Umeå University Odontological Dissertations, New series nr: 126

Structural and functional studies of streptococcal surface adhesins

Åsa Nylander

Department of Odontology

Umeå University, Sweden 2013

Responsible publisher under Swedish law: the Dean of the Medical Faculty

This work is protected by the Swedish Copyright Legislation (Act 1960:729)

ISBN: 978-91-7459-689-2

ISSN: 0345-7532

Elektronisk version tillgänglig på http://umu.diva-portal.org/

Printed by: Print & Media

Umeå, Sweden 2013

Till Mor

“42”

- Douglas Adams

“The true delight is in the finding out rather than in the knowing.”

- Isaac Asimov

i

Table of ContentsTable of Contents......................................................................................... i List of original publications ........................................................................ ii Abstract ..................................................................................................... iii Abbreviations ............................................................................................ iv Enkel sammanfattning på svenska ............................................................. v Introduction ............................................................................................... 1 Materials and methods ............................................................................. 17 Aim of thesis............................................................................................. 20 Result and discussion ............................................................................... 21

Structures of the C-terminal domain of the antigen I/II protein family (paper I &

IV) ......................................................................................................... 21 Structural and functional analysis of the N-terminal domain of the Streptococcus

gordonii adhesin Sgo0707 (paper II) .......................................................... 24 Protein expression and purification of Streptococcus pyogenes adhesin AspA

(paper III) .............................................................................................. 26 Conclusion ................................................................................................ 29 Acknowledgements .................................................................................. 30 References................................................................................................ 31

ii

List of original publications

This thesis is based on the following publications and manuscripts, referred

to by their roman numerals throughout the thesis.

Paper I Nylander Å., Forsgren N., Persson K. (2011) Structure of the

C-terminal domain of the surface antigen SpaP from the

caries pathogen Streptococcus mutans. Acta

Crystallographica Section F. Structural Biology and

Crystallization Communications. F67(1):23-26.

Paper II Nylander Å., Svensäter G., Senadheera D. B., Cvitkovitch D.

G., Davies J. R., Persson K. Structural and functional analysis

of the N-terminal domain of the Streptococcus gordonii

adhesin Sgo0707 PLoS ONE 8(5): e63768

Paper III Nylander Å., Hall M, Jenkinson H., Persson K. Expression

and purification of Streptococcus pyogenes adhesin AspA.

Manuscript

Paper IV Hall M., Nylander Å., Jenkinson H., Persson K. Structure of

the C-terminal domain of the Streptococcus pyogenes antigen

I/II-family protein AspA. Manuscript

iii

Abstract

The oral cavity is home to an array of microorganisms that are associated

with dental plaque. Some Gram-positive bacteria are common inhabitants of

the oral cavity and in order to colonize such a unique environment adhesion

becomes essential and is accomplish by adhesins expressed on the bacterial

surface. Adhesins can interact with host molecules or with structures on the

resident oral microbial flora. Members of the antigen I/II (AgI/II) protein

family are commonly found on the surface of oral streptococci and have the

unique feature that their putative adhesin domain is located in the centre of

the primary sequence. Crystal structures representing parts of the C-terminal

domains from two AgI/II members, SpaP from Streptococcus mutans and

AspA from Streptococcus pyogenes, were determined to 2.2 and 1.8 Å

resolution respectively. The structures are very similar and consist of two

domains with DEv-IgG folds. The proteins are stabilized by intramolecular

isopeptide bonds and tightly coordinated metal ions.

Another group of surface proteins is the microbial surface

components recognizing adhesive matrix molecules (MSCRAMMs) that have

their putative adhesin domain in the N-terminal, presented on a stalk

formed by multiples of repeated C-terminal domains. Sgo0707 from

Streptococcus gordonii is an example of this group of proteins and its N-

terminal domain was determined to 2.1 Å resolution. The structure consists

of two domains, N1 and N2, both of which adopt β-sandwiches. In the

Sgo0707 structure no isopeptide bonds or metal ions were detected. A

putative binding cleft is present in the N1 domain. Functional studies

revealed collagen type-1 and keratinocytes as possible binding partners.

In order to further characterize the AgI/II protein AspA from

S. pyogenes a long form of the protein, AspA-AVPC, was expressed and

purified. During the purification process it was observed that the protein

fragmented into two major parts. This process could be inhibited by the

addition of 0.5 mM EDTA during protein purification.

In conclusion, these studies have resulted in adding to the

knowledge of protein structures and function of streptococcal surface

proteins.

iv

Abbreviations

3D Three dimensional

AgI/II Antigen I/II

CLSM Confocal laser scanning microscopy

CM Cytoplasmic membrane

CW Cell wall

CWA Cell wall anchor

EDTA Ethylenediaminetetraacetic acid

EPS Exopolysaccharides

GCF Gingival crevicular fluid

GlcNAc N-acetylglucosamine

IgG-C Immunoglobulin G-constant

IMAC Immobilized metal affinity chromatography

IPS Intracellular polysaccharides

IPTG Isopropyl β-D-1-thiogalactopyranoside

LTA Lipoteichoic acids

MAD Multiple-wavelength anomalous dispersion

MALDI- TOF MS Matrix-assisted laser desorption/ionozation

time-of-flight mass spectrometry

MIR Multiple isomorphous replacement

MR Molecular replacement

MSCRAMM Microbial surface components recognizing

adhesive matrix molecules

MurNAc N-acetylmuramic acid

RMSD Root-mean-square deviation

S. Streptococcus

S-layer Surface layer

SAD Single-wavelength anomalous dispersion

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel

electrophoresis

Sec Secretory

SeMet Selenomethionine

SRP Signal recognition particle

Staph. Staphylococcus

TA Teichoic acids

Tat Twin arginine transport

vWFA von Willebrand factor A

Å Ångström (1 Å=0.1 nm)

v

Enkel sammanfattning på svenska

I munhålan finns det bakterier som bildar samhällen på tänderna, s.k. (tand)

plack. För att bakterier ska kunna kolonisera munhålan måste de snabbt

kunna fästa till molekyler och ytor, annars sväljs de ner i magsäcken där

magsyran oskadliggör dem. Bakterier uttrycker därför ytprotein som gör att

de kan fästa till molekyler i munhålan och på så sätt stanna där. Mer än 700

olika bakteriearter har påvisats i munhålan, men vanligtvis ligger

medelvärdet på ca 100 i en frisk individs mun. Olika streptokockarter är

vanliga i munhålan och de uttrycker flera ytproteiner. I denna avhandling

beskrivs; pili, antigen I/II, och så kallade MSCRAMM.

Mycket i kroppen är uppbyggd av proteiner, vilka består av aminosyror

bundna till varandra i kedjor. Proteinkedjan veckar sig i sekundära

strukturer som arrangerar sig i förhållande till varandra och ger proteinet en

tre dimensionell struktur (3D). Att ta reda på 3D strukturen av ett protein är

av intresse för att få ledtrådar till dess funktion vilket t.ex. kan leda till möjlig

läkemedelsdesign. Röntgenkristallografi är en metod för att bestämma

strukturen av ett protein, där man först uttrycker, renar och till sist

kristalliserar proteinet. Kristalliseringsprocessen är ofta tidskrävande och

inte alltid lyckosam, men om man lyckas få proteinkristaller, kan man genom

att bestråla kristallen med röntgenstrålar beräkna proteinets 3D struktur på

atomnivå.

Denna avhandling baseras i huvudsak på tre strukturer av olika

streptokockproteiner; SpaP från Streptococcus mutans, Sgo0707 från

Streptococcus gordonii, och AspA från Streptococcus pyogenes som jag här

sammanfattar väldigt kort. Strukturellt är SpaP från S. mutans och AspA

från S. pyogenes från antigen I/II familjen väldigt lika varandra (artikel I

och manuskript IV). De är båda uppbyggda av två domäner som består av

β-strängar, som i sin tur bildar en s.k. β-sandwich. När modellerna av

proteinerna jämförs genom att de placeras på varandra, sammanfaller de

flesta av de analyserade kolatomerna i huvudkedjorna, eftersom proteinerna

är strukturellt mycket lika. Strukturen av MSCRAMM-proteinet Sgo0707

från S. gordonii (artikel II) uppvisar även det två domäner, som båda

veckar sig i β-sandwichar. En av domänerna har en möjlig bindningsficka så

experiment utfördes för att ta reda på vad som kan tänkas binda till denna

ficka. Resultatet visade att collagen typ-1 (ett protein som bl.a. finns i senor

och ben) och keratinocyter (cellerna som täcker munhålans mjuka delar)

binder till Sgo0707 vilket visar att Sgo0707 är inblandad i vidhäftning.

Planen var också att kristallisera en längre form av AspA proteinet (AspA-

AVPC) när det upptäcktes att proteinet bröts ner till två mindre fragment,

vi

men orsaken till nedbrytningen är ännu inte känd. Vi märkte att om EDTA

(en molekyl som binder metalljoner) tillsattes till proteinet så uteblev

nedbrytningen. Studier fortgår för att få reda på betydelsen av denna

nedbrytning.

1

Introduction

Microbiology in the oral cavity

The oral cavity is the portal to the respiratory tract as well as to the gastro

intestinal tract and is therefore an important gateway into the body. The

acidic environment of the stomach is an effective defence mechanism against

ingested microorganisms. The human mouth consists of mucosal surfaces,

teeth, and saliva, which makes the oral cavity a unique environment. The

amounts of microorganisms present on the mucosal surfaces are quite low

due to the regular shedding of epithelial cells (desquamation). The papillary

structures of the tongue can harbour obligate anaerobes as these surfaces

give protection against the salivary flow and have low redox potential. The

non-shedding tissues of the teeth can accumulate a larger mass of

microorganisms than the mucosal tissue. The local environments of teeth

vary; the tooth is divided into a subgingival region (below the gingival

border), and a supragingival region (above the gingival border) (Marcotte

and Lavoie, 1998). The buccal (cheek) and lingual (tongue) surfaces of the

teeth have different salivary flow, and tongue movement on the lingual side

effects colonization. The occlusal surfaces (biting surface) and the surfaces

between the teeth (approximal surfaces) are more difficult to clean which

makes them more susceptible to microbial growth (Marsh and Martin,

2009). Saliva is produced by three major glands (parotid, submandibular

and sublingual) and a number of minor glands. Saliva contains bicarbonate

to facilitate buffering; calcium, and phosphate for the integrity of minerals in

the tooth; and proteins. Salivary proteins range in concentration and

comprise proline-rich proteins, amylase, statherin, histatin, mucins,

lysozyme, lactoferrin, peroxidase, and secretory IgA (Dodds et al., 2005;

Oppenheim et al., 1988; Slomiany et al., 1996). The saliva lubricates the oral

cavity, which is necessary for protection, speech, taste, chewing, and

swallowing (Mandel, 1987). The oral cavity is covered with saliva, and within

the salivary layer, a salivary pellicle is formed. The acquired enamel pellicle,

with so far 130 identified proteins, covers the teeth in the oral cavity

(Siqueira et al., 2007). The pellicle contains adsorbed salivary glycoproteins,

(e.g. gp340 (Loimaranta et al., 2005)) phosphoproteins, lipids (Al-Hashimi

and Levine, 1989; Slomiany et al., 1986), and other molecules. These

structures can be used for attachment and subsequent colonization by

microorganisms of the oral cavity. Hannig showed that the formation of the

pellicle is initiated immediately at the contact of a surface to saliva and the

thickness of the pellicle increases over time. After 24 hours the teeth’s lingual

surfaces is covered by a 100-200 nm thick pellicle, and a 1000-1300 nm

pellicle on the buccal surfaces (Hannig, 1999). Gingival crevicular fluid

(GCF) is a highly nutrient plasma exudate present in the gingival crevice.

2

GCF can be used as nutrients for resident bacteria, and contains members of

the immune defense (for example immunoglobulins and leucucytes). The

flow of GCF is slow in healthy sites, but increases in gingivitis and

periodontitis (Marcotte and Lavoie, 1998).

Biofilms and dental plaque

The oral cavity is home to a diverse range of microbial life; Gram-positive

and Gram-negative bacteria, fungi, mychoplasma, viruses, and protozoa.

Streptococcus, Actinomyces, Veillonella, and Neisseria are bacteria

commonly isolated from early dental plaque (Jenkinson and Lamont, 2005a;

Li et al., 2004). The initial contacts with host molecules during colonization

are weak (hydrophobic, van der Waals, and electrostatic interactions). If the

environment is suitable the interaction becomes stronger and specific

through bacterial adhesins (Nikolaev and Plakunov, 2007). The biofilm

continues to grow into a mature biofilm with different species and bacterial

exopolysaccharides (EPS) being incorporated (Branda et al., 2005).

Streptococcal and Actinomyces species are Gram-positive commensal, early

colonizers of the oral cavity that adhere to proteins, peptides or other

molecules adsorbed in the salivary pellicle. The early colonizers can alter the

environment by displaying new attachment sites for subsequent

microorganisms, changing the pH, metabolizing oxygen and thereby

changing the redox potential and by producing metabolites that can be used

as nutrients by other microorganisms (Jenkinson and Lamont, 2005b;

Marsh, 2006) . Late colonizers attaches to the new surfaces available to them

through the surfaces of resident bacteria. The majority of the bacteria in the

early oral biofilm are commensal, but as the biofilm grows, more pathogenic

bacteria accumulate. If dietary conditions are met (high sugar content in the

diet), the mutans streptococci group and lactobacilli can thrive resulting in

acidic metabolic products lowering the local pH causing enamel destruction

i.e. caries (Loesche, 1986; Marsh, 2006). The oral biofilm on teeth is known

as dental plaque, and consists of an array of microorganisms in a three

dimensional (3D) structure with EPS, channels, and cavities. In a biofilm, the

properties of the different species are less important; with the properties as a

community instead becoming essential. Since bacteria in a community

interact with each other in ways not yet fully understood, and the biofilm

itself provides shelter from the environment, the planktonic pure culture

format commonly implemented in laboratories results in that approximately

50% of the oral bacteria cannot be cultured. (Wade, 2002). In order to get a

better understanding of how microorganisms in dental plaque collaborate,

studies of pure cultures may need to be bypassed for the advances of other

growth techniques.

3

Gram-positive bacteria

The cell envelope (figure 1) of Gram-positive bacteria contains a

cytoplasmic membrane (CM) made up of a lipid bilayer, transport

proteins, ion pumps, and enzymes. The CM is responsible for electron

transport and energy production in the form of ATP (Murray et al., 2002).

Outside the CM the cell wall (CW) is located, which consists of a thick (30-

100 nm) peptidoglycan layer. Peptidoglycan consists of polysaccharide

chains, made up of repeating units of N-acetylglucosamine (GlcNAc) and N-

acetylmuramic acid (MurNAc) and the polysaccharide chains are cross-

linked (Ghuysen et al., 1965). Teichoic acids (TA) and Lipoteichoic

acids (LTA) are present in the peptidoglycan layer and are both polymers of

modified ribose or glycerol, linked by phosphate moieties (Armstrong et al.,

1958). TA is attached to peptidoglycan in the cell wall, and LTA is attached to

the lipid bilayer in the CM (Silhavy et al., 2010). The surfaces of some Gram-

positive bacteria are covered by a repeating unit of a (glyco) protein with a

molecular mass of 40-200 kDa. This self-assembled crystalline surface

layer (S-layer) is 5–25 nm thick with pores 2-8 nm in size, and is non-

covalently linked to other neighboring molecules and to the cell wall. The S-

layer gives the bacterium stability, acts like a sieve, and protects the

bacterium from the host immune defense system, however it is usually lost

during prolonged cultivation (Sara, 2001). Some Gram-positive bacteria have

a capsule composed of extracellular polysaccharides as the outermost layer

covering also the S-layer when present (Mesnage et al., 1998).

Figure 1: The Gram-positive envelope consist of; the CM (lipid bilayer) with

membrane proteins (pink); the cell wall with repeating units of GlcNAc

(blue) and MurNAc (purple) with cross-links (black lines), TA (orange) and

LTA (light green); the S-layer (grey pentagons), and the capsular

polysaccharide (green ovals). Parentheses around the S-layer and capsule

indicate that they are not always present. Adapted from Silhavy et al. (2010).

Stabilization of Gram-positive surface proteins

Proteins are stabilized in a number of ways (Kauzmann, 1959). The

hydrophobic effect is an important driving force when it comes to protein

4

folding. In short, hydrophobic side chains fold into the interior of the protein

while polar and charged side chains form the protein surface. Strong ionic

interactions (salt bridges) between residues with opposing charges will

contribute to the stabilization of proteins when present. Hydrogen bonds

in the protein interior or exterior between side chains, backbone, or water

molecules help to stabilize the protein. Intrinsic metal ions (commonly

iron, calcium, magnesium, or zinc) bind side chains or main chains and

participate both in function and stabilization of the protein. van der Waals

forces are transient forces that are weak but numerous, adding stability to

the proteins. Protein can also be stabilized by covalent bonds; disulfide

bonds are covalent bonds formed spontaneously between the sulphur atoms

of two adjacent cysteine residues during protein folding. Intramolecular

isopeptide bonds are covalent bonds that also can be spontaneously

formed between the nitrogen of a lysine side chain and the gamma carbon of

an asparagine/aspartic acid side chain. A catalytically essential glutamic- or

aspartic acid must be present in the vicinity for the isopeptide bond to form,

contributing with hydrogen bonds to the isopeptide bond (Kang and Baker,

2011; Kang et al., 2007) (figure 2). Isopeptide bonds are quite common in pili

or other elongated Gram-positive surface proteins.

Figure 2: Isopeptide formation. A) A prerequisite for spontaneous

intramolecular isopeptide bond formation is close proximity between a

lysine, and an asparagine (or aspartic acid). A glutamic acid (or aspartic acid)

must also be present to catalyze the bond formation. B) The formation of the

covalent isopeptide bonds between the side chain nitrogen from lysine and

the gamma carbon from asparagine result in the release of NH3 (ammonia).

The catalytically important glutamic acid hydrogen bonds (black dashed

lines) to a lysine hydrogen atom, and an asparagine oxygen. Adapted from

Kang and Baker (2011).

5

Streptococci

Streptococci are a family of spherical shaped Gram-positive bacteria that

grow in pairs or chains. Most species are facultative anaerobes and display

partial hemolysis (alpha-hemolysis) on blood agar. Viridans streptococci is a

collective term used for oral streptococci based on the green coloration

surrounding colonies created by the partial hemolysis on blood agar (the

latin word viridis meaning green) (Holman, 1916). To classify streptococcal

species Lancefield created a method for grouping cell wall antigens based on

surface carbohydrate composition; polysaccharides (group A, B, D, E, F, and

G), teichoic acid (D, and N), or lipoteichoic acid (H) (Lancefield, 1933; Nobbs

et al., 2009). To further classify streptococci 16s rRNA sequencing can be

used, where streptococcal species are divided into six groups; the salivarius,

mutans, mitis, and anginosus groups with members commonly isolated from

the oral cavity; and the pyogenes, and bovis groups which are not considered

oral streptococci (Nobbs et al., 2009). Oral streptococcal species are

acidogenic (produces acids) and aciduric (tolerate acids) to some extent.

During periods of excess dietary sugar streptococci are able to store sugars as

intracellular polysaccharides (IPS) for use as an energy source during times

of low dietary sugar supplies (Takahashi et al., 1991; van Houte et al., 1970).

Streptococci express a number of surface proteins containing an LPxTG-

motif in the C-terminus (described in coming sections); Streptococcus

gordonii strain CH1 expresses 20 (Nobbs et al., 2009); Streptococcus

mutans strain UA159 expresses 6 (Ajdic et al., 2002), and Streptococcus

pyogenes expresses 13 (Ferretti et al., 2001), based on whole genome

sequencing.

Streptococcus gordonii

S. gordonii is considered to be a commensal bacteria, meaning that the

bacteria benefits by the relationship to the host, while the host is unaffected.

This may be a truth with modification, as the colonization by S. gordonii in

the oral cavity benefits the host by outcompeting possible pathogenic

bacteria, and on the other hand displays surface molecules known to serve as

attachment sites for pathogenic bacteria (Brooks et al., 1997). S. gordonii

display an array of surface proteins, and can cause infective endocarditis

when spread to the blood stream (Douglas et al., 1993). S. gordonii produces

EPS from sucrose; which is incorporated into dental plaque (Branda et al.,

2005) and are able to catabolize arginine leaving ammonia as a product,

which raises local pH and is thus important for pH homeostasis (Burne and

Marquis, 2000).

Streptococcus mutans

The acidogenic, and aciduric S. mutans is implicated in dental caries, and

converts sugars to acidic products that are detrimental for the enamel

6

(Loesche, 1986). S. mutans are involved in plaque maturation by production

of EPS from sucrose (Branda et al., 2005).

Streptococcus pyogenes

S. pyogenes is a human pathogen responsible for mild (pharyngitis and

scarlet fever) to lethal (necrotizing fasciitis and streptococcal toxic shock

syndrome) conditions (Cunningham, 2000), and is not specific to the oral

cavity. The cells are 0.5-1.0 µm in diameter and are surrounded by a

hyaluronic acid capsule; creating a barrier to the host immune system

(Murray et al., 2002).

Gram-positive surface proteins require passage through the

cytoplasmic membrane

The countless proteins produced by cells have different objectives; some

fulfill their fate by remaining in the cytoplasm, while others are secreted into

the extracellular matrix for destination elsewhere. A group of proteins are

displayed at the cell wall, interacting with the environment. A majority of the

secreted proteins from Gram-positive bacteria will carry an N-terminal

signal peptide sequence destined for the secretory (Sec) pathway (van

Wely et al., 2001). Newly synthesized proteins will be bound by a signal

recognition particle (SRP) and transported to the CM (Keenan et al., 2001;

Zanen et al., 2006). There the unfolded protein will be transported through

the membrane by the aid of specific Sec proteins forming a CM channel

(translocon). During secretion through the CM the signal peptide will be

cleaved off by signal peptidase I. In Gram-negative bacteria surface proteins

are folded in the periplasmic space. Since Gram-positive bacteria lack a

periplasmic space, protein folding must take place elsewhere. A single

microdomain in the S. pyogenes CM, termed ExPortal, has been identified. It

corresponds to the location of the Sec translocon and may be of importance

for protein biogenesis (Rosch and Caparon, 2004; Rosch and Caparon,

2005). Another way to cross the CM is by the Twin-arginine transport

(Tat) pathway. Tat can transport folded proteins and is present in some

Gram-positive bacteria, but is not yet so well characterized (Dilks et al.,

2003).

Gram-positive surface proteins

The Gram-positive surface proteins can be divided into four groups based on

structure, function, and the method of attachment (Desvaux et al., 2006). 1)

Transmembrane proteins are amphipathic, with the hydrophobic part of

the protein embedded in the lipid bilayer, and the hydrophilic part

interacting with the cytoplasm and extracellular milieu. Transmembrane

proteins can traverse the membrane once or several times. (Lodish H, 2000).

2) Lipoproteins with a lipobox (specific sequence in the signal peptide

7

containing a cysteine important for lipid attachment) will be attached to the

CM. Lipoproteins are often involved in transport (Hutchings et al., 2009). 3)

Cell surface proteins with an LPxTG-motif (or equivalent) are attached to

the cell wall and will be described in more detail in the following sections. 4)

Cell wall binding proteins are associated to the cell wall by non-covalent

attachment to components (e.g. LTA) in the cell wall. They are commonly

amidases, involved in cell wall breakdown for cell growth and contain a

glycine-tryptophan (GW) repeat modules that associate with LTA (Scott and

Barnett, 2006).

The rest of this thesis will focus on Gram-positive bacterial cell surface

proteins containing an LPxTG-motif, facilitating cell wall attachment (table

1). This collection of proteins can be divided into three groups: pili, the

antigen I/II (AgI/II) protein family, and microbial surface components

recognizing adhesive matrix molecules (MSCRAMM).

Cell wall attachment

Proteins containing LPxTG-motifs will be translocated unfolded through the

CM by the Sec pathway. The signal peptide is cleaved off and the remaining

protein is retained at the CM by the C-terminal hydrophobic domain and the

positively charged tail. The CM-bound housekeeping enzyme sortase A

cleaves the LPxTG-motif between the T and G residues and a covalently

linked intermediate is formed (Mazmanian et al., 1999; Navarre and

Schneewind, 1994). A nucleophilic attack by the amino group (NH2) from the

CW precursor protein lipid II on the carbonyl (C=O) group of the acyl-

enzyme intermediate (Ruzin et al., 2002; Ton-That et al., 2000) results in

the protein-lipid II complex being incorporated into the CW (figure 3).

8

Figure 3: Sortase mediated cell wall attachment of surface proteins carrying

an LPxTG-motif. 1) The signal peptide is cleaved off during passage through

the CM. 2) The protein is retained at the CM by the hydrophobic side chains

traversing the CM and the positively charged side chains in the C-terminal. A

sortase cleaves the protein between the T and G residue. 3) A protein-sortase

intermediate is created, 4) and a nucleophilic attack by Lipid II on the

protein-sortase intermediate results in a protein-lipid II complex. 5) The

protein is incorporated into the cell wall. Adapted from (Hendrickx et al.,

2011; Scott and Barnett, 2006).

Pili (fimbriae)

Pili are elongated structures protruding 1-2 µm from the cell wall that are

present on the cell surface of some bacteria (Ton-That and Schneewind,

2003). Pili on Gram-positive bacteria usually consist of a minor base pilin

attached to the peptidoglycan in the cell wall, a major pilin polymerized into

the backbone (shaft) (figure 4A) and a minor tip pilin, containing an adhesin

domain, usually at the tip of the structure (figure 5A). Sortase Class C is pili

specific and polymerizes identical pili protein through a lysine residue in the

pili motif creating the elongated structure (Kang and Baker, 2012; Ton-That

and Schneewind, 2003). The structures of some of the backbone pilins on

Gram-positive bacteria have been solved by the use of X-ray crystallography

and they show the backbone pilin subunits adopting β-sandwich folds with

an immunoglobulin G-constant (IgG-C) fold or variations thereof, e.g. the

DEv-IgG and IgG-rev fold (figure 6). S. pyogenes backbone pili Spy0128

consist of two domains which both adopt an IgG-rev fold, with isopeptide

9

bonds covalently linking adjacent β-strands of the same sheet (Kang and

Baker, 2009; Kang et al., 2007) . β-sandwiches with stabilizing isopeptide

bonds is also seen in the structures of RrgB from Streptococcus pneumoniae

(El Mortaji et al., 2012; Paterson and Baker, 2011; Spraggon et al., 2010),

and BcpA from Bacillus cereus (Budzik et al., 2009). SpaA from

Corynebacterium diphtheria (Kang et al., 2009) and GBS80 from

Streptococcus agalactiae (Vengadesan et al., 2011) consist not only of β-

sandwiches and isopeptide bonds, but also stabilizing metal ions. Apart from

all the above mentioned stabilizing factors, FimA (Mishra et al., 2011) and

FimP (Persson et al., 2012) from Actinomyces oris also contain disulphide

bonds. The tip pilin Spy0125 from S. pyogenes (Pointon et al., 2010), RrgA

from S. pneumoniae (Izore et al., 2010) and GBS104 from S. agalactiae

(Krishnan et al., 2013) both show stabilizing isopeptide bonds. The top

domain of Spy0125 contains a thioester bond between a cysteine and

glutamine residue relevant for adhesion to host molecules. RrgA and GBS104

consist of a top domain with a von Willebrand factor A-type domain (vWFA)

where six α-helices form a barrel with a β-sheet in the center. The minor

base pilin GBS52 from S. agalactiae, known to mediate adhesion to lung

epithelial cells (Krishnan et al., 2007), and FctB from S. pyogenes (Linke et

al., 2010) are both believed to be located at the base of the protein and is

attached to the cell wall by housekeeping sortase A. The structure of GBS52

and FctB both consist of domains with IgG-rev folds but only GBS52 is

stabilized by an isopeptide bond (Kang et al., 2007).

Figure 4: Domain organization of different groups of surface adhesins with

signal peptide in dark grey, and the putative adhesin region in red/orange.

A) Pilin backbone subunit of Spy0125 from S. pyogenes; signal peptide, N-

terminal domain, and a C-terminal region (CTR). B) AgI/II AspA from S.

pyogenes; signal peptide, N-terminal domain, Alanine-rich domain, Variable

domain, Proline-rich domain, C-terminal domain, and a cell wall anchor

segment. C) MSCRAMM Sgo0707 from S. gordonii; signal peptide, non-

repetitive N-terminal domain, two repeat regions, a small non-repetitive C-

terminal region, and a cell wall anchor segment.

10

Figure 5: Models of cell wall anchored adhesins. A) Illustration of the S.

pyogenes pili, B) the AgI/II protein, C) MSCRAMM Sgo0707 from S.

gordonii. The backbone pilin in A is reduced in number of repeats.

Figure 6: Common β-sandwich folds in Gram-positive adhesins. A) The

IgG constant fold. B) The DE-variant IgG (DEv-IgG) fold has additional

modules between the D and E β-strands and can contain an isopeptide bond

between β-strand A and F. C & D) The two variants of the IgG-rev fold has

reversed order of the β-strands and can contain an isopeptide bond between

β-strand A and G. Adapted from (Vengadesan and Narayana, 2011).

11

Antigen I/II

Antigen I/II (AgI/II) is a family of surface proteins expressed by basically all

oral streptococci, and also some streptococci not specific to the oral cavity.

AgI/II facilitate colonization by mediating adherence to an array of host

molecules, such as the salivary agglutinin glycoprotein 340 (gp-340)

(Loimaranta et al., 2005; Zhang et al., 2006), fibronectin (Petersen et al.,

2002), and collagen (Heddle et al., 2003; Soell et al., 2010), as well as to

other microorganisms, Porphyromonas gingivalis (Daep et al., 2006; Park

et al., 2005), Candida albicans (Silverman et al., 2010) and Actionomyces

(Jakubovics et al., 2005). The protein consist of approximately 1500 amino

acids and have the following domain structure (figure 4B); an N-terminal

domain (N), an Alanine-rich repeat region (A), a Variable domain (V), a

Proline-rich repeat region (P), a C-terminal domain (C), followed by a cell

wall anchor (CWA) segment with an LPxTG-motif, a hydrophobic domain,

and finally a positively charged tail (Brady et al., 2010; Jenkinson and

Demuth, 1997). Despite the central location of the V-domain in the primary

sequence, the V-domain is presented at the tip of the protein with the

flanking A- and P-domains intertwining, forming a stalk (Larson et al.,

2010). The C- and the N-domains form the base of the protein at the cell

surface (figure 5B). The elongated protein model has a length of > 50 nm and

is stabilized in the C-domain by metal atoms (Ca2+) and isopeptide bonds.

The amino acid sequence identity between the full-length AgI/II proteins in

different streptococcal species ranges from 80% between S. gordonii SspA

and SspB, to only 27% between S. mutans SpaP and S. pyogenes AspA.

The first determined structure of the AgI/II protein family was the V-domain

from the caries pathogen S. mutans (Troffer-Charlier et al., 2002). The V-

domain of S. mutans is made up of a β-sandwich with eight β-strands in each

sheet. The V-domain from the commensal S. gordonii was solved (Forsgren

et al., 2009) and showed high structural similarity despite low sequence

identity (26%) to the V-domain from S. mutans. A putative binding pocket

with a bound metal ion is present in both structures. The C-domain of AgI/II

proteins consists of three domains, C1, C2, and C3. The C2-3 domains of SspB

from S. gordonii (Forsgren et al., 2010), SpaP from S. mutans (paper I)

(Nylander et al., 2011), and AspA from S. pyogenes (paper IV), and C1-3

domains of SpaP from S. mutans (Larson et al., 2011) have been determined

revealing a striking structural identity among the proteins. Each domain

forms a β-sandwich with DEv-IgG fold, with isopeptide bonds covalently

linking the β-sheets in the β-sandwich. All structures contained at least one

stabilizing metal ion modeled as Ca2+. S. gordonii SspB binds the minor

fimbriae Mfa1 on P. gingivalis but despite structural similarity, SpaP cannot

bind Mfa1 (Brooks et al., 1997).

12

Microbial Surface Components Recognizing Adhesive Matrix

Molecules (MSCRAMM)

The definitions of MSCRAMMs are microbial molecules located at the cell

wall that recognize ligands (for example collagen, laminin, fibronectin, and

fibrinogen) in the ECM with high affinity and specificity. The MSCRAMMs

share a domain organization with an N-terminal signal peptide, followed by a

non-repetitive sequence (putative adhesin domain). Next follows one or two

separate repeat regions, a cell wall-spanning domain with an LPxTG-sorting

motif, a hydrophobic membrane-spanning domain, and finally a positively

charged C-terminal tail (Patti et al., 1994) (figure 4C). The repeat regions of

MSCRAMMs are varied both in size and in number of repeats as well as in

the number of different repeat regions present. Some fibrinogen-binding

proteins (Staphylococcus aureus ClfA (Deivanayagam et al., 2002), Staph.

aureus ClfB (Ganesh et al., 2011), and Staphylococcus epidermidis SdrG

(Ponnuraj et al., 2003) have 200-300 serine-aspartic acid residue repeats in

the C-terminal. The Serine-rich repeat (SRR) proteins GspB and Fap1 from

S. gordonii and Streptococcus parasanguinis respectively, have an

approximately 2000 residue long segment of SRRs. The structure of the non-

repetitive N-terminal part, have been determined, and GspB consist of

domains with predominately β-sandwiches. (Pyburn et al., 2011). The non-

repetitive N-terminal domain of S. parasanguinis Fap1 can be divided into

two domains, the Nα-domain, consisting of α-helices (Garnett et al., 2012)

and the Nβ-domain, consisting of a β-sandwich (Ramboarina et al., 2010). S.

gordonii Sgo0707 has two repeat regions, one with 84 amino acids repeated

eight times, followed by an 88 amino acid sequence repeated five times

(paper II) (Nylander et al., 2013). MSCRAMMs are an important virulence

factor so the structure of these proteins can give vital information for

possible drug design. β-sandwiches are the most common feature of the N-

terminal putative adhesin domain in MSCRAMMs protein, especially of the

DEv-IgG fold. Staph. aureus display the collagen binding CNA protein on the

cell surface and structural studies on the N-terminal half (called A) reveled

β-sandwiches of the DEv-IgG fold (Symersky et al., 1997). CNA-A binds

collagen in a so called “hug model” by assuming an open conformation where

collagen is able to bind in between N1 and N2 (Zong et al., 2005). The C-

terminal half of the CNA protein, CNA-B (consist of three repeats, B1-3)

displays the IgG-rev fold (Deivanayagam et al., 2000). ACE from

Enterococus faecalis (Liu et al., 2007) and the N-terminal part of RspB from

the human pathogen Erysipelothrix rhusiopathiae shows two β-sandwiches

with the DEv-IgG fold (Devi et al., 2012). β-sandwiches can be seen in S.

pyogenes Epf (Linke et al., 2012), UafA from uropathogen Staphylococcus

saprophyticus (Matsuoka et al., 2010), the N-terminal part of ACP (NtACP)

from S. agalactiae (Auperin et al., 2005), and Spr1345-MucBD from S.

pneumoniae (Du et al., 2011). The authors of the structures of all the

13

MSCRAMMs above do not mention the presence of any isopeptide bond, or

they state that no isopeptide bonds were found. Only two structures seem to

contain metal ions, ClfA and UafA.

The M1 protein from S. pyogenes and protein G from the Lancefield group G

streptococci differ in structure to pili, AgI/II, and MSCRAMM mentioned in

this thesis, as they lack the commonly found β-sandwich folds. The N-

terminal part of the M1 protein forms an α-helical coiled coil (McNamara et

al., 2008). The structures of the three domains (B1-B3) of protein G from the

Lancefield group G streptococci (Achari et al., 1992; Derrick and Wigley,

1994; Gallagher et al., 1994) was determined individually and they all display

a β-sheet and an α-helix that share high structural similarities between them,

with a root-mean-square deviation (RMSD) of 0.4 Å, and sequence identity

ranging from 88-98%.

14

Table 1: Structures of Gram-positive surface adhesins determined by X-ray crystallography. Organism Type of protein Ligand Fragment PDB code Reference Comments Actinomyces oris Major pilin (type-

2) Asialofetuin, oral epithelial cells, and S. oralis.

FimA 3QDH Mishra 2011 DEv-IgG and rev-IgG folds. Isopeptide bond, disulphide bond and Zn2+. (Trypsin treated).

Actinomyces oris Major pilin (type-1)

FimP 3UXF Persson 2012 IgG-like fold. Isopeptide bond, disulphide bond and Ca2+.

Bacillus cereus Major pilin BcPA 3KPT 3RKP (Mutants)

Budzik 2009 Hendrickx 2012

Rev-IgG and IgG-like fold. Isopeptide bonds. (Trypsin treated).

Corynebacterium diphtheriae

Major pilin SpaA 3HR6 Kang 2009 DEv-IgG and rev-IgG folds. Isopeptide bond and Ca2+.

S. agalactiae Major pilin(type-1) GBS80 3PF2 Vengadesan 2011 DEv-IgG and rev-IgG folds. Isopeptide bonds and Ca2+. (α-chymotrypsin treated).

S. agalactiae Minor (tip) pilin (type-1)

GBS104 3TXA (N2-N3) Krishnan 2013 β-sandwich and a vWFA -type domain.

S. agalactiae Minor (base) pilin (type-1)

Lung epithelial cells GBS52 3PHS Krishnan 2007 Rev-IgG folds and

isopeptide bond. S. pneumoniae Major pilin RrgB 2Y1V El-Mortaji 2012 IgG-like fold and isopeptide

bonds. S. pneumoniae Minor (tip) pilin Host cells,

fibronectin, collagen, and laminin.

RrgA 2WW8 Izore 2010 β-sandwiches and a vWFA -type domain. MIDAS motif with Mg2+ and isopeptide bonds.

S. pyogenes Major pilin Spy0128 3B2M Kang 2007 IgG-like fold and isopeptide bonds.

S. pyogenes Minor (tip) pilin Spy0125 2XI9

Pointon 2010 IgG-rev fold. Thioester and isopeptide bonds.

S. pyogenes Minor (base) pilin FctB 3KLQ Linke 2010 Rev-IgG fold. Extended proline-rich tail.

15

Organism Type of protein Ligand Fragment PDB code Reference Comments S. mutans AgI/II AgI/II-V 1JMM Troffer-Charlier

2002 β-sandwich and a metal ion.

S. gordonii AgI/II SspB-V 2WD6 Forsgren 2009 β-sandwich and Ca2+. S. gordonii AgI/II P. gingivalis SspB-C2-3 2WZA Forsgren 2010 DEv-IgG folds. Isopeptide

bonds and Ca2+. S. pyogens AgI/II AspA-C2-3 Hall TBP DEv-IgG folds. Isopeptide

bonds and Ca2+. S. mutans AgI/II SpaP-C2-3 3OPU Nylander 2011 DEv-IgG folds. Isopeptide

bonds and Ca2+. S. mutans AgI/II PAc-C1-3 3QE5 Larson 2011 DEv-IgG fold. Isopeptide

bonds and Ca2+. S. mutans AgI/II A3VP1 3IOX Larson 2010 β-sandwich and Ca2+ in V-

domain, A- and P-domains form a stalk.

Enterococus faecalis

MSCRAMM Collagen ACE 2OKM (N2 d) 2Z1P (N1-N2 )

Ponnuraj 2007 Liu 2007

DEv-IgG fold on N1 and N2.

Erysipelothrix rhusiopathiae

MSCRAMM Collagen RspB-N 3V10 Devi 2012 DEv-IgG folds.

Staph aureus MSCRAMM Collagen CnaA 1AMX (N2) 2F6A(collagen)

Symersky 1997 Zong 2005

DEv-IgG fold on N1 and N2. Collagen-hug.

Staph aureus MSCRAMM CnaB (B1-B2 domains)

1D2O (B1) 1D2P (B1-B2)

Deivanayagam 2000

Rev-IgG folds, repeat region.

Staph aureus MSCRAMM Fibrinogen ClfA (N2-N3

domains) 1N67 2VR3 (N2-N3 + fibrinogen)

Deivanayagam 2002, Ganesh 2008

DEv-IgG fold on N2 and N3, metal ions.

Staph aureus MSCRAMM Fibrinogen, Cytokeratin 10, Dermokine.

ClfB (N2-N3 domains)

4F27 (Fibrinogen) 3AU0

Xiang 2012 Ganesh 2011

DEv-IgG folds.

Staph epidermidis MSCRAMM Fibrinogen SdrG (N2-N3 domains)

1R19 2RAL (Mutants)

Ponnuraj 2003 Bowden 2008

DEv-IgG folds.

Staph saprophyticus

MSCRAMM UafA 3IRP Matsuoka 2011 β-sandwiches and K+.

S. agalactiae MSCRAMM Heparin. NtACP 1YWM Auperin 2005 β-sandwich and a three α-helix bundle.

16

Organism Type of protein Ligand Fragment PDB code Reference Comments S. gordonii MSCRAMM Collagen,

Kerationocytes. Sgo0707-N 4IGB Nylander 2013 N1 consist of a β-sandwich,

N2 of a DEv-IgG fold. S. pyogenes MSCRAMM Human epithelial

cells Epf 4ES8 Linke 2012 β-sandwiches.

S. pyogenes MSCRAMM Fibrinogen M1 2OTO McNamara 2008 α-helices forming a coiled coil.

S. pneumoniae MSCRAMM Mucin Spr1345-MucBD

3NZ3 Du 2011 IgG-like fold.

Lancefield group G streptococci

MSCRAMM Serum albumin and Human IgG

Protein G 1PGA/B (B1) 1PGX (B2) 1IGD (B3)

Gallagher 1994 Achari 1992 Derrick and Wigley 1994

β-sheet and an α-helix.

S. gordonii SRR/MSCRAMM

Human platelets GspB 3QC5 Pyburn 2011 β-sandwiches and a metal ion.

S.parasanguinis SRR/MSCRAMM

Fap1 3RGU (NRα) 2X12 (NRβ)

Garnett 2012 Ramboarina, 2010

NRα consist of α-helices and NRβ of a β-sandwich.

17

Materials and methods

Cloning, protein expression and purification

The gene of interest is amplified using PCR with gene specific primers.

Ligation of the DNA-fragment into the lac operon of the expression vector is

done after the digestion of the vector and PCR product with appropriate

restriction enzymes. The vector contains the DNA code for six residues of

histidine, facilitating protein purification. Antibiotic resistance will be

relayed to the correct constructs of gene and vector and can thus be selected

on agar plats containing the correct antibiotic. Protein expression is usually

done in Escherichia coli expression strains and is induced by the addition of

isopropyl-β-D-thiogalactopyranoside (IPTG) which activates transcription of

the lac operon. The standard protein purification process implemented in

this thesis was done in two steps, first by immobilized metal affinity

chromatography (IMAC) where histidine residues in the his-tag of the

protein adheres to the nickel-matrix, facilitating purification through

affinity, secondly by size exclusion chromatography where the proteins are

fractioned according to size.

From crystal to structure

The two step purification protocol usually renders the protein of high enough

purity (95%) to be set up for crystallization trails; if the purity is not reached

additional purification steps can be performed, for example ion exchange

chromatography. The protein does not only need to be pure, it also must be

homogenous (all molecules must be identical), and monodisperse (exist as

monomers, dimers, or larger oligomers, not a mixture) in order to pack into

crystals.

Vapour diffusion is a common technique for obtaining crystals, where a drop

of protein is mixed with the well solution (mother liquor) and placed either

as a hanging drop, or sitting drop in air contact to the well solution. As the

well solution is diluted in the drop by the addition of the protein, equilibrium

will be reached through vapour diffusion of water from the drop to the well

solution. In order to initiate crystallization the protein must be

supersaturated and nucleation must occur. The growth of the crystal will

continue until the protein in the drop is depleted or if defects, or mechanical

stress renders crystal growth impossible. There are commercial

crystallization kits available to test the protein against an array of different

buffers, pH, precipitants, and additives.

18

Crystallography is a method to get a 3D image of a protein by subjecting it to

X-rays. The content of the protein crystal (the unit cell and the protein

electrons) will diffract the X-ray beam and the structure of the protein can be

calculated. Acquired crystals are mounted on loops and vitrified in a stream

of liquid nitrogen to limit radiation damage during data collection. Data

collection can be done at a synchrotron (possibility to vary the wavelength

and a sharper beam) or a home source (set wavelength). The mounted crystal

is subjected to X-rays, and the crystal is rotated in increments of usually 1.0

degree during which an image of the diffraction pattern is collected. Most of

the X-rays will go straight through the crystal, but approximately 5% of the

X-rays will be diffracted by the crystal. The diffracted beam will give rise to

spots on the detector and the positions and intensity of the spots will be

recorded. The amplitude of the diffracted wave can be calculated from the

intensities, but its phase is not recorded. This is the so called phase problem

of crystallography, and it must be solved in order to obtain an electron

density map. To solve the phase problem a number of techniques can be

implemented; multiple isomorphous replacement (MIR), multiple-

wavelength anomalous dispersion (MAD), single-wavelength anomalous

dispersion (SAD) and molecular replacement (MR). To use MIR data sets are

collected of the native protein crystal and also of crystals soaked in liquid

containing heavy atoms. The native data set is subtracted from the heavy

atom soaked data set, resulting in the intensities contributed by the heavy

atom, and the location of the heavy atom can be identified. This will enable

the phases of all intensities to be determined. MAD and SAD require the

introduction of heavy atoms (for example selenium) into the protein, usually

by substituting methionine with selenomethionine (SeMet) during protein

synthesis. The position of the heavy atoms can be identified and the phases

can be determined. If a homologous protein structure is known usually with

more than 30% sequence identity, MR can be used to get initial estimates of

the phases.

The bottleneck of X-ray crystallography is the requirement of diffracting

protein crystals. The ultimate method is to clone, express, purify, and

crystallize the full-length protein, but this is often only possible for smaller

proteins. For Gram-positive surface proteins this is usually difficult due to

the large size of the full-length protein. The large proteins can therefore be

divided into domains in order to reduce the size of the protein. The design of

constructs can be done by mimicking previously used domain borders in

already determined crystal structures and also by sequence analysis. During

work with AspA-AVPC from S. pyogenes N-terminal sequencing and matrix-

assisted laser desorption/ionization time of flight mass spectrometry

(MALDI-TOF MS) were used to analyze the fragments. N-terminal

sequencing is done by transferring the proteins separated by sodium dodecyl

19

sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to a PVDF

membrane. The protein is cleaved repeatedly at the N-terminal one amino

acid at the time and the leaving amino acid is determined. Since only amino

acids in the N-terminal part of the proteins are determined, MALDI-TOF MS

is preformed in order to get a more complete view of the fragments. In-gel

trypsin digestion is carried out and the peptides are subjected to a laser beam

that ionizes the fragments and the time it takes for the fragment to reach the

detector is dependent on the mass of the fragment.

20

Aim of thesis The purpose of this thesis was to determine the 3D structure of streptococcal

surface adhesins using X-ray crystallography techniques, and also to conduct

functional studies in order to elucidate possible binding partners to the

structurally determined adhesins.

Specific aims

Paper I Determine and study the structure of SpaP-C2-3 from the

caries pathogen S. mutans, and compare the structure to the

commensal SspB from S. gordonii.

Paper II Determine the structure and perform functional studies of the

MSCRAMM Sgo0707 from S. gordonii.

Paper III Express and purify AspA-AVPC from S. pyogenes, and

elucidate fragmentation behavior.

Paper IV Determine and study the structure of AspA-C2-3 from S.

pyogenes, and compare the structure to the homologous

proteins SpaP from S. mutans and SspB from S. gordonii.

21

Result and discussion

Structures of the C-terminal domain of the antigen I/II protein

family (paper I & IV)

During the last decade, 3D structures of domains of AgI/II proteins from oral

streptococci have been determined using X-ray crystallography. In order to

compare the known structure of the commensal S. gordonii SspB to the

caries implicated S. mutans SpaP (paper I) and the human pathogen S.

pyogenes AspA (paper IV) the structures of the truncated C-terminals from

these two proteins were determined using X-ray crystallography.

The structures of SpaP-C1136-1489 and AspA-C971-1306

The protein crystals of SpaP-C1136-1489 belonged to the P21212 space group and

contained six copies in the asymmetric unit. The monomeric structure

consisted of 354 residues and was solved by MR and refined to a resolution

of 2.2 Å. The AspA-C971-1306 protein crystals also belonged to the P21212 space

group and contained two molecules in the asymmetric unit. The structure

was solved by MR and refined to a resolution of 1.8 Å, and consisted of 336

residues. The 3D structures of SpaP-C1136-1489 and AspA-C971-1306 consist of

two domains; C2 and C3 (figure 7A and B). In C2 the β-strands form anti

parallel sheets from five and six β-strands resulting in a β-sandwich with a

DEv-IgG fold. Two short α-helices are located on top of the β-sandwich

forming the apex of the protein. An α-helix is present perpendicular to the β-

sandwich facing the surroundings. The C3 domain contains a DEv-IgG

folded β-sandwich constructed from two antiparallel sheets, each cosist of

five strands.

Stabilization of SpaP-C1136-1489 and AspA-C971-1306

SpaP-C1136-1489 and AspA-C971-1306 are stabilized in both domains by isopeptide

bonds (figure 7A and B). All four isopeptide bonds in the two structures are

formed between a lysine side chain in the first β-strand in one sheet, and an

asparagine side chain in the last β-strand of the other sheet, resulting in

covalently linking the two β-sheets in the β-sandwich, adding stability to the

β-sandwich and the protein as a whole. The essential aspartic acid is located

in strand C, from the same sheet as the asparagine residue is located. To

further stabilize the SpaP protein, two metal ions (modelled as calcium ions)

are bound in the vicinity of the isopeptide bonds in both domains (figure 7A).

The AspA protein only contains one metal ion, located in the C2 domain

close to the isopeptide bond (figure 7B).

22

Figure 7: A) The crystal structures of S. mutans SpaP-C2-3 and B) S.

pyogenes AspA-C2-3. Calcium ions and isopeptide bonds are represented by

grey sphere and black bonds, respectively. C) Superposition of SpaP (blue),

AspA (green), and SspB from S. gordonii (red).

The structures of SpaP-C1136-1489 and AspA-C971-1306 put in

perspective

The structures of SpaP-C1136-1489 and AspA-C971-1306 are very similar with an

RMSD of 1.84 Å on 332 aligned Cα and a sequence identity of 36%. Also S.

gordonii SspB-C are very similar to S. mutans SpaP-C1136-1489 with an RMSD

of 1.2 of 337 aligned Cα, and a sequence identity of 66% (figure 8). As the

RMSD suggest, the structures are very similar as shown by superposition of

the three structures (figure 7C). The isopeptide bonds and calcium ions are

located in identical positions. The SspB Adherence Region (BAR) is a

segment (1167 to 1250) in S. gordonii SspB involved in adherence to P.

gingivalis fimbria Mfa1 (Brooks et al., 1997). Studies with a synthetic peptide

of 27 residues from BAR in SspB (1167-1193) and the corresponding

sequence in SpaP (1242-1268) resulted in adherence between the SspB

peptide and Mfa1, but no adherence to SpaP, despite a 59% primary

sequence identity in that area. The different residues result in different

electrostatic surface charge of the protein in this area. Mutation of two amino

acids (1182 N to G, and 1185 V to P) in the SspB peptide to mimic the amino

acids in SpaP, showed no adherence. (Demuth et al., 2001). This implies that

this part of the BAR is involved in species specific interactions. The two

corresponding amino acids in AspA are identical to SpaP, so adherence to

Mfa1 is not to be expected.

23

AspA NLIQPTKTIVDDKGQSIDGKSVLPNSTLTYVAKQDFDQYKGMTAAKESVMKGFIYVDDYK

SpaP NYIKPTKVNKNENGVVIDDKTVLAGSTNYYELTWDLDQYKNDRSSADTIQKGFYYVDDYP

SspB LYPEGAMVNKNKEGLNIDGKEVLAGSTNYYELTWDLDQYKGDKSSKEAIQNGFYYVDDYP

* ** * ** ** * * **** ** *****

AspA DEAIDGHSLVVNSIKAANGDDVTNLLEMRHVLSQDTLDDKLKALIKASGISPVGEFYMWV

SpaP EEALELRQDLVKI-TDANGNEVTGV-SVDNYTSLEAAPQEIRDVLSKAGIRPKGAFQIFR

SspB EEALDVRPDLVKV-ADEKGNQVSGV-SVQQYDSLEAAPKKVQDLLKKANITVKGAFQLFS

** * * * * * * *

AspA AKDPAAFYKAYVQKGLDITYNLSFKLKQDF--KKGDITNQTYQIDFGNGYYGNIVVNHLS

SpaP ADNPREFYDTYVKTGIDLKIVSPMVVKKQMGQTGGSYENQAYQIDFGNGYASNIVINNVP

SspB ADNPEEFYKQYVATGTSLVITDPMTVKSEFGKTGGKYENKAYQIDFGNGYATEVVVNNVP

* * ** ** * * * * ********* * *

AspA ELTVHKDVF---DKEGGQSINAGTVKVGDEVTYRLEGWVVPTNRGYDLTEYKFVDQLQHT

SpaP KINPKKDVTLTLDPADTNNVDGQTIPLNTVFNYRLIGGIIPANHSEELFEYNFYDDYDQT

SspB KITPKKDVTVSLDPTS-ENLDGQTVQLYQTFNYRLIGGLIPQNHSEELEDYSFVDDYDQA

*** * * *** * * * * * * *

AspA HDLYQK-DKVLATVDITLSDGSVITKGTDLAKYTETVYNKETGHYELAFKQDFLAKVVRS

SpaP GDHYTGQYKVFAKVDIILKNGVIIKSGTELTQYTTAEVDTTKGAITIKFKEAFLRSVSID

SspB GDQYTGNYKTFSSLNLTMKDGSVIKAGTDLTSQTTAETDATNGIVTVRFKEDFLQKISLD

* * * * * ** * * * ** **

AspA SEFGADAFVVVKRIKAGDVANEYTLYVNGNPVKSNKVTTHT

SpaP SAFQAESYIQMKRIAVGTFENTYINTVNGVTYSSNTVKTTT

SspB SPFQAETYLQMRRIAIGTFENTYVNTVNKVAYASNTVRTTT

* * * ** * * * ** ** * * *

Figure 8: Sequence alignment of the AgI/II C2-3-domains, with the boxed 27

amino acid sequence involved in S. gordonii SspB adherence to P. gingivalis

fimbriae Mfa1. Stars indicate identical residues in all three sequences.

24

Structural and functional analysis of the N-terminal domain of the

Streptococcus gordonii adhesin Sgo0707 (paper II)

S. gordonii expresses many different proteins on the cell surface with which

it interacts with the environment. Sgo0707 was identified by Davies and co-

workers as a putative surface adhesin (Davies et al., 2009) and structural and

functional studies were implemented in order to determine the structure and

to elucidate possible binding partners.

Binding studies of Sgo0707

A S. gordonii Sgo0707 deletion mutant (ΔSgo0707) was constructed in order

to elucidate structures that interact with Sgo0707. Whole saliva, serum,

collagen type-1, and keratinocytes were coated on an ibiTreat µ-slide VI flow-

cell. The wild-type and ΔSgo0707 were added to the coated flow-cells and

adhered cells were stained and visualized with confocal laser scanning

microscopy (CLSM). The ΔSgo0707 showed a 30% reduction in adherence to

collagen type-1 and keratinocytes compared to the wild-type. Saliva and

serum-coated surfaces showed no differences in adherence between wild-

type and the deletion mutant ΔSgo0707. These data suggest that Sgo0707

may be involved in adherence of S. gordonii to collagen type-1, and

keratinocytes. The N-terminal domain of the Sgo0707 protein was shown to

be involved in adherence, by adding different concentrations of recombinant

Sgo0707-N protein to flow-cells covered by adhered collagen type-1,

adherence of wild-type cells was decreased.

The structure of Sgo0707-N

Protein crystals of Sgo0707-N36-456 belonged to the I222 space group and

contained four molecules in the asymmetric unit. The structure was solved

with SAD, using SeMet labeled protein, and was refined to a resolution of 2.1

Å. The structure of the N-terminal part of Sgo0707 can be divided into two

distinct domains, N1 and N2 (figure 9A). The N136-311 domain consists mainly

of β-strands forming a β-sandwich fold, and contains a putative binding cleft

of 333 Å3 (figure 9B and C), as determined by CASTp server, with mainly

negatively surface potential. According to a DALI server search, the variable

domain of AgI/II from S. gordonii (Forsgren et al., 2009), and S. mutans

(Troffer-Charlier et al., 2002) revealed to be the closest structural relatives,

with an RMSD of 3.7 Å , and approximately 10% sequence identity. The

N2312-456 consists of a β-sandwich with a DEv-IgG-fold with the B. cereus

backbone pilin BcpA (Budzik et al., 2009) as the closest structural relative

with an RMSD of 2.3 Å, and 15% sequence identity. The location of collagen

type-1, and keratinocytes adherence to Sgo0707-N is not known, it is

however possible that the putative binding pocket is involved. A collagen

type-1 fragment was used in web-based docking programs to elucidate

25

possible locations for adherence on the Sgo0707-N crystal structure. The

results indicate the putative binding pocket and the interface between N1 and

N2 (figure 9D) as possible collagen type-1 binding sites. The N-terminal part

of Sgo0707 is presented at the tip of a putative elongated protein with the

shaft constructed of repeat domains, whose structural and functional role is

unknown.

Figure 9: A) The structure of Sgo0707-N as a ribbon presentation, with the

N1 domain in pink, N2 in purple, and a putative binding pocket (X). B) The

N1 domain with negatively charged, polar, and aromatic residues involved in

the putative binding pocket, shown as green stick models. C) View of the

binding pocket tilted 90° into the paper plane. D) The second possible

binding site of collagen type-1 (X), view rotated 90° from figure A.

26

Protein expression and purification of Streptococcus pyogenes

adhesin AspA (paper III)

The human pathogen S. pyogenes causes an array of conditions, ranging

from mild to severe. Although S. pyogenes is not specific for the oral cavity,

it does express AspA, a member of the AgI/II protein family.

Cloning, protein expression and purification of AspA

The M28_spy1325 gene from S. pyogenes was amplified almost in full length

(AspA-AVPC) using PCR. Protein expression was done in E. coli BL21 (DE3)

cells and purification was carried out using IMAC followed by size exclusion

chromatography. Purification resulted in soluble protein.

Auto fragmentation of AspA-AVPC

During laboratory work with AspA-AVPC, it was noticed that the protein

fragmented as observed with S. mutans AgI/II proteins in earlier studies

(Kelly et al., 1989; Russell et al., 1980). The fragmentation of AspA-AVPC

occurred at both 4°C and 22°C, but was accelerated in warmer temperature

and resulted in two fragments, approximately 50 and 75 kDa (figure 10).

Cysteine and serine protease inhibitors were used during protein purification

which makes it unlikely that proteases of those families are responsible for

the fragmentation. Metalloproteases are however not inhibited during

purification. Studies made by Maddocks and co-workers indicated that

AspA-V binds a zinc ion, possibly through histidine residues as observed in

certain streptococcal zinc metalloprotease (Hase and Finkelstein, 1993;

Maddocks et al., 2011). The biological relevance for the fragmentation is

unclear. One possible reason may be to relocate, if the environment is no

longer optimal for the bacteria. Or it may be a consequence of

overexpression, and may not occur under normal biological conditions.

Conformational changes in the cell wall attached protein compared to the

soluble protein may also be an explanation.

27

Figure 10: Auto fragmentation of AspA-AVPC. A) After incubation for four

days. B) After incubation for 15 days. C) Chromatogram from purification of

fragmented AspA-AVPC showing two peaks. D) Size exclusion

chromatography fractions of purified fragmented AspA-AVPC.

N-terminal sequencing and MALDI-TOF MS

The fragments were N-terminally sequenced and subjected to MALDI-TOF

MS. The result showed that the 55 kDa fragment contains the C1-3-domain,

and the 67 kDa fragment starts in the His-tag in the N-terminal part of the

protein and ends in the P-domain.

Inhibition of auto-fragmentation

The protein stability in the presence and absence of EDTA during

purification was analyzed (figure 11). The conclusion of these studies was

that auto fragmentation was inhibited in the presence of 0.5 mM EDTA. In

order to find out if the addition of a metal ion could accelerate the

fragmentation, Zn2+ was added in a range of concentrations. No increase in

fragmentation was seen, however 0.5 mM Zn2+ appears to inhibit

fragmentation (figure 11A), but how the inhibition is accomplished is not

known, possibly by blocking an active site or causing conformational changes

to the protein. These results imply that EDTA sequesters some unknown

metal ion crucial for the auto fragmentation, or that EDTA itself physically

blocks fragmentation.

28

Figure 11: AspA-AVPC protein purified with or without EDTA and

supplemented with ZnCl2, followed by incubation in 22°C for two days. A)

AspA-AVPC purified without EDTA, with ZnCl2 added at different

concentrations. B) AspA-AVPC supplemented with EDTA at different

concentrations, and in the presence or absence of ZnCl2.

29

Conclusion

The determination of the C2-3 domain of SpaP from S. mutans (paper I),

and AspA from S. pyogenes (paper IV) using X-ray crystallography revealed

that the two shared highly similar protein structures, despite the low primary

sequence identity. They were also very similar to the already known structure

of SspB from S. gordonii. All three structures consist of two domains, C2 and

C3 which consist of DEv-IgG folds in each domain. SpaP and AspA both have

stabilizing isopeptide bonds in identical positions to SspB, and calcium ions

are present to further stabilize the proteins. Despite the structural similarity,

the amino acid sequence differ in such a way that SpaP cannot be bound by

P. gingivalis fimbria Mfa1 but SspB can. The structure of MSCRAMM S.

gordonii Sgo0707-N (paper II) revealed two domains, N1 and N2, with a β-

sandwich in each domain. No isopeptide bonds or metal ions were present in

the model. However the N1 domain consists of a putative binding cleft with

mainly negative surface potential. Binding studies was performed on wild-

type cells and on a Sgo0707 deletion mutant in order to elucidate possible

ligands. Binding studies using flow-cell techniques resulted in collagen type-

1, and keratinocytes as possible binding partners. Keratinocytes line the oral

cavity and are important ligands in the oral cavity, whereas collagen type-1

may be of interest when S. gordonii is spread to non-oral sites, as with

infective endocarditis. Expression and protein purification of AspA-AVPC

from S. pyogenes (paper III) resulted in a soluble protein that easily

fragmented into two pieces of 55 and 67 kDa in the absence of EDTA. The

biological relevance for the fragmentation is not clear, but one reason may be

to relocate when the environment is no longer suitable. Continued work with

AspA-AVPC to further elucidate the fragmentation is of outmost importance.

It would be of interest to clone, express, and purify individual domains, and

different combinations thereof to see whether or not fragmentation occurs,

and the putative metalloprotease could in that way possibly be identified. If

AspA turns out to be a metalloprotease, the V-domain is the most likely

candidate because of the bound metal ion and its location at the tip of the

protein. The three determined structures will add to the knowledge of Gram-

positive surface proteins generally, and streptococcal adhesins specifically.

But much more work need to be done in order to clarify the composition of

molecules involved in colonization within the oral cavity.

30

Acknowledgements

Det finns många människor som förtjänar min tacksamhet! Här nedan

nämner jag de jag inte glömt bort!

Först och främst vill jag tacka min handledare Karina Persson för din

hjälp med allt inom forskningen, men även för att du vid väl valda tillfällen

slängde ut orden: Slappna av!

Min bihandledare Elisabeth Sauer-Eriksson för hennes expertis!

Jobbarkompisarna på Strukturbiologin (dåtid); Nina, Malin, Ulrika,

Monika, Anders O, Anders Ö, Gitte, Kristoffer, Shenghua, Stefan,

(nutid); Karina, Liz, Lixiao, Aaron, Uwe, Tobias, Roland, Afshan och

Misgina. Med en särskilt tack till Christin som kan svara på det mesta!

Tack till Malin LP för all hjälp och stöd med allt mellan himmel och jord!

Tack till vår lilla grupp som vuxit sig större! Nina som la grunden, Michael

och Patrik (a.k.a. bildexperten) som fortsätter arbetet! Ett särskilt tack till

Michael som hjälpt mig med en massa saker, däribland korrekturläsning av

denna avhandling!

Jag hade privilegiet att tillhöra den Svenska Nationella Forskarskolan i

Odontologisk Vetenskap och har därigenom träffat en hel hög trevliga

människor! Våra kurser har varit berikande både akademiskt och socialt.

Särskilt tack till Anna Oldin för att du gjort mötena extra roliga!

Tack också till alla på Odontologin, särskilt tack till Jan O, Anders J,

Elisabeth G, Rolf, Bernard och Lillemor.

Livet består ju inte bara av jobb, så tack även till min familj och utökade

släkt! Tack till Siv, Hans, Björn och Niklas!

Tack till vänner som finns där för både toppar och dalar! Särskilt tack till

Åsa, Daniell och Hobbe!

Till min älskade Mattias, tack för att du alltid stöttat mig! Tack också för att

du plockat ner mig från väggarna när stressen blivit för påträngande!

(Som en lite parentes vill jag också tacka mig själv! BRA JOBBAT!!! )

31

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