membrane heme receptor HasR from Serratia marcescens
Dissertation
zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)
vorgelegt von
Dipl. Biol. Stefanie Becker, geb. Krieg
an der
Mathematisch - Naturwissenschaftliche Sektion Fachbereich Biologie
Tag der mündlichen Prüfung: 20.03.2012 1. Referent: Prof. Dr. Wolfram Welte
2. Referent: Prof. Dr. Winfried Boos
3. Referent: Prof. Dr. Stephan Nuÿberger
Zusammenfassung XI
Summary XIII
1 Introduction 1
1.1 The cell envelope of Gram negative bacteria . . . 1
1.2 Iron . . . 4
1.3 TonB dependent transporters . . . 6
1.3.1 Siderophore receptors . . . 6
1.3.2 Heme receptors . . . 8
1.3.3 The TonB-ExbB-ExbD complex . . . 9
1.3.4 Mechanisms of energy transfer and substrate transport . . . 10
1.4 Regulation . . . 11
1.4.1 Fur . . . 11
1.4.2 Sigma factors . . . 13
1.5 Heme acquisition in Serratia marcescens . . . 13
1.6 The heme acquisition system Has . . . 14
1.6.1 HasA . . . 16
1.6.2 HasR . . . 18
1.6.3 Interaction between HasA and HasR . . . 19
1.6.4 HasI-HasS . . . 21
1.6.5 HasB . . . 21
2 HasA-HasR 23 2.1 Motivation . . . 23
2.2 Methods . . . 23
2.2.1 Protein expression . . . 23
2.2.2 Protein purication . . . 24
3 HasR mutants 43
3.1 Isoleucine 671 . . . 43
3.1.1 Motivation . . . 43
3.1.2 Methods . . . 43
3.1.3 Results . . . 44
3.2 Mutants aected in contact area one . . . 49
3.2.1 Motivation . . . 49
3.2.2 Methods . . . 49
3.2.3 Results . . . 49
3.3 Mutants aected in contact area two . . . 52
3.3.1 Motivation . . . 52
3.3.2 Methods . . . 52
3.3.3 Results . . . 53
3.4 Heme binding histidines . . . 56
3.4.1 Motivation . . . 56
3.4.2 Methods . . . 56
3.4.3 Results . . . 56
4 HasR 61 4.1 Motivation . . . 61
4.2 Methods and results . . . 61
4.2.1 Purication of HasR alone . . . 61
4.2.2 Preparation of HisHasR . . . 62
4.2.3 Stability of HasR . . . 64
4.3 Summary of the results . . . 66
5 HasA-HasR-HasB 69 5.1 Motivation . . . 69
5.2.2 Purication . . . 71
5.3 Results . . . 72
5.3.1 Susceptibility of the holoHasA-HasR-HasB complex to ionic strength 72 5.3.2 Crystallization . . . 72
5.3.3 R733Q . . . 73
6 Discussion 75 6.1 HasA-HasR . . . 75
6.1.1 Comparison of HasR and ShuA . . . 75
6.1.2 Heme . . . 76
6.1.3 HasA . . . 79
6.1.4 B-Factors . . . 80
6.1.5 I671G . . . 83
6.1.6 H603A . . . 83
6.1.7 R297A-N800A . . . 84
6.2 HasR . . . 87
6.2.1 Crystallization . . . 87
6.2.2 Dps . . . 88
6.3 HasA-HasR-HasB . . . 89
6.4 Outlook . . . 89
6.5 Conclusion: model of HasA-binding and heme-transfer . . . 91
References 93
Abbreviations 109
Publications 111
Danksagungen 113
1.1 The cell envelope of Gram negative bacteria . . . 2
1.2 Structure of OmpF . . . 3
1.3 Structure of LamB . . . 4
1.4 Structure of heme b . . . 5
1.5 Structures of siderophore receptors . . . 6
1.6 Structures of siderophore receptors . . . 8
1.7 Structures of TonB . . . 10
1.8 The fur box and the Fur structure . . . 12
1.9 The heme acquisition system of Serratia marcescens . . . 15
1.10 Structures of HasA . . . 17
1.11 Domains of HasR . . . 18
1.12 Spectra of holoHasA, holoHasR and holoHasA-HasR . . . 20
1.13 Alignment of TonB and HasB . . . 22
2.1 HasA expression and purication . . . 25
2.2 HasR expression and purication . . . 26
2.3 Crystals of the HasA-HasR-complexes . . . 28
2.4 Fluorescence scan . . . 29
2.5 Results from SHELXC/D/E . . . 31
2.6 Maps from dierent stages of the structure determination process . . . 33
2.7 Structure of the HasA-HasR-heme complex . . . 35
2.8 Interface between HasA and HasR . . . 37
2.9 Mutations in the HasA-HasR interface . . . 38
2.10 Surface properties of the HasA-HasR-heme complex . . . 39
2.11 Electrostatic surfaces of the HasA-HasR-heme complex . . . 41
2.12 Comparison of the holo and apo complex . . . 42
3.1 Structure of the HasA-HasR(I671G)-heme complex . . . 46
4.2 Stability of HasR . . . 65
4.3 Stability of HasR . . . 65
4.4 Aminoacid sequence of HasR . . . 68
5.1 Models of the HasA-HasR-heme complex with TonB . . . 70
5.2 Induction and purication of HasB . . . 71
5.3 Stability of the holoHasA-HasR-HasB complex . . . 72
5.4 A crystal of the holoHasA-HasR(R733Q)-HasB-heme complex and the dirac- tion pattern of this crystal . . . 74
6.1 Comparison of HasR and ShuA . . . 77
6.2 Structural alignment of HasR and ShuA . . . 78
6.3 Comparison of HasA structures . . . 79
6.4 Comparison of B-Factors of HasR . . . 81
6.5 Comparison of B-Factors of HasA . . . 82
6.6 Heme binding site in HasA-HasR(H603A)-heme and HasA-HasR(I671G)- heme . . . 84
6.7 Snapshots illustrating the course of the targeted molecular dynamic simu- lation . . . 86
6.8 Structure of Dps . . . 88
1.1 Heme anities of HasA mutants . . . 20
2.1 Data statistics for HasA-HasR-heme and HasA-HasR . . . 30
2.2 Renement statistics for the wild type HasA-HasR complexes . . . 32
2.3 List of H-bonds and salt bridge in the HasA-HasR interface . . . 36
3.1 Data statistics for the HasA-HasR(I671G)-heme complex and the HasA- HasR(I671G)-bisheme complex. . . 45
3.2 Renement statistics for the HasA-HasR(I671G)-heme complex and the HasA-HasR(I671G)-bisheme complex. . . 48
3.3 Statistics for the HasA-HasR(P669A)-heme complex. . . 51
3.4 Statistics for the HasA-HasR(R297A-N800A)-heme complex. . . 53
3.5 List of H-bonds and salt bridge in the HasA-HasR(R297A-N800A) interface 54 3.6 Statistics for the HasA-HasR(H603A)-heme complex. . . 59 6.1 Conditions yielding crystals of the HasA-HasR(R733Q)-HasB-heme complex 89
Die hier vorgelegte Arbeit beschäftigt sich mit kristallographischen Untersuchungen an dem Auÿenmembranrezeptor HasR aus Serratia marcescens.
S. marcescens ist ein opportunistisches Pathogen und einer der häugsten Kranken- hauskeime. Dabei ist einer der bestimmenden Faktoren für seine Virulenz die Fähigkeit, Eisen aus dem Wirtskörper aufzunehmen. Dieses ist hauptsächlich in Form von Häm ver- fügbar. Das hier untersuchte Häm-Aufnahme-System besteht aus dem Auÿenmembran- rezeptor HasR, der freies Häm oder Häm von dem extrazellulären Häm-Binde-Protein HasA aufnehmen kann. HasA seinerseits kann freies Häm oder an Hämoglobin oder den Hämoglobin-Haptoglobin-Komplex gebundenes Häm binden. Die für den Transport durch die Auÿenmembran des Häms benötigte Energie wird von dem periplasmatischen Protein HasB an den Rezeptor übertragen. Die hier gezeigten Untersuchungen konzentrieren sich auf den Rezeptor HasR und seine Interaktionen mit HasA und HasB. Zu Beginn dieser Arbeit war noch keine Struktur eines Häm-Rezeptors bekannt.
In dieser Arbeit wurden HasR und mehrere HasR-Mutanten im Komplex mit HasA kristallisiert und mehrere 3D-Strukturen bestimmt, die verschiedene Stadien der Bindung der beiden Proteine zeigen. Versuche, HasR allein oder im Komplex mit HasB zu kri- stallisieren, blieben bisher erfolglos.
HasR besteht aus einem 22-strängigen β-barrel mit langen extrazellulären Loops und kurzen periplasmatischen Turns. Die die Membran durchspannende Pore des Proteins ist von einer sogenannten Plug-Domäne verschlossen. Das lösliche extrazelluläre Protein HasA bindet an den Rezeptor und überträgt dabei das Häm. Sowohl die Bindung der beiden Proteine als auch die Übertragung des Häms erfolgen spontan und benötigen keine zusätzliche Energie.
In dieser Arbeit schlagen wir, basierend auf den Strukturen, und unter Einbeziehung funktioneller Daten ein Modell vor, das beschreibt, wie die beiden Proteine schrittweise miteinander interagieren und dabei das Häm übertragen wird: Der erste Kontakt der bei- den Proteine wird hauptsächlich durch elektrostatische Interaktionen verursacht und führt
wird dann aufgrund der sterischen Behinderung einer Aminosäure von HasR aus seiner Bindetasche in HasA verdrängt und von dem Rezeptor gebunden. Dieser letzte Schritt in dem Prozess der Bindung der beiden Proteine und damit gekoppelten Übertragung des Häms ist in der Struktur des wildtyp HasA-HasR-Häm-Komplexes zu sehen. Während oder nach der Bewegung des Häms von HasA zu HasR verändert eine Aminosäure von HasA ihre Konformation so, dass ein Zurückwandern des Häms zu HasA unterbunden wird. Dies zeigt der Vergleich der Strukturen des wildtyp Komplexes mit und ohne Häm.
Dieser mehrstuge Prozess verhindert vermutlich, dass das Häm beide Proteine verlässt und wieder an die Umgebung abgegeben wird. Das hier erstellte Modell könnte all- gemeine Gültigkeit besitzen für Protein-Protein-Interaktionen, die mit der Übertragung eines Liganden gekoppelt sind. Dies ist zum Beispiel auch der Fall bei Rezeptoren, die ihren Liganden direkt von Wirtsproteinen übernehmen.
Unsere Arbeit wird von mehreren kooperierenden Gruppen unterstützt und ergänzt.
Philippe Delepelaire und Mitarbeiter aus der Arbeitsgruppe für Biologische Membra- nen am Institut Pasteur in Paris führten alle genetischen, mikrobiologischen und bio- chemischen Studien an HasA und HasR durch, die die Grundlage für die strukturellen Untersuchungen bilden und unsere Ergebnisse funktionell ergänzen. Nadia Izadi-Pruneyre und Mitarbeiter aus der Arbeitsgruppe NMR an Biomolekülen am Institut Pasteur in Paris stellten uns NMR-Untersuchungen an HasB und ITC-Messungen an den Inter- aktionen zwischen HasA und HasR und zwischen HasB und HasR zur Verfügung. Simon Becker aus der Arbeitsgruppe von Kay Diederichs (AG Bioinformatik, Fachbereich Bio- logie, Universität Konstanz) führte mit Unterstützung von Thomas Exner (Fachbereich Chemie, Universität Konstanz) molekulardynamische Studien basierend auf den Kristall- strukturen durch.
The work presented here is focused on crystallographic studies on the outer membrane receptor HasR from Serratia marcescens. S. marcescens is an opportunistic pathogen which has become one of the prevalent organisms causing hospital-acquired infections.
One of its main virulence factors is its ability to acquire iron from its host, primarily in form of heme. The receptor HasR can take up free heme or heme bound to the hemophore HasA, a soluble heme-binding protein secreted by S. marcescens. HasA can acquire heme from hemoglobin or hemoglobin-haptoglobin. The transport of heme through the outer membrane requires energy, which is provided by the periplasmic protein HasB. Here, HasR was studied alone as well as regarding its interactions with the hemophore HasA and the energy-providing protein HasB. When this work was started, no structures were known for any member of the heme receptor family.
In this work, HasR and several mutants were crystallized in complex with HasA and heme and several 3D-structures determined providing insights into dierent stages of binding of the two proteins and heme transfer from HasA to HasR. Attempts to crystallize HasR alone and in complex with HasB have so far not been successful.
HasR consists of a 22-strandedβ-barrel closed by an N-terminal globular domain. The β-strands are connected by long and exible extracellular loops and short periplasmic turns. The soluble extracellular hemophore HasA binds to HasR and transfers heme to the receptor. This process of binding and heme transfer occurs spontaneously and does not require energy.
Based on the structures and in accord with functional data, we propose a model de- scribing a four-step process of protein-protein binding coupled with heme transfer. In the rst step, HasA contacts HasR mainly due to electrostatic interactions and binds in a rst transition complex. This complex is seen in a mutant crystal structure. In the second step, the proteins rearrange and HasA closes in on HasR to cover a larger interface. In this step, one of the two heme coordinating loops of HasA becomes disordered, but the heme is still bound to HasA. This step is trapped in another mutant crystal structure. In
heme being lost to the environment.
This model might be generally valid for ligand transfer from a protein with a higher to one with a lower ligand anity, as is the case, for example, for receptors interacting directly with host proteins (e.g. hemoglobin, transferrin, lactoferrin).
All studies were performed in tight cooperation with several collaborators: Philippe Delepelaire and coworkers in the Bacterial Membranes Unit at the Institut Pasteur in Paris did the genetic, micobiological and biochemical work which provide the functional examinations complementing our structural studies. Nadia Izadi-Pruneyre and coworkers in the Unit of NMR of Biomolecules at the Institut Pasteur in Paris provided NMR studies on HasB and ITC measurements on the interactions between HasB and HasR as well as HasA and HasR. Simon Becker in the group of Kay Diederichs (Fachbereich Biologie, Universität Konstanz) and Thomas Exner (Fachbereich Chemie, Universität Konstanz) performed molecular dynamics simulations based on our structures.
1.1 The cell envelope of Gram negative bacteria
The cell envelope of Gram negative bacteria has a rather complex composition of three distinct compartments (see Fig. 1.1): the inner membrane, the periplasmic space and the outer membrane [11, 39].
The inner membrane is a symmetric bilayer consisting of a mixture of phospholipids. In E. coli it is made up of 75 % phosphatidylethanolamine, 20 % phosphatidylglycerol and 5 % cardiolipin [111]. These cell membranes are relatively impermeable to hydrophilic compounds, whereas hydrophobic compounds can permeate the membrane. The inner membrane thus constitutes an eective barrier for all hydrophilic compounds. To allow uptake of these solutes transport proteins are embedded in the membrane. Active trans- port can be energized either by ion gradients across the membrane, most importantly the proton motive force (pmf) or by highly energized molecules such as ATP. Integral inner membrane proteins are mainly composed of apolar α-helices.
The periplasm between the inner and outer membrane is an aqueous compartment that contains membrane-derived oligosaccharides, soluble binding proteins belonging to inner membrane transport systems, chaperons and enzymes. In addition, it contains a thin peptidoglycan layer providing stability.
The outer membrane, in contrast to normal cell membranes, is not symmetric with re- spect to its lipid composition. The inner monolayer is composed of phospholipids as is the inner membrane. The outer layer, however, is almost exclusively made up of lipopolysac- charides (LPS) [110]. This LPS layer is much more stable than phospholipids and also much less permeable to hydrophobic compounds. It is signicantly less uid than the phos- pholipid layer. This is mainly due to the saturated fatty acid chains, which are packed more densely than the partly unsaturated, kinked fatty acid chains of phospholipids, and to the negatively charged saccharides of the core region, which often are additionally stabilized by divalent cations. The outer membrane therefore constitutes a very eec-
Figure 1.1: The cell envelope of Gram negative bacteria (from [110]).
Circles depict the polar head groups of glycerophospholipids (phosphatidylethanolamine in red and phosphatidylglycerol in yellow). Ovals and rectangles depict sugar residues (labeled in the gure. MDO: membrane-derived oligosaccharides, KDO: 3-deoxy-D-manno-octulosonic acid).
tive barrier, that also renders the bacteria very insensitive to detergents and hydrophobic antibiotics.
On the other hand, it also presents an additional barrier that has to be overcome in nutrient import and excretion. The outer membrane generally contains approximately 30-40 % protein. Outer membrane proteins almost exclusively formβ-barrels. It has been hypothesized that this is due to the fact thatβ-strands are less hydrophobic than helices:
In order to be incorporated into a membrane, in a β-barrel only every second amino acid needs to have a hydrophobic sidechain. Transmembraneα-helices on the other hand consist almost exclusively of hydrophobic amino acids. Therefore, hydrophobic helical proteins could not be transported through the inner membrane but are incorporated into it [109].
Three classes of proteins can be found in the outer membrane: porins, specic channels and active transporters.
(a) View perpendicular to the membrane plane (top ex- tracellular medium, bottom periplasm)
(b) View from the extracel- lular side
Figure 1.2: Crystal structure of the E. coli OmpF trimer (PDB-code 2OMF).
In view (b) the loop can be seen that folds back into the channel and restricts the diameter of the pore.
Porins areβ-barrels composed of 8 -18 strands and leave open pores of up to 20 Å diam- eter. The inner wall of the pores is predominantly hydrophilic, so hydrophilic compounds up to a size of approximately 600 Da can diuse freely through the outer membrane. The porins form trimers in the outer membrane with one pore formed by each monomer. A well studied example is E. coli OmpF (see Fig. 1.2) [31] which has 16 β-strands per monomer. The wide pore is narrowed by an extracellular loop that is folded towards the channel interior and forms anα-helix. In a small region the pore is, therefore, narrowed to about 7×11 Å. This feature ensures high diusion rates without loss of the sieving func- tion: if the whole pore was restricted to this diameter, diusion would be much slower, but this small region is sucient to prevent diusion of larger molecules.
The specic channels are 18-stranded β-barrels, that also form trimers with each monomer forming one pore. But in contrast to the porins, they do not simply allow passage of all molecules up to a certain size but are permeable to specic molecules or groups of molecules. An example for specic channels is the E. coli maltoporin LamB [114], which also functions as adhesion site and entry point to phage lambda. As can be seen in Figure 1.3, three of the extracellular loops fold back into the channel to constrict its diameter. The interior is lined by several aromatic residues that accommodate maltose and maltodextrins.
(a) View perpendicular to
the membrane plane (b) View from the extracel- lular side
Figure 1.3: Crystal structure of the E. coli LamB trimer (PDB-code 1MAL).
The only family of active transporters across the outer membrane that has been found so far are the TonB dependent transporters. Active transport is necessary for nutrients that are bigger than 600 Da or are present in the medium in concentrations too low to allow ecient uptake purely by diusion. There is, however, no direct energy source present at the outer membrane: there is no ATP in the periplasm and, because of the presence of the porins, no proton gradient across the outer membrane. Therefore, TonB dependent transporters use the energy provided by the proton motive force across the inner membrane to energize transport. They will be described more closely in section 1.3.
1.2 Iron
Iron is an essential micronutrient for almost all organisms, and all organisms require approximately the same concentration of 0.3 - 10µM iron for growth [129]. It plays a vital role for synthesis of DNA, RNA and chlorophyll, in electron transport, oxygen metabolism and nitrogen xation, and also as a cofactor for many enzymes such as catalase.
The only organisms known so far that do not need iron are some Lactobacillus species [5] and Borellia burgdorferi [106]. In these organisms the role of iron as a cofactor is lled for example by manganese or cobalt.
Despite its abundance on earth, iron is scarcely available to living organisms [100]. In anaerobic environments, it is present as Fe2+, which is soluble under physiological pH, and microorganisms can acquire it easily. In aerobic conditions, however, it is oxidized
to Fe3+, which forms insoluble iron hydroxides, leaving the available iron concentration below 10−18M [34]. This concentration is much lower than the above stated minimum requirement for microorganisms and, therefore, too low for passive uptake [46].
The concentration of free iron within animal or plant host organisms is even lower. Iron is toxic to cells as it promotes the formation of reactive oxygen species, most importantly in the Fenton reaction: F e2++H2O2 → F e3++OH−+OH·. Therefore, iron is bound to iron carrier proteins or complexed in heme.
Intracellular iron is primarily bound to ferritin and myoglobin or as a cofactor of en- zymes, such as catalases. In extracellular uids, all iron is bound to transferrin or lacto- ferrin. Both proteins are present in such high concentrations that they are iron-loaded to only 30 %. This ensures that free iron is bound by the respective proteins immedi- ately. Keeping the concentration of free iron so low is also thought to be one of the host's mechanisms to ght infections [128].
Because of these limiting iron supplies, bacteria had to develop sophisticated mecha- nisms to acquire iron. Virtually all aerobic bacteria synthesize and secrete low molecular weight compounds, termed siderophores, that bind Fe3+ with very high anity and in turn are taken up by specic receptors. In case of Gram negative bacteria, the transport of the iron-loaded siderophores through the outer membrane is performed by TonB de- pendent receptors (described in section 1.3) and energized by the proton motive force of the inner membrane.
Figure 1.4: Structure of heme b
Many siderophores of host living organisms are able to extract the iron from transferrins and lactoferrins.
A second way for bacteria to capture iron from host proteins is by direct interaction of the outer membrane receptor with the iron carrier protein and subsequent uptake of the iron alone. This is the case in transferrin and lactoferrin receptors.
A third iron source present in mammalian hosts is iron in heme and bound to heme carrier proteins. Heme is an aromatic molecule composed of a porphyrin ring system with an iron atom in the middle. Porphyrins are composed of four pyrroles con- nected by methine bridges. The pyrrol subunits can be substi- tuted in dierent ways, resulting in several naturally occurring
heme types. The most common type is heme b, which is shown in Figure 1.4. Heme receptors will be described in sections 1.3.2 and 1.6.
(a) FecA (PDB code 1KMP) (b) FecA with bound ferric
citrate (PDB code 1KMO) (c) FpvA with bound fer- ric pyoverdine (PDB code 2IAH)
Figure 1.5: Crystal structures of FecA and FpvA.
The citrate and pyoverdine molecules are shown in red, the FecA switch helix in orange and the FpvA N-terminal extension in green.
1.3 TonB dependent transporters
TonB dependent transporters (TBDTs) are the only system known so far for active trans- port across the outer membrane. As there is no direct energy source available at the outer membrane, the energy from the proton motive force across the inner membrane is trans- duced to the outer membrane by the TonB-ExbB-ExbD complex (see section 1.3.3). The energy is most probably transferred by direct interaction between TonB and the receptor, as TonB can be crosslinked to FepA [121].
For a long time it was assumed, that TonB dependent receptors only transport iron and cyanocobalamin. However, recent ndings suggest that other nutrients are transported by this mechanism as well, such as nickel, cobalt, copper, and even carbohydrates [101, 113].
1.3.1 Siderophore receptors
Three-dimensional structures of several siderophore receptors have been determined (ex- amples in Fig. 1.5). These include the E. coli transporters FhuA [47, 95] for ferrichrome,
FepA [22] for enterobactin, FecA [45] for ferric citrate, BtuB [27] for Vitamin B12 and cyanocobalamin, and the Pseudomonas aeruginosa receptors FpvA [28] for pyoverdine and FptA [29] for pyochelin.
A common fold that can be deduced from these structures consists of a 22-stranded antiparallel β-barrel with short periplasmic turns and longer extracellular loops. In con- trast to the porins, the barrel is not open to allow diusion through the pore, but closed by an N-terminal globular domain termed the "plug" (also called "cork" or "hatch").
The β-barrel in the receptors of known structure is about 55 - 70 Å high and has an elliptical shape about 35 and 45 Å wide [46]. In all structures, the barrels protrude from the external surface of the outer membrane, but the length of the extracellular loops varies and seems to be related to the size of the transported substrate. Two girdles of aromatic residues can be found that determine the exact position of the barrel in the membrane.
The plug is formed by a mixed four-stranded β-sheet that is tilted by approximately 45◦ relative to the plane of the outer membrane [46]. The ligand binding site is located on the extracellular side of the plug and inside the barrel. In case of FecA the extracellular loops close the binding pocket completely and the citrate is solvent-inaccessible [45].
All TonB dependent receptors have a sequence N-terminally of the plug that is called TonB box and is thought to interact with TonB. This sequence shows only weak conserva- tion and the structure of this motif seems to be more important than the sequence itself.
In many of the ligand-free receptor structures the TonB box lies within the barrel and is not easily accessible from the periplasmic side, whereas in the ligand bound receptors it is unfolded and reaches into the periplasm. In complex with TonB, it folds into a β-strand that forms an intermolecularβ-sheet with TonB (see section 1.3.3).
Figure 1.6 shows the two siderophore receptors that have been crystallized in complex with a C-terminal TonB fragment, namely FhuA [104] and BtuB [120]. In both works, the TonB fragments are of comparable length and are in the same conformation, i.e. can be superimposed well onto each other. They are, however, not in the same position to the respective barrels: if the receptors are superimposed onto each other, the TonB fragments are in dierent positions.
Some TBDTs have an additional N-terminal domain that is required for regulation of the respective operon. Examples for this class are E. coli FecA, Pseudomonas aeruginosa FpvA and Serratia marcescens HasR. The structure of this domain is (of all structures solved so far) only visible in FpvA (see Fig. 1.5 (c)) [131].
(a) FhuA with TonB C-
terminus (PDB code 2GRX) (b) BtuB with TonB C- terminus (PDB code 2GSK)
Figure 1.6: Crystal structures of the siderophore receptors FhuA and BtuB in complex with C-terminal TonB fragments of around 80 amino acids.
The intermolecularβ-sheets between TonB (cyan) and the TonB box of the receptor (blue) can be seen. The substrates are indicated in red.
The two receptors are shown in the same orientation and it can be seen, that the TonB fragments are not in the same position with respect to the receptors.
1.3.2 Heme receptors
Heme receptors, together with transferrin and lactoferrin receptors, form a family closely related to but distinct from the siderophore receptors.
All heme receptors show a highly conserved amino acid region with the motifs FRAP and NPNL and a conserved histidine between them ([15], also see 6.1). Siderophore receptors do not have this region, while some transferrin and lactoferrin receptors have the FRAP motif and the histidine residue but not the NPNL motif [15]. The function of these motifs is unknown.
The heme receptors can be divided into two groups. In the rst group, the recep- tor interacts directly with the substrate (heme) or host proteins (hemoglobin, albumin, hemopexin, transferrin, lactoferrin). Examples for this type of receptors are the Yersinia enterocolitica HemR [124] and the Shigella dysenteriae ShuA [135].
In the second group, the bacteria secrete a protein that catches the heme and shuttles
it to the receptor. These soluble heme binding proteins have been termed hemophores in analogy to the siderophores [66, 91]. Examples for these receptors can be found in Serratia marcescens and some Yersinia and Pseudomonas species.
The Serratia marcescens hemophore dependent heme acquisition system will be de- scribed in section 1.6.
1.3.3 The TonB-ExbB-ExbD complex
E.coli TonB is a 239 residue protein consisting of three domains: a short hydrophobic N-terminal domain anchoring it in the cytoplasmic membrane, an extended periplasmic domain with an unusually high proline content, and a globular C-terminal domain. Struc- tural data are only available for the C-terminal domain which has been investigated both by crystallography and NMR. Interestingly, the oligomeric state of the C-terminal frag- ments strongly depends on their length: fragments of 93 amino acids and longer are monomeric in solution, whereas fragments of 86 amino acids and shorter form dimers [73]. Both the monomeric [74] and dimeric [25, 73] forms have been crystallized (see Fig.
1.7). The NMR structure (Fig. 1.7 (a)) of the monomeric form [116] shows a compact globular fold with a 4-strandedβ-sheet and two helices packed to one side. In the crystal structure (Fig. 1.7 (b)) strand three is longer and takes the place of the fourth strand of a second molecule. The same place is taken by a strand formed by the TonB box of FhuA and BtuB in the respective complex structures (see section 1.3.1). The crystal structures of the short fragments show an intertwined dimer (see Fig. 1.7 (c)). The physiological relevance of this domain swapped form is not clear, but both the monomeric and dimeric forms interact with FhuA [73].
ExbB [41] is a 244 aa protein with three putative transmembrane helices, a very small cytoplasmic and a larger periplasmic domain [70].
ExbD contains 141 residues and comprises a small N-terminal cytoplasmic domain, a single transmembrane helix, and a C-terminal periplasmic domain [49].
The three proteins form tight complexes in the inner membrane and all three are nec- essary for function. In E. coli, ExbB and ExbD are homologous to TolQ and TolR [40]
and in many cases can complement each other. If both ExbB and TolQ or ExbD and TolR are absent, no energy-dependent transfer across the outer membrane occurs. Many experimental results show the importance of the complex of all three proteins both for their function and their stability. TonB interacts with ExbB and ExbD in vivo and in vitro [17, 80, 81]. Both ExbB [48, 122] and ExbD [3] have to be present to stabilize TonB
(a) NMR structure of TonB (PDB code 1XX3): C- terminal fragment of 136 amino acids (only 88 could be assigned)
(b) Crystal structure of a monomeric TonB (dimer in the asu (PDB code 1UO7)):
C-terminal fragment of 92 amino acids
(c) Crystal structure of an intertwined dimer of TonB (PDB code 1IHR):
C-terminal fragment of 77 amino acids
Figure 1.7: Structures of the TonB C-terminal domain: the monomeric forms in solution and in the crystal show the same conformation. The relevance of the intertwined dimer in (c) is not clear.
and ExbB also stabilizes ExbD [122].
ExbB and ExbD form homodimers and -trimers in the membrane [64] and the ratio of TonB:ExbB:ExbD present in the E. coli membrane has been determined to be 1:7:2 [62].
The exact composition of the complex during proton translocation or TonB energization is, however, not clear.
1.3.4 Mechanisms of energy transfer and substrate transport
TonB-dependent transporters compete for a limited number of TonB complexes [65, 69]. It has been hypothesized that the occupancy of the transporter is signaled to the periplasmic side. In FhuA and FecA, for which ligand free and ligand bound structures are available, a so-called switch helix is found at the N-terminus of the plug (see Fig. 1.5 (a) and (b)).
Upon substrate binding, this helix unwinds and the TonB box reaches further into the periplasm. This is thought to be the signal for TonB to bind and transfer energy. The C-terminal part of TonB binds weakly to all proteins [71], which suggests that it scans the outer membrane for occupied receptors to which it then transfers its energy [63]. It is not known, however, how this energy transfer is accomplished.
Two hypotheses have been proposed. The so-called "shuttle model" suggests, that the hydrophobic N-terminus of TonB can ip between the inner and outer membrane. In the inner membrane it interacts with ExbB-ExbD and thereby acquires an energized confor- mation. The protein then ips to the outer membrane where the C-terminus binds to the receptor and transfers the energy [79, 107]. This model is based mainly on membrane
fractionation experiments in which TonB was found in both membranes [85].
According to the "pulling model" the N-terminal helix of TonB remains in the cytoplas- mic membrane and the protein stretches to span the whole periplasm. The C-terminus binds to the TonB box of the receptor and exerts a pulling force on the plug domain causing the release of the substrate into the periplasm [26]. The latter model is strongly supported by the observation, that a TonB construct unable to leave the cytoplasmic mem- brane is fully active [71]. A special form of the pulling model is the so-called "propeller model" [25], which has been put forward mainly to explain the observation of dimeric TonB-fragments in crystals (see section 1.3.3). Here, ExbB and ExbD rotate two TonB molecules around each other which leads to a twisting of the two periplasmic domains and a concomitant shortening resulting in a pulling force on the C-terminus, which is bound to the receptor plug. However, a sucient shortening of the periplasmic domain can also be achieved with a monomeric TonB under the assumption that the proline rich region of the periplasmic domain changes its conformation from a polyproline I to a polyproline II helix in response to the rotation [38]. The idea of ExbB and ExbD rotating the TonB molecule is attractive since ExbB and ExbD have homology to MotA and MotB, respectively, the motor proteins of the E. coli agellum.
The exact mode of substrate transfer through the receptor is also not understood. A number of hypotheses have been formulated that could explain this transfer. The plug could leave the barrel completely keeping its conformation [96] or undergo conformational changes within the barrel [26] or it could be pulled slowly into the periplasm and unfold during this pulling. This last hypothesis is supported by experiments [20, 24] and molecu- lar dynamic simulations [55] showing that the force necessary for slow unfolding is orders of magnitude below that of unplugging the receptor in one step. The same calculations also show, that the interaction between TonB and the TonB box as well as the TonB C-terminus itself are stable enough to withstand those pulling forces.
1.4 Regulation
1.4.1 Fur
Fur is an important global regulator in many Gram negative and also some Gram positive bacteria [44, 98]. It has rst been found in E. coli mutants in which all iron uptake genes are expressed constitutively. These mutants were named fur (for f erric uptake
of the presence or absence of iron [33]. In the presence of intracellular iron, Fe -Fur binds to DNA sequences in the upstream region of iron regulated genes and blocks transcrip- tion. The fur consensus sequence GATAATGATAATCATTATC [130] was traditionally interpreted as a palindrome of two 9 bp inverted repeats and one unmatched base in the middle similar to classical bacterial repressors [108]. However, the same sequence can also be interpreted as three 6 bp repeats of the sequence NATA/TAT [44] (see Fig. 1.8 (a)). The latter interpretation also gives a possible explanation to the observation that dierent genes which are under the control of the fur repressor need dierent levels of iron depletion for repression: Three repeats seems to be the minimum, but there can be more and their relative orientation seems to be of less importance [43]. Thus, the number of Fur dimers which can bind to a given fur box would depend on the number of repeats of this 6 bp motif and determine the degree to which the operon is repressed.
The crystal structure of Pseudomonas aeruginosa Fur [105] shows a dimer with an N- terminal DNA-binding domain and a C-terminal dimerization domain. Also, two metal binding sites (occupied by zinc in the crystal) can be found (Fig. 1.8 (b)).
(a) Two interpretations of the fur box as a palindrome (top) or a repetition of a 6 bp motif (bottom) (from [44]).
(b) The crystal structure of Pseu- domonas aeruginosa Fur (PDB code 1MZB) shows a dimer with two DNA- binding domains (blue), two dimeriza- tion domains (green) and two possible iron binding sites (red spheres indicate Zn2+atoms).
Figure 1.8: The fur box and the Fur structure
In the absence of intracellular iron, the Fur dimer looses its ability to bind to the operon and transcription of the Fur regulated genes can occur. Most probably, a conformational change of the protein, dependent on whether or not iron is bound to it, is responsible for its DNA binding. However, as no structure is available of Fur without a bound metal, this conformational change is not known in detail.
Fur is a repressor for all TonB dependent receptors, TonB, ExbB-ExbD and Fur itself.
In addition, it plays a role in virulence, colonization and biolm formation [23].
1.4.2 Sigma factors
In addition to the negative regulation by the iron bound Fur repressor, some of the iron and heme uptake systems are positively regulated by so-called extracytoplasmic function sigma factors (ECFσ). These ECFσfactors are usually controlled by a membrane bound anti-sigma factor [60]. The presence of the substrate is signaled from the N-terminal extension of the receptor to the anti-sigma factor in the inner membrane, which in turn releases the ECFσ factor into the cytoplasm to start transcription of the operon [16].
These two regulatory systems together allow ne-tuning of expression in response to available iron sources: if the intracellular iron concentration is low, Fur is released from the DNA and the sigma / anti-sigma pairs are expressed. If then an iron source is encountered, the respective sigma factor is released and directs the RNA polymerase to the corresponding operon [18]. This signaling cascade requires energy provided by the TonB-ExbB-ExbD complex (see 1.3.3) [72].
1.5 Heme acquisition in Serratia marcescens
Serratia marcescens is an enterobacterium that colonizes various biotopes such as water, soil, plants, insect gut, rodents, and humans, and it is also known to cause diseases [54]. In the last decades, it raised increasing problems as an opportunistic pathogen especially in hospitals [56], and many strains are resistant to multiple antibiotics [61]. Main virulence factors are the biosynthesis of LPS, hemolysin and the iron uptake systems [84].
S. marcescens synthesizes several siderophores and siderophore receptors in response to the respective environment. In vertebrate hosts, it uses heme and heme bound to hemoglobin and hemopexin as an iron source.
S. marcescens possesses two heme uptake systems [8, 9]. The Hem system is closely related to the Yersinia enterocolitica Hem system and consists of the receptor HemR [123],
The second system, Has, consists of the receptor HasR, which alone can take up free heme or heme bound to hemoglobin, and a hemophore HasA, which strongly increases the eciency of heme uptake.
The two systems complement each other: the Hem system requires only moderate iron depletion for induction and can take up heme at concentrations of 10−6M or higher, whereas the Has system is activated at much stronger iron depletion and can transport heme at concentrations of 10−7M [8]. HemR recognizes heme and hemoglobin, whereas HasR, together with HasA, can also take up heme from hemopexin [8].
1.6 The heme acquisition system Has
Figure 1.9 shows a schematic representation of the Has system: the hemophore HasA is secreted to the extracellular medium by a specic ABC exporter of the Type I Secretion System: the ABC protein HasD [92], the inner membrane MFP protein (membrane fusion protein family) HasE [92] and the outer membrane protein HasF [13] of the TolC family.
In the medium, HasA binds free or hemoglobin bound heme and shuttles it back to the receptor HasR. Upon binding heme is spontaneously transferred from holoHasA to apoHasR as has been shown by UV-Vis absorption spectra (see Fig. 1.12 and section 1.6.3) and resonance Raman spectroscopy [67].
Heme-loaded (holo) and heme-free (apo) HasA both bind to HasR [93] in a 1:1 stoi- chiometry [67] and with similar anities (apparent Kd 5×10−9M) and independently of TonB [90].
Both binding of HasA and transfer of heme from the hemophore to the receptor do not require energy [67, 93], whereas transport across the outer membrane as well as dis- sociation of HasA from HasR require energy provided by the HasB-ExbB-ExbD complex [88].
Heme is then transported across the inner membrane by the binding protein dependent ABC transporter of the hem system, HemTUV.
(a) HasA is secreted to the extracellular medium by a Type I Secretion System where it binds heme (either free or captured from hemoglobin) and returns it to the outer membrane receptor HasR.
(b) Genetic organization of the has operon.
Figure 1.9: The heme acquisition system of Serratia marcescens (from [126], more details in the text)
.
a low spin ferric state [66]. Iron in the heme-hemophore complex is in the oxidized form with a very low redox potential [66], reecting its high solvent accessibility.
HasA binds free heme with a very high anity (5.3×1010M−1 [37]) and also heme bound to hemoglobin, hemopexin, and hemoglobin-haptoglobin [8]. Heme is transferred from hemoglobin to HasA probably by passive transfer due to the higher anity rather than by a protein-protein interaction [91, 93]. The Ka of HasA for heme is among the highest reported for heme binding proteins. Myoglobin has a higher anity (Ka around 1014M−1 [59]) and HasA cannot extract heme from myoglobin [51]. Moreover, no stable complex can be found between HasA and hemoglobin by analytical ultracentrifugation [93].
As is typical for proteins secreted by the Type I Secretion System, HasA has no N- terminal signal peptide but a C-terminal targeting sequence that shows similarities to other proteins secreted by ABC exporters. These targeting sequences show weak sim- ilarities in the C-terminal 50 residues [92], and the extreme C-terminus, consisting of a negatively charged residue followed by several hydrophobic amino acids (ELLAA in HasA), has to be exposed [132]. The secreted protein recognizes the ABC protein, which then interacts with the MFP and then with the outer membrane protein [87]. In E. coli, HasF can be replaced by TolC (the hemolysin exporter) or PrtF (the Erwinia chrysan- themi protease exporter)[12, 92].
The fact that the targeting sequence is located at the C-terminus implies that HasA has to be fully synthesized before secretion. In the cytoplasm, it is kept in an unfolded (or partly folded) state by SecB [36]. Folded HasA in the cytoplasm inhibits secretion of newly synthesized HasA molecules [36].
During or after secretion, in S. marcescens, the HasA protein is cleaved by the S.
marcescens metalloprotease PrtSM: the active protein in S. marcescens is only 17 kDa as compared to 19 kDa in E. coli [92].
The crystal structure of holoHasA (see Fig. 1.10 (a)) [6] shows an original α/β-fold with a 5-stranded β-sheet on one side and four-α helices on the other side. The heme is bound by residues H32 and Y75 on two loops, A1 and A2 respectively, connecting the two parts and highly exposed to the solvent. Y75 is stabilized by a hydrogen bond
Figure 1.10: Structure of HasA (adapted from [134]):
(a) crystal structure of holoHasA, (b) NMR structure of apoHasA, (c) secondary structure elements of HasA.
H32 lies on loop A1, Y75 and H83 on loop A2.
from H83 [133]. H83 can serve as an alternative iron ligand in the absence of Y75 or of both, Y75 and H32 [37]. Heme binding for all three single mutants, all possible double mutants and the triple mutant has been determined [37]: the anity of H32A is reduced 5-fold as compared to the wild type, whereas that of Y75A as well as H83A is reduced 400-fold. The double mutants bind heme with strongly reduced anities (103-fold for H32A-H83A, 104-fold for Y75A-H83A and 106-fold for H32A-Y75A). The triple mutant shows no detectable heme binding. Together, these results suggest that Y75 is a stronger ligand than H32, which ts to the observation that in the crystal structure the distance between the H32Nand the iron is longer (2.3 Å) than that between the Y75Oη and the iron (2 Å).
An NMR structure of apoHasA in solution is available (Fig. 1.10 (b)) [134], which shows no major changes in the secondary structure elements compared to holoHasA.
Figure 1.11: Domain organization of HasR: signal sequence gray, N-terminal extension green, TonB box pink, plug orange andβ-barrel blue
Only loop A1 is moved by about 30 Å as compared to the holoHasA structure to widely open the heme binding site.
1.6.2 HasR
The receptor HasR has weak sequence homology to the siderophore receptors, for example it shares 18 % identity with BtuB, 17 % with FpvA and 16 % with FecA.
HasR is a 92 kDa protein and was predicted to have the same overall fold as the known siderophore receptors, i.e. a C-terminalβ-barrel closed by a plug. In addition, HasR, like FecA and FpvA has an N-terminal extension necessary for signaling. This signal domain interacts with the anti-sigma factor HasS in the inner membrane. HasR expressed without the N-terminal extension is fully functional for hemophore binding and free and hemophore bound heme uptake but does not induce transcription of the has operon [14]. The domain organization of HasR is shown in Figure 1.11.
Alignments of the heme receptors reveal that the hemophore dependent heme receptors have longer extracellular loops compared to the other heme receptors and the siderophore receptors. Those longer loops could be better suited to bind the proteins than the shorter loops which apparently are sucient for binding siderophores.
The barrel alone is inserted into the outer membrane and functions as a heme specic channel [94]: heme is eciently transported across the outer membrane, but this heme transport is not TonB dependent: the conductivity of the outer membrane is not increased in the presence of the HasR barrel, and the cells are not more sensitive to detergents or large antibiotics. These ndings indicate that the heme is indeed taken up by diusion.
HasR binds heme with an anity constant ofKa = 5×106M−1 and apo and holoHasA with Ka >109M−1 and apparent Ka= 5×107M−1, respectively [67].
The two histidines conserved in the heme receptors are present in HasR at amino acid positions 189 in the plug (H1) and 603 in loop L7 of the barrel (H2). Both single
mutants are still able to take up heme, but higher heme concentrations are needed for growth, whereas the double mutant shows no heme uptake [67]. All three mutants are able to bind HasA (with lower anities), but the heme is not transferred [67]. The single mutants are unable to acquire heme from wild type holoHasA, but can still take up heme from HasA mutants with reduced heme anities (see Table 1.1).
1.6.3 Interaction between HasA and HasR
The function of the hemophore is to increase the eciency of heme uptake especially at very low heme concentrations. HasR alone can take up heme at concentrations of 10−5M but together with HasA at 10−7M [8]. The eect of HasA can also be seen when hemoglobin is used as a heme source: HasR alone allows growth at hemoglobin concentrations of10−4M or higher, whereas concomitant expression of HasA reduces the required hemoglobin concentration to 10−6M [51]. Heme uptake from the hemophore, however, requires more energy and more TonB complex than free heme uptake [88].
The anity between the two proteins is very strong and in vitro the two proteins after binding cannot be separated under non denaturing conditions. Two of the β-strands of HasA, S3 and S5, form two independent binding sites (also see section 6.1). Mutations in one of them only reduce the anity slightly but mutations in both abolish binding to HasR [86].
The heme is spontaneously transferred from the high anity binding site on HasA to the lower anity binding site on HasR in vitro, indicating that this heme transfer does not require energy [67]. The characteristics of heme in UV-Vis absorption spectra are very sensitive to the environment of the heme and can, therefore, serve as an indicator for its binding partners. In case of HasA and HasR, the spectra look quite dierent. Figure 1.12 shows typical spectra of holoHasA and holoHasR: the very prominent peak in the blue region is at 406 nm in holoHasA but at 411 nm in holoHasR. Also, the small peaks in the 500-650 nm region, which are indicative of the spin state of the iron, are quite dierent:
in holoHasA, four peaks can be seen at 492, 537, 567 and 620 nm, in holoHasR the bands at 492 and 620 nm almost disappear, whereas the other two are increased and shift to 533 and 560 nm.
The third spectrum in Figure 1.12 is one of a complex resulting from mixing holoHasA with apoHasR. It exactly matches the one from holoHasR, indicating that the heme is spontaneously transferred during complex formation.
Table 1.1 summarizes the heme anities of the HasA mutants and also the ability of
Figure 1.12: Typical spectra of holoHasA, holoHasR and a complex of holoHasA and apoHasR (from [67]). The spectra indicate that heme is transferred from holoHasA to apoHasR upon complex formation.
E. coli strains producing HasR and HasR mutants to grow with the HasA mutants as the only heme source.
HasA Heme anity HasRWT HasRH2 HasRH1 HasRH1H2
WT 5.3×1010M−1 + - - -
H32A 1010M−1 + - - -
H83A 2×108M−1 + + - -
Y75A 108M−1 + + - -
H32H83AA 2.3×107M−1 + + - -
Y75H83AA 1.8×106M−1 + + + -
H32Y75AA 6×104M−1 + + + -
Table 1.1: Heme anities of the HasA mutants and growth of E. coli producing HasR and HasR mutants with the HasA mutants as the only heme source (taken from [67]).
1.6.4 HasI-HasS
The genes for the sigma and anti-sigma factors hasI and hasS lie in the has operon up- stream from hasR. A fur box is present upstream from the hasI start codon, and HasI and HasS are expressed under iron limitation [112]. Expression of HasI is not autoregu- lated, but HasI boxes have been identied upstream from hasS and hasR [14]. The ECFσ factor HasI is a 19 kDa protein that has 30 % identity to E. coli FecI and 26 % identity to Pseudomonas putida PupI. The anti-sigma factor HasS has a molecular weight of 35 kDa and shares 24 % and 27 % identity with FecR and PupR, respectively.
The signal for sigma factor release from the membrane is the presence of both HasA and heme bound to the receptor. The region on HasA necessary for signaling is in β-strand S3, one of the two β-strands involved in binding to HasR [35].
1.6.5 HasB
Serratia marcescens, in addition to TonB, has a second TonB like protein, HasB, encoded in the operon [103].
HasB is specic to HasR and not to heme TBDTs [9], i.e. it does not energize S.
marcescens HemR.
HasB is not functional in E. coli, however, a two amino acid addition downstream from residue 24 in the HasB transmembrane anchor renders it functional in E. coli. The corresponding region in E. coli TonB interacts with ExbB [103]. Also, HasB is functional in E. coli if the S. marcescens ExbB and ExbD are expressed as well (P. Delepelaire, personal communication).
Structure determination of HasB by NMR is currently being undertaken [83]. A com- parison of secondary structure elements of HasB and TonB shows dierences especially in the N- and C-terminal regions (see Fig. 1.13).
Figure 1.13: Alignment of the C-terminal domains of S. marcescens HasB and E. coli TonB (from [83]). The secondary structure elements of both proteins are indicated: arrows depict β-strands, cylinders α-helices. Asterisks indicate conserved residues.
2.1 Motivation
The rst and main focus of this work was on determining the structure of the hemophore- receptor complex. Not only has there never before been a structure available of a heme receptor, but also, there was no structure available of a TonB dependent receptor in complex with an extracellular protein.
As described in section 1.6.3, the heme is spontaneously transferred from HasA to HasR during binding. This process does not require energy even though the anity of HasA for heme is 10 000 times higher than that of HasR for heme.
We expected the structure of the complex to give us ideas as to how this transfer is accomplished.
Also, as the alignments of receptors revealed that the extracellular loops of heme recep- tors, and especially of the hemophore dependent heme receptors, are signicantly longer than those of the siderophore receptors, we assumed they might be exible when not in contact with the respective binding partner and therefore easier to crystallize in the complex than alone.
2.2 Methods
2.2.1 Protein expression
HasA
HasA was expressed with an N-terminal HisTag in plasmid pQE32 in E. col PAP105 cells [94]. PAP105 pHis6HasA cells were grown in liquid cultures in LB medium supplemented with 100µg/ml ampicillin at 37◦C and 250 rpm. At an OD600nm of 0.5 protein expression was induced by addition of 100µM IPTG. After 3 h of induction cells were pelleted at 10 000 g for 5 min and resuspended in 50 mM Tris pH 7.5, 300 mM NaCl, 10 mM imidazole.
Cells from E. coli popc4420(pFR2) (an E. coli MC4100 derivative devoid of the major outer membrane proteins OmpF, OmpC, LamB) and carrying the pFR2 plasmid con- taining the hasr gene under the control of the paraBAD promoter, were grown in 300 liter fermenters in M9 minimal medium supplemented with 0.2 % casamino acids, 0.4 % glycerol, 60µM iron citrate at 30◦C. At OD600nm of 0.5, arabinose was added to a nal concentration of 40 mg/liter to induce expression of the HasR receptor for 3 hours. The whole culture was quickly chilled to 10◦C and centrifuged. The cell pellet was quick-frozen in liquid nitrogen and kept at -80◦C until use. Alternatively the same medium with a 5 times higher concentration of concentrated carbon and nitrogen sources was used to increase the cell yield per liter and reduce culture size. In that case 15 - 20 g of wet cell pellet / liter were routinely obtained. All cultures were grown at the Recombinant Proteins and Antibodies Platform of the Institut Pasteur. An example for the expression is shown in Figure 2.2 (a).
The cells were broken by French Press, membranes were resuspended in water, quick- frozen in liquid nitrogen and sent to Konstanz on dry ice where they were kept at -80◦C until use. Up to this step all work was done by Philippe Delepelaire at the Institut Pasteur, Paris who sent the cells to Konstanz on dry ice. All further steps were done by the author. The same sharing of work was maintained with all HasR mutants.
2.2.2 Protein purication
HasA
In the cells described above HasA was expressed in the cytoplasm. Thus, frozen cells were thawed and disrupted by French Press at 16 000 psi. After centrifugation for 1 h at 75 000 g, the supernatant containing HasA was ltered through a 0.2µm lter (Peske) and the protein was puried by liquid chromatography.
In the culture conditions described above, between 5 and 20 % of the HasA is already loaded with heme during expression. Therefore, to obtain holoHasA extra hemin had to be added during purication. To obtain apoHasA, the existing mixture of holo and apoHasA had to be separated.
(a) Coomassie-stained SDS-PAGE: before and after induction and after Ni-column ("puried")
(b) Example for the elution prole of the
Q-Sepharose-column (c) Spectrum of frac-
tions 5-9 from the exam- ple in (b)
Figure 2.1: An example for the process of HasA expression and the purication of apoHasA
For purication of holoHasA the cell extract was loaded on a 1 ml HiTrapFF column (GE Healthcare) loaded with Ni2+ in 50 mM Tris pH 7.5, 300 mM NaCl, 10 mM imidazole and after washing with the same buer, HasA was eluted in one step with 200 mM imidazole.
The purity as judged from SDS-PAGE was over 99 % (see Fig. 2.1 (a)). The protein was then dialysed or diluted to reduce the imidazole concentration to approximately 20 mM.
To load the HasA with heme, a solution of hemin dissolved in NaOH and diluted in Tris buer without NaCl was mixed with HasA in a molar ratio of at least 2. HasA immediately binds its ligand due to the high anity. Excess heme was removed either by geltration using a Bio-Gel P-60 column (Biorad) or by passing the HasA-heme mixture over a small desalting column (NAP5 column, GE Healthcare).
For purication of apoHasA the cell extract was loaded on a 1 ml Q-SepharoseFF column (GE Healthcare) equilibrated with 50 mM Tris pH 7.5. After washing in the same buer, the protein was eluted with a linear gradient from 0 to 1 M NaCl over 30 column volumes.
ApoHasA elutes at a slightly lower salt concentration than holoHasA (see Fig. 2.1 (b)).
The separation is not complete but the heme saturation in the rst fractions is below 5 % (see Fig. 2.1 (c)): to estimate the fraction of HasA loaded with heme, the calculated extinction coecients 280 = 21 000 M−1 (for the protein) and 406 = 100 000 M−1 (for the HasA-bound heme) were used. Together with Lambert-Beer's Law Aλ =λ·c[protein]·d this allows an estimation of the fraction of loaded versus unloaded HasA.
The apoHasA containing fractions were further puried by anity chromatography as described for holoHasA.
(a) Expression of HasR:
red circle: HasR after 3 h of induction.
(b) Solubilization of HasR: part of the HasR cannot be solubilized (i.e. is still in the pellet).
(c) Outcome of the purication of HasA-HasR: after anity chro- matography and gelltration the purity is around 95 %.
Figure 2.2: Example for the expression of HasR and the purication of the HasA-HasR complex
The protein is very stable, both in its holo and apo form: it can be kept for weeks at 4◦C or frozen in liquid nitrogen and kept at -80◦C without any detectable degradation.
HasR
All HasR constructs were solubilized with n-tetradecyl-N,N-dimethyl-3-ammonio-propane- sulfonate (ZW3-14) using a two step protocol (see Fig. 2.2 (b)). The suspended mem- branes were treated with 0.25 % ZW3-14 in a Tris buer at pH 8.5 and in presence of protease inhibitor cocktail (complete EDTA-free, Roche) and rotated for 30-60 min on ice. After centrifugation at 100 000 g for 1.5 h the supernatant contained mainly inner membrane proteins and was discarded. The pellet was resuspended in Tris pH 8.5 with protease inhibitor and ZW3-14 was added to a concentration of 4 %. After another 2 h of incubation, the remainig membrane fragments and insoluble proteins were pelleted at 100 000 g for 1.5 h and the supernatant ltered through a 0.45µm lter.
HasA-HasR complex
For the preparation of heme containing complexes this mixture of solubilized proteins was mixed with the puried holoHasA and kept at 4◦C for several hours. The complex forms spontaneously in solution and was puried by anity chromatography using the HisTag on HasA. The protein was loaded on a 5 ml HiTrapFF column (GE Healthcare) loaded with Ni2+ in 50 mM Tris pH 7.5, 150 mM NaCl, 0.02 % ZW3-14, washed in the same buer and eluted with 200 mM imidazole. The eluted protein was concentrated by ultraltration (Vivaspin6 or Vivaspin20 with molecular weight cuto of 100 kDa) to approximately 1 ml and further puried by size exclusion chromatography (100 ml Sephacryl-S300) in 50 mM Tris pH 7.5, 0.02 % ZW3-14. The elution proles of the gel ltration showed no dierence whether 0, 50 or 150 mM NaCl was used. The purity of the complex after gelltration as judged by SDS-PAGE was around 95 % (see Fig. 2.2 (c)). After gelltration the detergent was exchanged by binding the protein to a 1 ml Q-Sepharose FF column (GE Healthcare) and washing with at least 100 ml of a buer containing the desired detergent.
The protein was eluted in the same buer but complemented with 400 mM NaCl and then concentrated and repeatedly diluted to reduce the salt concentration to below 20 mM. The pure protein complex could be concentrated to at least 50 mg/ml without precipitation.
To form apo complexes, HasR was rst puried by anion exchange chromatography (5 ml Q-Sepharose column, GE Healthcare) using a linear gradient over 30 column volumes from 0 to 1 M NaCl in 50 mM Tris pH 7.5, 0.02 % ZW3-14 with protease inhibitors and then mixed with puried apoHasA to form the HasA-HasR complex which was puried as described for the holo complex.
2.2.3 Crystallization
Initial crystallization screenings were performed with Nextal kits (Quiagen) in sitting drops using a pipetting robot to mix 100 nl of reservoir solution with 100, 200 and 400 nl of protein solution and equilibrating against 100µl of reservoir. Fine screenings were performed in hanging drops mixing 2µl of protein solution with equal volumes of reservoir solutions and equilibrating against 1 ml of reservoir.
The proteins were used in concentrations between 20 and 26 mg/ml in 50 mM Tris pH 7.5 and detergent at 2-3× the cmc. Detergents tested were octyltetraoxyethylene (C8E4), octylpentaoxyethylene (C8E5), octylpolyoxyethylene (C8POE), octyltrioxyethylene (C8E3), decyltetraoxyethylene (C10E4), decylpentaoxyethylene (C10E5), propyl-dicyclohexyl-α- mal-
(a) Crystals of the holo complex (b) Crystals of the apo complex Figure 2.3: Example pictures of crystals of the HasA-HasR-complexes
toside (PCCαM), Lauryl-dimethylamine-oxide (LDAO) and n-octyl-β-D-glucopyranoside (βOG). The best crystallization results with respect to yield and size were obtained with 0.6 %C8E4.
Crystals were obtained in several conditions but only one gave nice cubic single crystals [76]. The best diracting crystals were grown with NaCl as precipitant (in concentrations of 1.8 to 2.5 mM) at pH 7.5 to 8.5. Some constructs needed addition of 100 mM K2HPO4. Crystals grew within 10-14 days at 18◦C to an average size of about 0.2×0.2×0.05 mm.
Crystals were soaked in articial mother liquor containing 25 % glycerol for a few seconds prior to freezing in liquid nitrogen.
Although the crystals seemed large, the diraction was too weak to give any reections using a rotating Cu anode, probably due to the high solvent content and the rather large unit cell. Therefore, all data were collected at the synchrotrons at the Paul Scherrer Institut (PSI) in Villigen and the ESRF in Grenoble.
2.2.4 Structure solution
holoHasA-HasR
The rst structure to be determined was that of the HasA-HasR-heme complex. Although it was expected (and later conrmed by the structure) that the 3D structure of HasR was very similar to those of the siderophore receptors, the sequence similarity was too low for molecular replacement [76]. Therefore, selenomethionine labeled HasR was produced using an E. coli strain in which methionine synthesis was repressed. This labeled HasR
Figure 2.4: Fluorescence scan of the HasA-HasR-heme crystal used for phasing.
The energies suggested by the scan and indicated below the graph were used for data collection.
behaved exactly as the wild type in purication, complex formation and crystallization.
A uorescence scan (see Fig. 2.4) of a crystal conrmed the presence of selenomethio- nine and allowed determination of peak and inection wavelengths.
A complete MAD dataset was collected at the SLS beamline X06SA to a resolution of 3 Å. Table 2.1 shows the data statistics for all three wavelengths. The diraction statistics were good to around 3.1 Å resolution (see Tab. 2.1), whereas the anomalous signal was usable to 5 Å only (see Fig. 2.5).
Data processing
Data were processed with XDS [68] to a resolution of 3.1 Å in space group F222 with unit cell axes a = 157, b = 163 and c = 595. Due to a beamline problem, the data collection of the rst dataset (remote high) stopped after 135◦. After the beamline was in operation again, a second dataset was started to collect the missing 55◦. However, during data processing it became clear, that this second part scaled badly with the rst, indicating that the beam was not yet stable. Therefore, this second part was not used for phasing.
Redundancy 2.8 (2.1) 3.6 (2.8) 3.7 (2.9) 13.8 (9.4) 6.5 (4.9) Rmeas [%] 11.0 (32.8) 15.5 (40.7) 16.1 (46.0) 22.5 (102.1) 16.6 (98.1) Rmrgd-F [%] 13.3 (38.8) 14.9 (39.9) 15.8 (45.4) 14.7 (59.1) 16.7 (77.7)
Table 2.1: Data statistics for the three wavelength MAD dataset of the wild type HasA-HasR- heme complex and the native datasets for the wild type HasA-HasR-heme and HasA-HasR complexes
This explains the lower completeness and redundancy of the remote high data compared to the other two. The other statistics however are good, which can be explained by the high symmetry space group. The R-Factors and the signal-to-noise ratio are even best for the remote high dataset. This could be explained by the fact, that this dataset was collected rst. However, the crystal was big enough so it could be moved to a new spot for each of the other two datasets, which should have reduced this eect. The data were scaled with XSCALE with the remote high data set as reference.
Phasing
The programs SHELXC/D/E [115, 117119] were used for substructure determination using the GUI hkl2map [102].
The statistics from SHELXC suggested, that the anomalous signal was usable to 4.5-5 Å (see Fig. 2.5 (a)).
The easiest way to determine the optimal resolution cuto for the substructure deter- mination, is to simply run SHELXD with dierent input values and compare the results (see Fig. 2.5 (b)). In this case, it was run with inputs from 4 to 6 Å in 0.1 Å steps and the correlation coecient (CC) between observed and calculated structure factors of the best solution was noted for each input. The CC was highest for 5 Å.
A phasing attempt was undertaken with SHELXE and as seen in Fig. 2.5 (c) the correct hand can be determined. However, the density from SHELXE was very dicult to interpret and therefore initial phases were calculated with SHARP [19]:
The rst 16 of the heavy atom sites from SHELXE were used as input and SHARP was run with renement of sites and their occupancies, renement of f0 and f00 and solvent attening assuming 70 % solvens content. The resulting map was much more detailed
(a) SHELXC
(b) SHELXD
(c) SHELXE
Figure 2.5: Results from SHELXC/D/E
than that from SHELXE.
After one round of density modication using PIRATE [32], several programs were tried for automated model building. Of all programs tested only RESOLVE [125] gave usable results. It built approximately 20 % of the cα trace of the barrel and large parts of HasA. Also, and maybe even more importantly, it gave a map that was considerably more clear allowing manual model building in COOT [42] to complete the model of HasR.
Also, HasA could be placed in this map using PHASER [97].
Renement was done by REFMAC [99], CNS [21] and PHENIX [1, 2].
The nal R-Factors reached with these Se-Met-data were Rwork = 27% and Rfree = 30 % at 3.1 Å resolution.
Later, a native dataset was collected at the ESRF beamline ID23-1 to a resolution of 2.7 Å and used for nal renement by PHENIX. The renement statistics are shown in Table 2.2.
model composition
protein residues 1832 1832
heme atoms 86
water molecules 57 19
B-factors
HasR 95.7 80.7
HasA 112.1 95.4
heme 90.7
deviation from ideal values
bond lengths [Å] 0.01 0.01
bond angles [◦] 0.61 1.13
Ramachandran plot
favored regions [%] 92.4 89.6
allowed regions [%] 99.2 99.5
Table 2.2: Renement statistics for the wild type HasA-HasR complexes
apoHasA-HasR
Proteins for the apoHasA-HasR complex were obtained as described in section 2.2.2. The complex was crystallized in the same condition (50 mM Tris pH 8.0, 2 M NaCl) as the holo complex and gave similar looking crystals (except for the color, see Fig. 2.3 (b)). One crystal diracted to 3 Å resolution (see Table 2.1) and could be processed in the same space group (F222 with a = 158, b = 165, c = 597). After processing with XDS, the test set for the Rfree reections was transferred using the program CAD from the CCP4 suite.
The data were rened with PHENIX against a model from the holo complex containing only the protein chains and the model was adjusted manually in COOT. The renement statistics of the resulting model are shown in Table 2.2.
