjusted to

45% saturation in ammonium sulfate Precipitated was performed after 30 min by

stirring The precipitated protein was collected by centrifugation for 30 min at

9780× g at 4°C and dissolved in 25 mM Tris-HCl buffer (pH 7.5) containing 3 mM

β-mercaptoethanol Subsequently further purified on a Bio-Gel P60 column (2.5 x

50, Biorad) equilibrated in the 25 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, and 3 mM β-mercaptoethanol (buffer A) XoPDF protein were collected at eluted fractions and diluted 5 times with buffer A and loaded on to a UNOPTMP Q6 column (Biorad) for ion-exchange chromatography purification XoPDF was then applied onto a Bio-Gel P100 column (Biorad) For crystallization, XoPDF protein was dialyzed into storage buffer containing 25mM Tris (pH7.5), 15 mM NaCl, 3

mM β-mercaptoethanol The concentration of pure protein was 8 mg ml-1

Proteine purity was examined using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Figure 2.1) (Ho, Hong, Ngo, & Kang, 2014; P T Ngo et al., 2008)

Figure 2.1 Purified XoPDF is shown on a 15% SDS-PAGE gel Lane M1, XoPDF (~ 21kDa) Lane P, prestained protein ladder (Fermentas)

2.3 Crystallization, da t a collection, and structure determination of substrate- or inhibitor-bound XoPDF

Crystals of XoPDF were grown using the hanging drop vapor diffusion method

in a 24-well plate (SPL Life Sciences, Korea) as previously published (P T Ngo et

al., 2008) Crystals of XoPDF grew in the reservoir solution contained 0.05 M cadmium sulfate, 0.1 M 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) pH 7.5, and 2.0 M sodium acetate trihydrate (H P Ngo et al., 2016) 0.9

μl of protein solution was added to 0.9 μl of reservoir solution in the hanging drops Hexagonal-pillar-shaped crystals were observed after 1 day For cryoprotection, the

fully grown crystals were added to a mixture of reservoir solution and 20% (v/v)

glycerol and subsequently flash-cooled at 100 K in liquid nitrogen (H P Ngo et al., 2016) For MA- or MAS-bound and actinonin-bound structures, native crystals

were soaked in the reservoir solution with 10 mM of substrate

(N-formylmethionine, fMA or N-formylmethionine-alanine-serine, fMAS) or actinonin for less than 1 h, and flash-cooled at 100 K in liquid nitrogen using the same cryoprotectant as that used for the native crystals X-ray diffraction data were collected at the beamline BL-6A at the Photon Factory, High Energy Accelerator Research Organization (KEK), Japan, and at the beamline 4A at the Pohang Light Source (PLS), South Korea Native crystals were diffracted to 2.6 Å resolution, while MA-, MAS-, and actinonin-bound crystals diffracted up to 1.9 Å resolution (H P Ngo et al., 2016) The XoPDF protein was crystallized in hexagonal space

group P6122 Data were integrated and scaled using DENZO and SCALEPACK, respectively (Otwinowski & Minor, 1997a) The phase of native XoPDF was obtained by molecular replacement (MR) with Phaser in the CCP4 program package

(McCoy et al., 2007) PDF from Leptospira interrogans (PDB ID: 1sv2, 42.3%

sequence identity) was used as a search template Model building and electron density interpretation were performed using the program COOT (Emsley, Lohkamp, Scott, & Cowtan, 2010) The structure was refined using the CCP4 program Refmac5 (Murshudov et al., 2011) The determined native XoPDF structure was used as a template to solve the substrate-bound complex structures by restrained refinement in the Refmac5 program All structures were validated using WHATIF

(Vriend, 1990) and SFCheck (Brat, Boles, & Wiedemann, 2009) Graphic

presentations were created using Pymol (Schrodinger, 2010)

determination of fragment chemical compound-bound XoPDF

Fragment library compounds of 342 at 20 mg/each were purchased from ChemBridge Corporation (San Diego, CA, USA) and Enamine LLC (Monmouth

Jct., NJ, USA) and dissolved in 100% DMSO at a final concentration of 1 M

Fragment cocktails were prepared by mixing 5 compounds in mutually distinct shapes and chemical properties to a final concentration of 200 mM per compound Native crystals were first isolated and equilibrated in mother liquors consisting of

0.05 M cadmium sulfate, 0.1 M HEPES pH 7.5 and 3.0 M sodium acetate trihydrate (H P Ngo et al., 2016) Fragment cocktail solutions were soaked to the drop at concentration ranging from 10 mM All soaking steps were carried at room temperature while incubation steps were carried out at 286K from 6 h to 24 h The fragment chemical cocktails-soaked crystals were flash-cooled at 100 K in liquid nitrogen using the same cryoprotectant as that used for the native crystals X-ray diffraction data were collected up to 2.0 Å resolution The fragment chemical compound (FCC)-bound structures were determined in the same way with MA-, MAS-, and actinonin-bound structures After we found extra positive electron density of putative chemical compound in the fragment chemical cocktails-soaked crystal structure, the identity of the positive density was confirmed again by soaking all the constituting compounds of the specific cocktail solution separately and determination of the complex structures (H P Ngo et al., 2016)

XoPDF crystallized in space group P6122 The unit-cell parameters were a = b

= 59.0 Å , and c= 266.3 Å , respectively The space group was derived by indexing (Otwinowski & Minor, 1997b) and data-collection statistics are provided

auto-in Table 1 Accordauto-ing to the Matthews coefficient calculation (Mathews, 1968), there

is one molecule in the asymmetric unit, corresponding with VM of 3.50 Å Da-1 and solvent content of 64.9%, respectively Molecular replacement (MR) using Phaser

in CCP4 program package (McCoy et al., 2007) with peptide deformylase from

was successful shows one monomer in the asymmetric unit

The gene encoding for AbPDF was cloned from A baumannii genomic DNA

The sequences of the oligonucleotide primers were designed based on the data on

genome sequences of other A baumannii from the NCBI website Forward (5‟-

CCC CCC CAT ATG GCC TTA TTA CCT ATT TTA AG -3‟) and reverse (5‟- CCC GGA TCC TTA ACG TTT TAC CGC AAC TTT TTC -3‟) primers were designed

to introduce NdeI and BamHI restriction sites (bold), respectively (Thien-Hoang Ho,

2017) The PCR-amplified DNA fragments were purified, inserted, and cloned into the pET11aHT vector, which contains an N-terminal 7×His tag followed by a

Tobacco etch virus (TEV) protease-cleavage site The expression vector AbPdf was transformed into E coli BL21(DE3), as described previously (Thien-

pET11aHT-Hoang Ho, 2017)

E coli BL21 (DE3) cells containing pET11aHT-AbPdf were grown at 310 K in

LB medium containing 50 g ml-1 ampicillin until the OD600 reached 0.6 The

protein expression of AbPDF was induced with 0.5 mM IPTG The cells were

cultured at 310 K After overnight growth, cells were then harvested by centrifuging

at 6000 × g for 20 min at 277 K The cell pellets were resuspended in ice-cold lysis buffer (25 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 mM imidazole and 3 mM -mercaptoethanol), sonicated and centrifuged (Thien-Hoang Ho, 2017) The soluble

fraction was applied onto Ni-NTA resin (Bio-rad) and purified and dialysis as described previously (Thien-Hoang Ho, 2017) The His-tag was cleaved by TEV protease at 277 K in an overnight reaction AbPDF was purified again using Ni-NTA resin (Bio-rad) Further purification was carried out on a HiTrap Q Anion-exchange column (GE Healthcare) equilibrated in buffer A (25 mM Tris-HCl, pH 7.5, 15 mM NaCl, and 3 mM β-mercaptoethanol) AbPDF was washed and eluted

with a gradient of 0 to 100% buffer B (25 mM Tris-HCl, pH 7.5, 1 M NaCl, and 3

mM β-mercaptoethanol) (Thien-Hoang Ho, 2017) The homogeneity of purified

protein was checked via SDS-PAGE (Fig 1) And concentrated to 4.5 mg ml-1 using Centrifugal Filters (Amicon ® Ultra-15, MWCO 10 kDa)

AbPDF protein was screened for crystallization on a 96-Well Intelliplate (Art Robbins) at 287 K 0.5 µl protein solution (4.5 mg ml-1) was added to 0.5 µl reservoir solution in a sitting drop 96-Well Intelliplate (Art Robbins) at 287 K Crystals of AbPDF grew in the MD1-46 (MorpheusTM) kit after two weeks with a

precipitant solution consisting of 0.03 M MgCl2, 0.03 M CaCl2, 15% (v/v)

PEG550MME, 15% (w/v) PEG 20000, 0.1 M Tris (base)/ Bicine pH 8.5 Crystals

from the sitting drop were mounted and flash-cooled in a cold nitrogen-gas liquid at

100 K with the cryoprotectant solution (0.03 M MgCl2, 0.03 M CaCl2, 15% (v/v) PEGMME, 15% (w/v) PEG 20000, 0.1 M Tris (base)/ Bicine pH 8.5, and 20%(v/v)

glycerol) (Thien-Hoang Ho, 2017)

X-ray data were collected at 100 K using the ADSC Q315r detector on

beamline 5C of the Pohang Light Source (PLS), Republic of Korea X-ray diffraction data to 2.4 Å resolution were collected for the AbPDF crystal (Fig.3) Diffraction data were collected from a single crystal with 1° oscillation per frame for a total of 316° at a 300 mm crystal to detector distance and with 3 sec exposure

per frame The raw data were processed and scaled using DENZO and SCALEPACK,

respectively (Zbyszek Otwinowski, 1997)

3 Chapter 3 Results

The XoPDF protein was purified to the level of a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis The crystal structure of XoPDF was determined at 2.6Å resolution (Table 1) There was one molecule in the asymmetric unit and electron density maps for all residues of XoPDF were clearly observed except for several residues in the C-terminus Similar to other PDF structures, XoPDF adopted an α+β fold with two α-helices, eight β-strands, and four

310 helices (Figure 3.1a) Helix α2 was at the central position and surrounded by

eight anti-parallel β-strands organized into three sides

Figure 3.1 Native structure of XoPDF (a) Overall structure of XoPDF

Signature motif 1 is shown as blue, motif 2 as cyan, motif 3 as purple, and CD loop

as orange Red sphere is a cadmium ion (b) Metal binding site A cadmium ion is bound by three signature motifs Metal coordination is shown with black dashed lines

PDFs have three conserved short stretches of amino acids of motif 1, motif 2, and motif 3, which are located physically close to each other to coordinate active

site metal ion (Figure 3.1b) Helix α2 carried conserved motif 3, HEXXH (HEYDH

in XoPDF), which is essential for metal coordination and substrate activation (Figure 3.3) The strong electron density map suitable for metal ions was found at the active site As cadmium ions were present at a high concentration in the crystallization buffer, the strong density was interpreted as a cadmium ion, which was pentacoordinated and bound with the side chains of Cys99, His141, and His145, as well as two water molecules with the distances of 2.4, 2.2, 2.2, 2.3, and

2.5, respectively (Fig 3.2a)

Figure 3.2 Metal binding and coordination of XoPDF (a) Cd2+ ion in the metal site of MAS-bound XoPDF structure with 2Fofc and FoFc maps (b) Zn2+ ion

in the metal site of MAS-bound XoPDF structure with 2Fofc and FoFc maps 2Fofc map (blue mesh) is contoured at 1.0 σ and Fofc map (green mesh) at 5.0 σ (c) Superimposed metal sites of Cd2+-bound XoPDF (purple), Co2+-bound EcPDF (PDB ID: 4AZ4; cyan), Ni2+-bound EcPDF (PDB ID: 4AL2; yellow), Zn2+-bound EcPDF (PDB ID: 1XEM; green) and Fe2+-bound EcPDF (PDB ID: 1XEN; orange)

C

B

A