KOJETIN, DOUGLAS J. Structural Characterization of Two-Component Signal
Trans-duction Proteins and Calbindin D28k . (Under the direction of Professor John Cavanagh.)
The studies described involve the structural analysis of proteins involved in signal
trans-duction pathways. In the first study, the metal binding properties of the initiation of
sporu-lation response regulator Spo0F was studied using a variety of biophysical and biochemical
techniques. The experiments show that most of the divalent metals studied, including and
Ca2+, Mg2+ and Mn2+, which display primarily 1:1 binding, allow for favorable conditions for phosphotransfer between Spo0F and its cognate kinase KinA. In contrast, Spo0F binds
up to three Cu2+ ions and the presence of this metal does not allow for the phosphotrans-fer reaction to occur. In the second study, a comparative modeling study of the OmpR
sub-family of response regulators from B. subtilis and E. coli was performed and used to
suggest the possibility of sub-classes within this related domain family based on regions of
the response regulator regulatory domain that is known to interact with cognate four-helix
bundle HisKA/Hpt domains. In the third study, the structural refinement of the
four-helix bundle LuxU phosphotransferase from V. harveyi is described using a combination
of dipolar couplings and water-based explicit refinement. In the fourth and last study, the
development of a solution structure of Ca2+-loaded calbindin D28k, an EF-hand calcium
binding protein, and the interaction between peptides derived from ran-binding protein M
Proteins and Calbindin D28k
by
Douglas John Kojetin
a dissertation submitted to the graduate faculty of
north carolina state university
in partial fulfillment of the
requirements for the degree of
doctor of philosophy
biochemistry
raleigh
2005
approved by:
Dr. John Cavanagh
chair of advisory committee
Dr. Dennis T. Brown Dr. William L. Miller
Douglas John Kojetin was born in Indianpolis, Indiana to John and Marsha Kojetin
on February 7th, 1977. His interest in science likely began in part thanks to his mother,
Marsha, who was a nurse at a local hospital in Indianapolis. Inspired from his high school
chemistry teacher, who always recalled his undergraduate studies as a chemistry major with
fond recollections, he pursued undergraduate studies in chemistry and obtained a B.S. in
chemistry with an emphasis in biological chemistry from Purdue University. Douglas joined
Dr. John Cavanagh’s research group at Purdue during his last year of undergraduate study.
This experience, combined with a summer internship at the Indiana University Medical
Center, further inspired Douglas to pursue graduate studies. Douglas began his graduate
studies at North Carolina State University in the laboratory of Dr. John Cavanagh, where he
was exposed to many disparate projects and his passion for scientific research was realized.
• American Foundation for Aging Research Fellowship. 2002-2005.
• A.R. Main-Becton Dickinson Departmental Award for Outstanding Graduate Achievement. North Carolina State University, Department of Molecular and Structural Biochemistry, Raleigh, NC, 2003.
Publications
1. Kojetin, D.J., Thompson, R.J., Benson, L.M., Naylor, S., Waterman, J., Davies, K.G., Opperman, C.H., Stephenson, K., Hoch, J.A. and Cavanagh, J. Structural analysis of divalent metals binding to theBacillus subtilis response regulator Spo0F: the possibility for in vitro metalloregulation in the initiaion of sporulation. Accepted to Biometals.
2. Venters, R.A, Coggins, B.E., Kojetin, D., Cavanagh, J. and Zhou, P. (4,2)D Pro-jectionreconstruction experiments for protein backbone assignment: application to human carbonic anhydrase II and calbindin D28K. J. Am. Chem. Soc., 127(24):
8785-95, 2005.
3. Ulrich, D.*, Kojetin, D.J.*, Bassler, B.L., Cavanagh, J. and Loria, J.P. Solution structure and dynamics of LuxU from Vibrio harveyi, a phosphotransferase protein involved in bacterial quorum sensing. J. Mol. Biol., 347(2): 297-307, 2005.
4. Kojetin, D.J., Thompson, R.T. and Cavanagh, J. Sub-classification of response regu-lators using the surface characteristics of their receiver domains. FEBS Lett., 554(3): 231-6, 2003.
5. Lutz, W., Frank, E.M., Craig, T.A., Thompson, R., Venters, R.A., Kojetin, D.J., Cavanagh, J. and Kumar, R. Calbindin D28KInteracts With Ran-Binding Protein M:
identification of interacting domains by NMR spectroscopy. Biochem. Biophys. Res. Commun., 303(4): 1186-92, 2003.
6. Helton, T.D., Kojetin, D.J., Cavanagh, J., Horne, W.A. Alternative splicing of a
β4 subunit proline-rich motif regulates voltage-dependent gating and toxin block of Cav2.1 Ca2+ channels. J. Neurosci., 22(21): 9331-9, 2002.
Dr. John Cavanagh. You have created a dynamic working environment that makes research both fun and interesting – it makes the job almost feel like a hobby. You have been a great mentor and have made available a wide variety of projects and resources that have, in total, made this experience completely satisfying and gratifying. Most of all, thank your friendship – past, present and future.
Richele Thompson. If John is the engine of the lab, you are the oil that keeps everything running smoothly (throwing aside oil changes, leaks, etc.; OK, bad metaphor, go figure). You have kept me sane during the stressful times and always pointed me in the right direction. I could not have come this far without your guidance and friendship.
Dr. Ronald Venters. You have been instrumental and influential not only on the projects that we collaborated on, but also on life in general. You have been a great friend and mentor.
Dr. Benjamin Bobay. Thanks for the many stimulating conversations, pushing me to do better, not forcing the Captain on me, and for being a great friend.
David Kordys. You have worked just as hard, and quite possibly harder, than I have on the calbindin project. Thanks for the many, many samples and for your friendship over the years.
Cavanagh Lab Group. I’d like to thank the entire lab for their support over the past years, in particular those who have assisted me along the way.
Collaborators and Colleagues. Dr. Steven Naylor and Linda Benson from the Mayo Clinic; Dr. James Hoch and Dr. Keith Stephenson from the Scripps Research Institute; Dr. Charles Opperman and Jenora Waterman from NC State University; Dr. Rajiv Kumar and his lab at the Mayo Clinic; Dr. Patrick Loria and Dr. Dagny Ulrich from Yale University; Dr. Bonnie Bassler from Princeton University; Dr. William Horne at Colorado State University; Dr. Ronald A. Venters, Dr. Pei Zhou and Brian Coggins from Duke University; Dr. Geoffrey Mueller from NIEHS; and Dr. David McK. Bird from NC State University. Thank you for your assistance, input and contributions on the projects that I’ve had the
My Committee. Dr. John Cavanagh, Dr. Dennis Brown, Dr. William Miller, Dr. Charles Opperman, and Dr. Ronald Venters. Thank you for your guidance and mentoring over the past years.
NMR Facilities at NC State University and Duke University, in particular Dr. Hanna Gracz, Mr. Eddie Bearfoot at NC State University and Dr. Len Spicer, Dr. Tony Ribeiro and Dr. Don Mika at Duke University.
Dr. Michael B. Goshe and Erik Soderblomat NC State University for assistance in checking sample quality through mass spectrometry, in particular to Dr. Goshe for great conversations during the time we shared office space.
Dr. Mark Rance and the NMR Facility at the University of Cincinnati for assistance in the collection of calbindin D28k aromatic NMR data. I look forward to my
future postdoctoral studies under your guidance and friendship.
Dr. Kenneth W. Dunn and Dr. Carrie Phillips at the Indiana University School of Medicine for giving me an early opportunity to experience the joys of research.
My Family. Thank you for all of your support over my entire life.
Erica. I have gained the most from this experience; you have given the most. Thank you for being patient – I appreciate it more than you’ll ever know.
List of Tables ix
List of Figures x
List of Symbols xii
List of Abbreviations xiii
1 Introduction 1
1.1 General concepts of signal transduction . . . 1
1.2 Two-component signal transduction . . . 3
1.2.1 Histidine protein kinases . . . 7
1.2.2 Response regulators . . . 11
1.3 EF-hand calcium-binding proteins . . . 14
1.3.1 The EF-hand domain . . . 14
1.3.2 EF-hand proteins: buffers and sensors . . . 20
1.4 Aims of this work . . . 20
2 Metal Binding Studies of Spo0F 22 2.1 Foreword . . . 22
2.2 Abstract . . . 23
2.3 Introduction . . . 23
2.4 Methods . . . 27
2.5 Results . . . 29
2.5.1 µESI-MS . . . 29
2.5.2 NMR spectroscopy . . . 32
2.5.3 Phosphotrasnfer between KinA∼P and Spo0F . . . 42
2.5.4 Sporulation inB. subtilis and P. penetrans . . . 42
2.6 Discussion . . . 47
3 Comparative Modeling of the OmpR Sub-family of Response Regulators 53 3.1 Foreword . . . 53
3.2 Abstract . . . 53
3.5 Results . . . 61
3.5.1 Determination of Optimal Modeling Parameters . . . 61
3.5.2 Modeled receiver domains of the OmpR sub-family of response regu-lators fromB. subtilis and E. coli . . . 65
3.6 Discussion . . . 74
4 Structural Refinement of the Phosphotransferase LuxU 78 4.1 Foreword . . . 78
4.2 Abstract . . . 79
4.3 Introduction . . . 79
4.4 Methods . . . 85
4.5 Results . . . 86
4.6 Discussion . . . 90
5 Structural Characterization of Ca2+-Loaded Calbindin D28k 92 5.1 Foreword . . . 92
5.2 Abstract . . . 93
5.3 Introduction . . . 94
5.4 Methods . . . 95
5.5 Results . . . 99
5.5.1 Backbone, side-chain and NOE assignment of Ca2+-loaded calbindin D28k . . . 99
5.5.2 Structure of Ca2+-loaded calbindin D28k . . . 110
5.5.3 Characterization of the Interaction between Calbindin D28k and its Effectors: myo-inositol monophosphatase and ran-binding protein M 118 5.6 Discussion . . . 122
6 Conclusions 128 6.1 Conclusions . . . 128
A Nuclear Magnetic Resonance Spectroscopy 131
B Calcium-Loaded Calbindin D28k Chemical Shift Table 144
Bibliography 160
1.1 Some members of the EF-hand protein family. . . 21
2.1 Detectable protein-metal complexes in µESI-MS metal concentration-based titration experiments . . . 29 2.2 Summary of the effect of copper on B. subtilis growth and spore formation. 44 2.3 Summary of the effect of copper on P. penetrans growth and spore formation. 47
3.1 B. subtilis and E. coli OmpR sub-class response regulators used as target sequences. . . 59 3.2 PSI-BLAST results for the response regulatory domain of ArcA from E. coli. 62 3.3 Optimal modeling parameter assay for the response regulatory domain of
ArcA from E. coli . . . 63 3.4 Optimal modeling parameter assay for the response regulatory domain of
PhoB fromE. coli . . . 65 3.5 Model statistics. . . 68 3.6 Sub-classes within the OmpR sub-family from B. subtilis and E. coli. . . . 74
4.1 Structural statistics for LuxU . . . 89
5.1 Calbindin D28k NMR Experiments. . . 96
5.2 NOE reference distances used in calbindin D28k structure calculations. . . . 97
5.3 Statistics for the ten lowest energy structures of calbindin D28k. . . 111
1.1 Two-component paradigm for sensory signaling via communication modules. 4
1.2 Examples of simple and complex two-component pathways. . . 6
1.3 Schematic representation of the histidine kinase core domains. . . 8
1.4 Structures of conserved dimerization domains in histidine protein kinases. . 9
1.5 Structures of conserved kinase domains in histidine protein kinases. . . 10
1.6 Structures of conserved HPt domains in histidine protein kinases. . . 11
1.7 Structures of conserved regulatory domains in response regulators. . . 13
1.8 Structures of effector domains in response regulators. . . 15
1.9 The EF-hand domain. . . 16
1.10 A sequence logo of the EF-hand domain in the Pfam database. . . 16
1.11 Various types of EF-hand motifs. . . 18
2.1 Spo0F-metal complexes analyzed usingµESI-MS. . . 30
2.2 Changes observed in regions of1H-15N HSQC spectra of Spo0F from B. sub-tilis as a consequence of metal titration. . . 34
2.3 Locations of diamagnetic metal-induced perturbations to Spo0F fromB. sub-tilis followed using1H-15N HSQC NMR. . . 38
2.4 Locations of paramagnetic metal-induced line broadening to Spo0F from B. subtilis followed using1H-15N HSQC NMR. . . 39
2.5 Minimum chemical shift difference titration curves for D11. . . 40
2.6 Metal dependance on the phosphotransfer between KinA∼P and Spo0F. . . 43
2.7 Sequence alignment andα4/β5/α5 surface similarity of Spo0F proteins. . . 45
2.8 Cu2+ dependence ofP. penetrans endospore formation. . . 46
2.9 Spo0F structures detail the position of H101 with respect to its active state. 50 3.1 A structural example of an OmpR sub-family response regulator: DrrB from T. maritima. . . 56
3.2 Response regulator:four-helix bundle co-crystal structures. . . 57
3.3 Color-coded hydrophobic gradient scale. . . 61
3.4 Modeled vs. solved structure of PhoB from E. coli. . . 66
3.5 Superimposition of modeled regulatory domains. . . 69
3.6 Hydrophobic surface characteristics of the B. subtilis comparative models. . 70
3.7 Hydrophobic surface characteristics of the E. coli comparative models. . . . 71
ative models. . . 75
3.10 Color-gradient hydrophobic surface characteristics of theE. coli comparative models. . . 76
4.1 The hybrid HSL-two-component quorum sensing circuit of V. harveyi. . . . 81
4.2 Sequence alignment ofV. harveyi LuxU and other Hpt proteins. . . 82
4.3 Actual and predicted secondary structure of LuxU . . . 83
4.4 Example of long-range NOE connectivities in an α-helix and β-strand. . . . 84
4.5 Solution NMR structure of LuxU. . . 87
4.6 Comparison of the 10 lowest energy ensembles of LuxU. . . 88
4.7 Summary of NOE distance restraints. . . 88
4.8 Comparison of LuxU to other Hpt protein domains. . . 90
4.9 Consensus sequence of residues in the active site of Hpt domains. . . 91
5.1 Outline of the general strategy used for calbindin D28k data analysis and structure calculations. . . 100
5.2 Summary of peak intensity increases between 25◦C and 50 ◦C. . . 102
5.3 15N-TROSY-HSQC temperature titration of15N-leucine calbindin D28k. . . 103
5.4 Examples of planes from deuterated NOESY experiments of calbindin D28k. 105 5.5 Connectivities observed to V62 HN and methyl protons in the deuterated NOESY data. . . 106
5.6 Examples of planes from protonated NOESY experiments of calbindin D28k. 108 5.7 Connectivities observed to I118 HN and Hδ1# methyl protons in the proto-nated NOESY data. . . 109
5.8 Secondary structure and observed NOE types for Ca2+-loaded calbindin D28k.113 5.9 Ten lowest energy structural ensemble of Ca2+-loaded calbindin D28k. . . . 114
5.10 1H-15N-TROSY-HSQC of Ca2+ calbindin D28k. . . 116
5.11 Peptide binding followed by 2D1HN-15N TROSY-HSQC of calbindin D28k. 120 5.12 Residues perturbed by LASIKNR peptide binding to calbindin D28k. . . 121
5.13 Ran-binding protein M peptide titration results mapped onto the ten lowest energy structural ensemble of Ca2+-loaded calbindin D28k. . . 123
A.1 Spin, magnetic dipole orientations and energy levels. . . 132
A.2 Triple resonance experiments for resonance assignment of 15N/13C-labeled proteins. . . 140
A.3 Scalar coupling constants for proteins. . . 141
Symbols
˚
A angstrom
δ chemical shift
∆δ change in chemical shift
~ Planck’s constant
Abbreviations & Nomenclature
4HB four-helix bundle motif
AI-1 autoinducer molecule 1
AI-2 autoinducer molecule 2
COSY correlation spectroscopy
HAP hispartyl-aspartyl (His-Asp) phosphorelay system
HisKA histidine phosphotransfer domain (dimeric)
Hpt histidine phosphotransfer domain (monomeric)
HSL homoserine lactone signalling molecule
Hz hertz; per second
K kelvin
kcal kilocalorie (103 of a Calorie)
µESI-MS Microelectrospary ionization mass spectrometry
MD molecular dynamics
HSQC heteronuclear single quantum correlation
NOE Nuclear Overhauser effect
NMR Nuclear magnetic resonance
Pi inorganic phosphate
PDB protein data bankor protein data bank id
ppm parts per million
RMS root mean square
RMSD root mean square deviation
RR response regulator
SANI susceptability anisotropy
SVD singular value decomposition
TAD torsion angle dynamics
TCS Two-component systems
TCST Two-component signal transduction
TROSY Transverse Relaxation Optimized Spectroscopy
A. aeolicus Aquifex aeolicus A. thaliana Arabidopsis thaliana B. subtilis Bacillus subtilis B. pertussis Bordetella pertussis C. crescentus Caulobacter crescentus C. vibrioides Caulobacter vibrioides M. tuberculosis Mycobacterium tuberculosis N. punctiforme Nostoc punctiforme
S. cervisiae Saccharomyces cervisiae S. melioti Sinorhizobium melioti S. pneumoniae Salmonella pneumoniae S. typhimurium Salmonella typhimurium R. meliloti Rhizobium meliloti T. maritima Thermotoga maritima V. cholerae Vibrio cholerae V. harveyi Vibrio harveyi
V. parahaemolyticus Vibrio parahaemolyticus V. vulnificus Vibrio vulnificus
Introduction
1.1
General concepts of signal transduction
Signal transduction is arguably the most important of all cellular mechanisms. Signal
transduction is defined as a process by which a stimulus is transformed from one chemical
form to another [1]. Signal transduction pathways, where multiple proteins are linked in
a complex regulatory network, are responsible for controlling numerous cellular functions
and responses and are primarily responsible for the prosperity of the cell. Abnormalities
in these pathways, generally caused by protein mutations, are the origin of practically all
diseases including cancer, immune and neurodegenerative disorders, as well as antibiotic
resistance and toxin production. Consequently, many research efforts are focused on
dis-secting signaling pathways within all cell types in order to fully understand them from the
cellular to sub-atomic scales.
Stimuli that activate signal transduction pathways vary as drastically as the types of
responses that are activated in response to these stimuli. In signal transduction systems,
stimuli are often referred to as messengers. First messengers are the primary messengers,
which are secreted from cells and bind to extra-cellular receptors upon reaching a
thresh-old concentration [2]. Second messengers are produced as a result of the first messenger
binding to the receptor and generally relay messages from one location within the cell to
another [3]. Messenger molecules, which influence the activity of enzymes, are wide-ranging
and include amino acids, peptides, proteins, nucleic acids, fatty acids, lipids, hormones,
neurotransmitters, metals and small molecules, such as methyl and phosphoryl groups.
Sig-nal transduction pathways can vary drastically between different cell types or organisms,
may be activated by one or multiple stimuli, may activate one or multiple cellular responses
or mechanisms, and may act synergistically with and be regulated by competing pathways.
Evolution has allowed for duplication and modification of signal transduction pathways
through the use of conserved, modular structural domains and motifs [3]. Smaller structural
domains act as building blocks used to construct larger, multi-domain proteins through a
process called domain shuffling. The considerable similarities in sequence and structure
between proteins within and between the hierarchical taxonomy trees allow the function of
a protein of unknown function to be inferred, provided there are sequential and structural
similarities to a protein of known function. Although in general there can be a high degree
of sequence and structural conservation between proteins within some cell types, there are
notable differences between the two major groups of kingdoms, prokaryotes and eukaryotes.
One example involves the type of kinases involved in phosphorylation signal transduction
cascades [4]. Prokaryotes primarily utilize His-Asp phosphotransfer systems, also referred
to as a two-component system, where phosphate is transferred from a conserved histidine
on a protein kinase to another protein containing an aspartic acid residue in its active site.
On the other hand, these signal transduction systems are rare in eukaryotes, which
pri-marily use Ser/Thr/Try phosphorylation pathways. There are a few cases where His-Asp
phosphotransfer systems have been eukaryotic systems [5–8] and Ser/Thr/Tyr kinases and
phosphatases in prokaryotic systems [9]. Eukaryotic kinases in two-component systems are
primarily hybrid proteins that contain both components of the His-Asp phosphotransfer
system. On the other hand, hybrid kinases are rare in prokaryotes, where the majority of
two-component systems consist of individual proteins [10]. One similarity between
eukary-otic and prokaryeukary-otic two-component signal transduction pathways is that both use these
signaling pathways to regulate the expression of specific genes. However, whereas the end
gene expression, two-component pathways in eukaryotic systems regulate other downstream
signaling pathways that directly regulate gene expression [4].
The following sections contain background information about two types of signaling
systems used in bacterial and eukaryotic signal transduction that are the subject of
stud-ies in this dissertation: two-component signal transduction and EF-hand calcium binding
proteins.
1.2
Two-component signal transduction
In order to adapt and survive in the presence of environmental stresses, bacteria must
possess the capability to sense and respond to signals within their environment. It
there-fore comes as no surprise that evolution has provided a simple and effective means for
bacteria to recognize specific stresses and coordinate a response – the two-component signal
transduction pathway [11]. These signaling systems are present in both Gram-positive and
Gram-negative bacteria, as well as other organisms including archaea, fungi, unicellular
eu-karyotes and higher plants [12]. More than forty of these systems have been observed in the
well-characterized bacterial systemsB. subtilis and E. coli [13, 14]. Even more astonishing
is the prediction of well over one-hundred two-component signal transduction pathways in
the draft genome of N. punctiforme [15]. Essentially all processes regulating metabolic
and physiological states in bacteria are controlled by two-component signal transduction.
The list of known processes controlled by two-component signal transduction pathways is
quite extensive and includes cell differentiation, cell cycle regulation, chemotaxis, small
molecule homeostasis, production of and resistance to antibiotics, quorum sensing, genetic
competence, regulation of transport, virulence and pathogenicity [16]. It was once said that
without two-component signal transduction ’bacteria would be rendered the equivalent of
deaf, dumb and blind’ [11].
While the so-called output, or process controlled by two-component signal transduction
pathways may differ tremendously, all are comprised of similar core domains that are
input domain "transmitter" "receiver" output domain
input signal
output signal
sensor kinase response regulator
P
Figure 1.1: Two-component paradigm for sensory signaling via communication modules. Sensory information flows through non-covalent controls exerted by one domain on an-other (dashed arrows) and through phosphorylation reactions between the transmitter and receiver domains. Adapted from Hoch and Silhavy [11]
respond to various stimuli. Two-component signal transduction systems can be generally
described as two modular proteins that act as an ’on-off’ circuit. These signaling pathways
contain a ’transmitter’ in the form of a histidine protein kinase and a ’receiver’ in the form
of a response regulator protein (Figure 1.1) [11, 17]. An environmental signal stimulates
the histidine protein kinase through its variable N-terminal sensing domain, causing an
autophosphorylation event to occur in its C-terminal kinase domain. The ATP-dependent
autophosphorylation event catalyses the formation of a phosphate group attached to a
con-served histidine residue on the histidine protein kinase, which enables the histidine protein
kinase to be recognized its cognate response regulator. The phosphate group is then
trans-fered from the histidine protein kinase to a conserved negatively charged residue, typically
an aspartic acid residue, on the response regulator protein. Phosphorylation of the response
regulator causes an activation event that allows it to perform a specific output, which can
include gene regulation, enzyme activity or binding to a downstream protein. Because of
the means by which the two proteins are phosphorylated, the two-component signal
trans-duction is also known as the hispartyl-aspartyl (His-Asp) phosphorelay system, or HAP
system.
in Figure 1.1 and may integrate multiple signals, proteins or output into the sensory
cir-cuit. Figure 1.2 shows some examples of complex two-component signal transduction
path-ways. The complexity builds upon the traditional two-component signal transduction
sys-tem, an example of which includes the EnvZ/OmpR system involved in osmoregulation in
E. coli (Figure 1.2a) [18]. The most common example of this complexity is the
phosphore-lay, or multi-component phosphorephosphore-lay, which links multiple two-component pairs together
in a single pathway. The two-component signal transduction phosphorelay leading to the
initiation of sporulation in B. subtilis (Figure 1.2b) [19] is one such pathway that contains
two pairs of two-component ’switches’. This pathway has been extensively studied and is
discussed further in Chapter 2.3. Another type of multi-component two-component signal
transduction system is the three-component system, such as the pathway leading to
pro-duction of virulence factors in V. cholerae (Figure 1.2.c), where the interaction between
two histidine protein kinases, ToxR and ToxS, act in combination to activate the response
regulator ToxT (reviewed in [20]). There are also pathways that use dual sensors/regulators
(Figure 1.2d), such as the histidine protein kinases NarL and NarP work in combination
to provide strict control of nitrate and nitrite metabolism through the response regulators
NarX and NarQ [21]. Other complex systems include a quorum sensing multichannel system
inV. harveyi that regulates bioluminescence (Figure 1.2h) [22]; the use of ’typical’ hybrid
phosphorelay systems: the E. coli Arc pathway regulating anaerobiosis (Figure 1.2f) [23]
and theS. cervisiae Sln1→Ypd1→Ssk1 pathway involved in osmotolerance (Figure 1.2e);
and ’atypical’ hybrid phosphorelay systems: the E. coli RcsC → YojN → RcsB pathway,
Figure 1.2g. Perhaps the most complex of all two-component systems involves the
regula-tion of the cell cycle inC. crescentus [24]. This pathway utilizes multiple histidine protein
kinases, one of which is linked in a phosphorelay, to control the master response
regula-tor CtrA. In addition, the function of CtrA is spatially oscillated ’on-off’ and is regulated
through time- and space-dependent means through another response regulator, GcrA [25].
There are also modes of complexity that extend beyond the traditional organization of
D
YojN RcsB
g
RcsC
H
H D
EnvZ
H D
OmpR a
D
ToxR ToxT
c
ToxS
H
H D
H D
NarQ
NarX
NarL
NarP d
H D
KinABCDE
D
Spo0F
H
Spo0B Spo0A
b
D
Sln1
H D H
Ypd1 Ssk1
e
ArcB
H D H
ArcA
f D
H D
H D
D LuxQ
LuxN LuxO
h H
LuxU
or crosstalk, which has been reported to occur in more than one HAP system. A
well-documented example of crosstalk involves the response regulator PhoB [26]. The histidine
protein kinase PhoR, which is activated by Pi, and PhoB comprise the phosphate
regu-lon in E. coli. However, two other pathways that activate PhoB which are independent of
Pi/PhoR have been discovered. This includes the catabolite sensing histidine protein kinase
CreC and the acetyl phosphate sensing histidine protein kinase EnvZ. An additional cross
regulation involving the VanS involved in vancomycin resistence has also been reported [27].
Another method of complexity involves protein phosphatases, which dephosphorylate
re-sponse regulators and act as a means of control of the ’on-off’ nature of the sensory switch in
some systems. One example of this involves a symbiotic relationship between the initiation
of competence and sporulation in B. subtilis, where phosphatase production is controlled
by an amplification of the competing process [28]. In this case, both pathways function
to sense, respond and control the overall wellness of the cell. Phosphatases that inhibit
sporulation are produced as a result of favorable conditions in a competing pathway, the
initiation of competence. There are more variations than those listed here, but it is easy to
understand both the aspects of simplicity and complexity of two-component signal
trans-duction. Evolution has duplicated and adapted this highly conserved signaling circuit, yet
individual pathways are controlled by disparate signals in order to perform many diverse
functions.
1.2.1 Histidine protein kinases
The first component of the two-component switch is the histidine protein kinase
(re-viewed in [4, 16]). The histidine protein kinase family of proteins has been divided into two
major classes based on their domain organization: class I and class II (Figure 1.3) [29–31].
Class 1 histidine protein kinases contain the active site histidine within the
homodimer-ization domain followed by a C-terminal catalytic core domain. Class 2 histidine protein
kinases contain the active site histidine within the phosphotransfer (HPt) domain found
H
sensor
TM
Dim
F G1 N G2
Class I
H
F G1 N G2
Dim
HPt
Class II
Figure 1.3: Schematic representation of the histidine kinase core domains: Sensor domain, transmembrane domain (TM), dimerization (Dim) domain, histidine containing phospho-transfer (HPt) domain, and kinase ATP-binding domain. Histidine kinases are categorized into two classes according to the location of the conserved active site histidine (H box) and the ATP-binding domain (N, G1, F, and G2 boxes). Adapted from Tomomori et al. [31]
domain rather than proximal as in the case of the class 1 proteins. These proteins range
in size from<40 kDa to>200 kDa [4]. Three-dimensional structures of individual domains
of histidine protein kinases have been developed, which reveal the strong conservation of
particular domains within these proteins [4, 32].
Figure 1.4 shows examples of the conserved dimerization domain. These domains form
a four-helix bundle through homodimerization of two-stranded coiled coils [33]. The site of
phosphorylation that contains the active site histidine residue is termed the H-box.
Phos-phorylation of this domain occurs in trans; the kinase domain of one subunit in the
ho-modimer phosphorylates the H-box histidine in the opposing subunit [11]. Phosphatase
activity of histidine protein kinases is mediated by the dimerization domain [11, 34].
Addi-tionally, this domain can also be found in phosphotransferase proteins, such as Spo0B from
B. subtilis [35].
Figure 1.5 shows examples of the conserved kinase domain. These domains are
a
b
c
Figure 1.4: Structures of conserved dimerization domains in histidine protein kinases. Dimerization domains of (a) EnvZ fromE. coli (PDB: 1JOY) [37], (b) Spo0B fromB. sub-tilis (PDB: 1IXM) [35] and (c) CheA fromT. maritima (PDB: 1B3Q) [38] are highlighted in green.
cleft defined by the boxes N, G1, F and G2.
Figure 1.6 shows examples of the conserved HPt domain. These domains also form
four-helix bundle structures, have an active site histidine, and typically transfer the phosphate
between proteins in phosphorelays. Whereas the dimerization domain forms a four-helix
bundle through homodimerization two-stranded coiled coils, the four-helix bundle formed
by the HPt domain is one contiguous peptide.
The variable sensor domains are typically found at the N-terminus of histidine protein
kinases and are responsible for detecting environmental stimuli. These domains show a
great deal of sequence and structural variation, which affords specificity in ligand/stimulus
binding across various two-component signal transduction pathways. Transmembrane
his-tidine protein kinases contain linker domains, which vary in length from 40 to>180 amino
acids [36]. These domains are thought to be coiled-coil domains, they directly precede the
active site H-box, and are critical for propagation of the signal originating from the sensor
a
b
d
c
a
b
c
Figure 1.6: Structures of conserved HPt domains in histidine protein kinases. HPt domains of (a) ArcB fromE. coli (PDB: 1FR0) [43], (b) Ypd1 fromS. cerevisiae (PDB: 1QSP) [44] and (c) CheA P1 domain fromS. typhimurium (PDB: 1I5N) [45] are highlighted in blue.
1.2.2 Response regulators
The second component of the two-component switch is the response regulator (reviewed
in [4]). These proteins generally contain two general domains, regulatory and effector,
although other possibilities exist, such as single domain and three domain proteins. The
N-terminal regulatory domain is approximately 125 residues in length and forms a doubly
wound α/β domain with a central five-stranded parallel β-sheet surrounded by five α -helices [4, 46, 47]. This domain contains an active site Asp pocket that consists of three
conserved aspartic acid residues, as well as to two other conserved threnoine and lysine
residues proximal to the Asp pocket [48–50]. The active site aspartic acid residues are
required for phosphorylation and dephosphorylation [51]. A divalent cation required for
phosphorylation binds in the Asp pocket in an octahedral coordination involving the nearby
residues and three water molecules [52, 53]. Phosphorylation in the Asp pocket is thought
to occur by means of a bipyramidal pentavalent phosphorus transition state [52, 54]. There
are a considerable number of regulatory domain structures solved by x-ray crystallography
and NMR spectroscopy (Figure 1.7). These structures show a high degree of structural
notably, in the conformation of loops surrounding the active site Asp pocket [56] and in the
position ofα-helix 4 and theα4/β5/α5 [57].
Studies have shown that the regulatory domains exist in a conformational flux, sampling
between the inactive and active, or phosphorylated, structural forms [55, 77].
Phosphoryla-tion of the regulator in the Asp pocket is thought to tip the flux from the inactive state to
the active state. The most notable structural changes between the apo and phosphorylated
states are found in the loops surrounding the Asp pocket, as well as the topology ofα1 and the β4-α4 loop surface [59, 78–80]. Moreover, these regions that experience changes as a result of phosphorylation have been shown to be important in protein-protein interactions,
including inter-domain interfaces [56, 81] and target protein interactions [69, 82–93].
The C-terminal effector domains display a modest degree of diversity across all response
regulator proteins. Examples of these domains are shown in Figure 1.8. The function of this
domain generally is that of DNA-binding in the activation or repression of the transcription
of specific genes, although some domains have catalytic functions, such as the
methyltrans-ferase domain of CheB [4, 94]. Despite the apparent diversity of effector domains, there are
three general groups of response regulator-based transcription factors: the OmpR, NarL
and NtrC sub-families [4]. The OmpR sub-family, the largest of all response regulator
groupings, contains a C-terminal winged-helix DNA-binding motif (Figure 1.8a) [95–98].
These domains contain a recognition helix that interacts with the major groove of DNA
and surrounding loops, orwings, that make contacts to the DNA minor groove. The
tran-scriptional activity of OmpR sub-family response regulators involves interactions with RNA
polymerase. However, it appears that individual proteins in this sub-family targets a
dif-ferent polymerase [99–102]. The NarL sub-family contains a C-terminal helix-turn-helix
DNA-binding motif that is approximately 60 residues (Figure 1.8b) [81]. This domain
con-tains four helices, one of which is referred to as the DNA recognition helix. The NtrC
sub-family contains two domains C-terminal to the regulatory domain: a medial ATPase
a b c d e
f g h i j
k l m n o
p q r s t
Figure 1.7: Structures of conserved regulatory domains in response regulators. Regu-latory domains of (a) PhoB from E. coli (PDB: 1B00) [58], (b) FixJ from R. meliloti
(PDB: 1DBW) [59], (c) ETR1 from A. thaliana (PDB: 1DCF) [60], (d) Rcp1 from Syne-chosystis PCC6803 (PDB: 1I3C) [61], (e) CheY from E. coli (PDB: 1JBE) [62], (f) RcpB from Calothrix PCC7601 (PDB: 1K66) [63], (g) RcpA from Calothrix PCC7601 (PDB: 1K68) [63], (h) DrrD from T. maritima (PDB: 1KGS) [64], (i) DivK from C. crescentus
(PDB: 1MB3) [65], (j) PhoP from B. subtilis (PDB: 1MVO) [66], (k) MicA fromS. pneu-moniae (PDB: 1NXP) [67], (l) NtrC fromA. aeolicus (PDB: 1NY5) [68][68], (m) Sln1 from
to the structure highlighted in Figure 1.8b). Dimers of NtrC sub-family response
regula-tors oligomerize into octamers upon phosphorylation, which stimulates ATP hydrolysis and
provides energy for open complex formation and activation of transcription [103–110].
1.3
EF-hand calcium-binding proteins
Intracellular calcium concentrations regulate many cellular processes, including
nu-cleotide metabolism, muscle contraction, cell-cycle control, differentiation and signal
trans-duction, in a wide variety of cell types [111, 112]. The EF-hand is a common motif present
in Ca2+ binding proteins that enables a protein to sense fluctuations in intracellular Ca2+ concentrations by means of a high affinity Ca2+-binding site. At low Ca2+ concentrations, in the range of 10−7-10−8 M, EF-hand containing proteins are generally not bound to Ca2+
and are subsequently inactive. Upon an influx of Ca2+, which raises intracellular Ca2+ concentrations in the range of 10−5-10−6 M, EF-hand containing proteins bind to Ca2+ and
become active. Thus, the EF-hand acts as a Ca2+ dependent switch, where the ’on’ or ’off’ status depends on the amount of Ca2+ available in the cell.
1.3.1 The EF-hand domain
The EF-hand domain is the most common type of calcium-binding motif among all
calcium binding proteins. The EF-hand is one type of domain that falls within the
so-called DxDxDG motif present in many calcium binding proteins [113]. EF-hand domains
consist of a helix-loop-helix structure with a fold such that the structure can be symbolically
represented using the index finger to represent the first helix and the thumb to represent the
second helix (Figure 1.9a [114]). The loop serves as the Ca2+ binding site, and the residues directly involved in Ca2+ binding fall within a conserved consensus sequence consisting of an extension of the DxDxDG motif (Figure 1.10) [113]. The EF-hand was named after the
Ca2+ binding protein parvalbumin, where this domain was shown to involve helices E and F.
a
b
c
d
Figure 1.8: Structures of conserved effector domains in response regulators. Highlighted in red are (a) the winged-helix domain of the OmpR sub-family response regulator DrrD from
T. maritima (PDB: 1KGS) [64], (b) the helix-turn-helix domain of the NarL sub-family response regulator NarL from E. coli (PDB: 1RNL) [81], (c) the σ54 activation/ATPase
a
b
c
Figure 1.9: The EF-hand domain. (a) A symbolic representation of the EF-hand domain. Helix E windows down the index finger, whereas helix F winds up the thumb of a right hand. When the calcium ion binds, helix F moves from the closed (apo-protein, light grey) to the open (holo-protein, dark grey) conformation. (b) The geometry of the calcium ligands. At positions X and Y, we usually find the side-chains of aspartic acid or asparagine; the side-chains of aspartic acid, asparagine or serine are found at Z and a peptide carbonyl oxygen lies at−Y.−X is usually a water molecule and−Z is a conserved bidentate ligand, glutamic acid or aspartic acid. (c) The modified EF-hand loop in p11 (S100A10), showing the network of stabilizing hydrogen bonds. Adapted from Lewit-Bentley and R´ety [114].
of X•Y•Z•−Y•−X••−Z, consisting of loop positions 1, 3, 5, 7, 9 and 12, respectively. These
residues coordinate the Ca2+ ion in an pentagonal bipyramidal coordination scheme from seven oxygen atom ’ligands’ (Figure 1.9b and Figure 1.11) [114, 117]. The letters X, Y, Z,
−X,−Y and−Z denote the six ligands that are involved in metal ion coordination, whereas
the dots denote other residues present in the Ca2+-binding loop. The atoms that coordinate the Ca2+ ion are conserved in the canonical Ca2+-binding loop [118], and hydrogen bonds between residues in the Ca2+-binding loop allow for the proper formation and stabilization of the loop structure (Figure 1.9c). Residues at positions 1, 3 and 5 provide monodentate
side-chain oxygen ligands, while the residue at position 12 is a bidentate oxygen ligand. The
residue at position 1 is thought to stabilize the Ca2+ loop through hydrogen bonds to the residues at loop positions 4, 5 and 6 through its side-chain carboxylate oxygens [119]. This
residue exhibits (φ,ψ) angles characteristic of a γ-turn and terminates the first α-helix, historically referred to asα-helix E. The residue at position 7 provides an oxygen ligand by means of its backbone carbonyl oxygen, while residue 9 hydrogen bonds to a water molecule
that acts as an additional Ca2+ ligand. A highly conserved glycine residue at position 6 does not participate in metal ion coordination, but, as previously mentioned, participates
in a hydrogen bond with the residue at position 1. It also provides a flexible point to
introduce a sharp turn in the Ca2+-binding loop, allowing for the proper structure of the loop to be assumed [119]. The second helix, historically referred to as α-helix F, starts at loop position 10 such that the last three residues in the Ca2+-binding loop (positions 10-12) make up the first three residues of the second helix. The residue at position 12 is usually a
glutamic acid residue that participates as a bidentate ligand. It has been noted that when
an aspartic aside residue is found at position 12, the Ca2+-binding loop adopts a smaller, more compact structure that may be involved in the reduced binding affinity for Ca2+ and increased binding affinity of different cations [120]. In this case, the aspartic acid side-chain
may participate as a monodentate ligand, which changes the geometry of the loop from
Figure 1.11: Various types of EF-hand motifs. The canonical EF-hand consists ofα-helix E, (residues 1−10), loop around the Ca2+ ion (10−21) andα-helix F (19−29). Residue
1 is often Glue (’E’); the insides of the helices usually have hydrophobic residues ’n’. The side-chains of five residues, approximated by the vertices of an octahedron (X, Y, Z,−X, & −Z) provide oxygen atoms to coordinate calcium; residue 16, ’#’ at−Y, bonds to the Ca2+
In non-canonical EF-hand Ca2+-binding loops, also called the pseudo EF-hand, the Ca2+-binding loop is comprised of 14 residues [118]. Figure 1.11 shows an example of the non-canonical EF-hand sequence found in the S-100 protein [117]. In contrast to canonical
Ca2+-binding loops, the non-canonical loop utilizes several backbone carbonyl oxygen atoms in the coordination of Ca2+. Despite this difference in Ca2+ ligand type, non-canonical loops still display pentagonal bipyramidal coordination geometry. Residues 1, 4, 6 and
9 participate in Ca2+ ion coordination through their backbone carbonyl oxygen, whereas residue 11 contributes through an interaction with a water molecule. Residue 14 provides
a bidentate ligand and is usually a glutamic acid residue, as its longer side chain facilitates
the positioning of its side-chain oxygen atoms in the chelation of the Ca2+ ion. Other EF-hand non-canonical variants have been observed, which are also shown and described
in Figure 1.11 [117].
EF-hand domains are often found in pairs, and there are significant contacts between
the two domains [119]. The most notable interaction is the anti-parallel β-sheet formed utilizing residues at positions 7 to 9 in the Ca2+-binding loops. A strong hydrogen bond is present between residues at position 8 and 8’ (the ’ indicating the second EF-hand in
the pair), bridging the backbone carbonyl and amide groups within very close proximity.
There are additional strong interactions in the anti-parallelβ-sheet between the Hαatoms of residues at positions 7 and 9’, as well as between the residues at positions 9 and 7’.
Other strong interactions are found between the side-chains of the four helices (A-D) in the
pair of EF-hands (A and B are the first and second helices of the first EF-hand; C and
D are the first and second helices of the second EF-hand). The most notable interactions
are between helices A and D, as well as helices B and C. Weaker interactions are observed
within the individual EF-hand between helices A and B, as well as C and D. The conserved
hydrophobic residue at position 8 points down into the hydrophobic core of the EF-hand
1.3.2 EF-hand proteins: buffers and sensors
Proteins containing EF-hand domains can be classified into two general groups: Ca2+
buffers and Ca2+ sensors [111, 118]. Table 1.1 lists a few members of each of these groups [111]. These groups have distinct biological functions and structural consequences
upon Ca2+ binding. Ca2+ buffer proteins are involved in Ca2+ buffering and transport within the cell. These proteins do not undergo a significant structural change upon binding
Ca2+. Ca2+ sensor proteins have regulatory roles within the cell, which may include binding to a downstream protein or act as a transcriptional regulator of specific genes. In contrast
to Ca2+ buffer proteins, these proteins undergo a significant structural change in response to Ca2+ binding [121]. This conformational change typically exposes a previously buried hydrophobic surface through which the Ca2+ sensor uses to perform its regulatory activity. This distinct difference in response to Ca2+ binding between Ca2+ buffer and Ca2+ sensor proteins is often used to imply the biological function of EF-hand containing proteins of
unknown function.
The binding of Ca2+ binding to Ca2+ sensor proteins causes a structural change from a Ca2+ free ’closed’ conformation, where the helices of each EF-hand are somewhat anti-parallel, to a Ca2+’open’ conformation, where the helices are more perpendicular in na-ture [111, 122–124]. It is thought that the concerted movement ofα-helix E and F’ (helices A and D), with respect to F and E’ (helices B and C), are responsible for the high
coopera-tivity of Ca2+ binding between the linked EF-hand sites. Ca2+ buffers, on the other hand, retain the Ca2+ free ’closed’ conformation upon binding Ca2+.
1.4
Aims of this work
The work presented in this dissertation described the studies performed on four
dis-tinct projects. Chapter 2 describes the study and effect of the metal binding properties
of the initiation of sporulation response regulator Spo0F using solution state NMR
Table 1.1: Some members of the EF-hand protein family.
Protein # of EF-hands M.W. (kDa) Function
Ca2+ sensors
Calmodulin 4 17 Modulator of enzymes and proteins
Troponin C 3-4 17 Modulator of muscle contraction
Calcineurin B 4 19 Regulatory subunit of protein phosphatase 2B
Myosin light chains 4 19 Modulator of muscle contraction
Recoverin 4 23 Modulator in retinal rod cells
S-modulin 4 23 Recoverin homologue
Visinin 4 22 Modulator in retinal cone cells
VILIP 4 22 Visinin-like protein
Neurocalcin 4 22 Modulator in brain cortex and cerebellum
Hippocalcin 4 23 Modulator in hippocampus
Frequenin 4 22 Modulator of K+ channel
Caltractin 4 20 Microtuble organization
Calpain large subunit 4 30 Protease catalytic domain
S100 proteins 2 10-12 Modulator of enzymes and proteins
Ca2+ buffers
Parvalbumin 3 12 Ca2+ buffering and transport
Calbindin D9k 2 9 Ca2+ buffering and transport
comparative models for the OmpR sub-class response regulators fromB. subtilis andE. coli
to provide insights into the inter-relatedness and classification of this close family of
pro-teins. Chapter 4 describes the efforts to refine the solution structure of the quorum sensing
phophotransferase LuxU fromV. harveyi using solution state NMR spectroscopy. Chapter 5
describes the development of a high-resolution solution structure and the effect of peptide
Divalent Metal Binding Studies of the Response
Regulator Spo0F from
B. subtilis
2.1
Foreword
The research outlined in this chapter was performed in collaboration with several groups.
Dr. Steven Naylor and Linda M. Benson of the Biomedical Mass Spectrometry &
Func-tional Proteomics Facility at the Mayo Clinic performed the µESI-MS experiments. Drs. James A. Hoch of the Division of Cellular Biology at the Scripps Research Institute and Dr.
Keith Stephenson, now at the School of Biochemistry and Microbiology at the University of
Leeds performed the phosphotransfer experiments between KinA and Spo0F. Dr. Charles
H. Opperman and Jenora Waterman of the Department of Plant Pathology at North
Car-olina State University performed the metal-based experiments in P. penetrans. Richele
Thompson of the Department of Molecular and Structural Biochemistry at North Carolina
State University performed the metal-based experiments in B. subtilis and assisted in the
NMR data collection and manuscript preparation. Dr. John Cavanagh of the Department
of Molecular and Structural Biochemistry at North Carolina State University assisted in
the NMR data collection and manuscript preparation. I was responsible for the sample
preparation, NMR data collection, analysis and preparation of the manuscript. The work
outlined in this chapter will be published in the journal Biometals.
2.2
Abstract
The metal binding properties of the initiation of sporulation response regulator Spo0F
were studied by µESI-MS and NMR spectroscopy. The µESI-MS studies reveal that an assortment of divalent metals bind to Spo0F in varying relative affinities and stoichiometries.
1H-15N-HSQC NMR studies confirm the binding characteristics observed in the µESI-MS
studies and structurally pinpoint the regions affected by metal binding. The titration of the
divalent metals Ca2+, Mg2+ and Mn2+ predominantly reveal binding effects in the active
site Asp pocket – the site of phosphorylation. Additional lower-affinity binding effects
are observed in the α4/β5/α5 surface region at higher, possibly biologically irrelevant, concentrations. Titration of Cu2+ revealed high-affinity binding effects in three distinct structural locations: (i) the active site Asp pocket, (ii) a site directly opposite of the
Asp pocket and 3) the surface region comprising the α4/β5/α5 interface, including the residue H101. Phosphotransfer experiments between KinA∼P and Spo0F in the presence
of Mg2+ supports phosphotransfer, while phosphotransfer in the presence of Cu2+ fails to support phosphotransfer. Additional experiments reveal that the presence of Cu2+ at low
concentrations in the sporulation growth media inhibits spore formation in B. subtilis. A
similar Cu2+-dependent regulation of spore formation is observed in P. penetrans, a close relative to theBacillus spp. of bacteria that undergo sporulation. The results suggest that,
depending on the type of divalent metal ion present,in vitro phosphorylation of Spo0F by
its cognate histidine protein kinase KinA can be inhibited.
2.3
Introduction
During the transition from exponential, or vegetative, cell growth to the stationary
phase, B. subtilis may undergo a process called sporulation. This process leads to the
formation of a dormant, environmentally resistant spore and occurs in response to nutrient
deprivation, high cell density, and input from cell cycle signals such as Krebs cycle, DNA
are seven stages of sporulation that result in the transformation of a vegetative cell into
a mother cell and a forespore. Although both of these cell types have identical genomic
content, each has cell-specific RNA polymerase sigma factors that regulate and induce the
expression of different genes [127]. The decision for the bacterium to undergo sporulation is
an important one. Sporulation is a complex, energy-intensive process that involves extensive
changes in gene expression and cellular morphology. Therefore,B. subtilis utilizes a simple
and precise mechanism for sensing and initiating sporulation: the two-component signal
transduction phosphorelay (reviewed in Chapter 1.2).
The phosphorelay leading to the initiation of sporulation inB. subtilis, otherwise known
as stage 0, has been extensively studied and used as a model in the study of other
com-plex two-component systems [11, 19, 125]. Current models of the sporulation phosphorelay
detail that the pathway is initiated by one of five histidine protein kinases: KinA, KinB,
KinC, KinD, and KinE. KinA has been shown to be the primary kinase in the initiation
of the sporulation phosphorelay in vitro. Activated in response to presently unidentified
environmental signals, the kinase autophosphorylates on a conserved histidine residue in
an ATP-dependent manner. The phosphoryl group is then transfered from Kin[A-E]∼P
to the response regulator Spo0F. Spo0F∼P transfers the phosphoryl group to the
phos-photransferase Spo0B, which is subsequently transfered from Spo0B∼P to the response
regulator Spo0A. Spo0A∼P is a transcription factor that binds to a conserved DNA
se-quence, TGNCGAA, termed a Spo0A box, and positively regulates transcription of itself
and genes required for subsequent stages of sporulation, including those required for axial
filament formation, asymmetric division, and the sigma factorsσF and σE. Spo0A∼P also negatively regulates transcription of a handful of genes including the transition state
regu-lator abrB, the spo0E phosphatase that dephosphorylates Spo0A∼P, and thespo0H gene
encoding the sigma factorσH.
Other processes include the production of products involved in generating nutritional
re-sources and inhibiting cell growth of potential competitors [125]. This includes the
develop-ment of genetic competence, which enables the bacterium to bind and internalize exogenous
DNA for purposes of either nutrition or repair of damage to DNA [128]. Although genetic
competence is not a subject of this research, and therefore not reviewed in entirety, it is
re-lated to the sporulation process. The signal transduction pathways leading to the initiation
of sporulation and competence are independent, competing pathways that are
intercon-nected [125]. Some genes required for sporulation are also required for the development of
competence. In addition, genes which are regulated by the sporulation pathway inhibit the
development of competence, and vice versa. For example, the progression of the pathway
leading to the development of competence results in the expression of the genes that act as
phosphatases on proteins involved in the initiation of sporulation pathway, such as RapA
and RapE [28]. Other phosphatases that act on the sporulation pathway, including RapB,
Spo0E and YnzD, are expressed during the early, middle or late stages of vegetative growth
conditions, which similar to competence is antithetical to sporulation. Additionally, YisI is
expressed during the transition from exponential and stationary phases [129].
The response regulator Spo0F represents a central node of control for the initiation of
sporulation phosphorelay and has been extensively studied biochemically and structurally.
Spo0F is phosphorylated by one of five kinases (Kin[A-E]), transfers the phosphate to
the downstream phosphotransferase (Spo0B), and can become dephosphorylated by one
of three phosphatases (RapA, RapB or RapE) [11, 19, 125]. Spo0F is a single domain
response regulator with a structure that adopts the conserved regulatory domain (α/β)5
fold [55, 73, 130]. It has a centralβ sheet core consisting of five parallelβ-strands, flanked by five αhelices, two on one side (α-helix 1 and α-helix 5) and three on the other (α-helix 2,α-helix 3 andα-helix 4). Phosphorylation of Spo0F requires a divalent metal ion, usually described as Mg2+, that binds in the conserved active site Asp pocket consisting of D10, D11 and D54. D11 is considered to be the principle residue in divalent metal binding, and
ion in the Asp pocket assists in phosphoryl catalysis and ensures the negative character of
the pocket is offset, thereby allowing the negative phosphate moiety to approach. Other
conserved residues, such as T82 and K104, are important for the catalysis and transfer of
phosphate as well.
Alanine scanning mutagenesis of 79 out of 124 residues in Spo0F identified mutations
that produced alternative in vivo sporulation phenotypes [90, 131]. These Spo0F mutants
were further analyzedin vitrofor their effect on protein:protein interactions with its cognate
kinase, KinA, or the phosphotransferase Spo0B. These studies, when compared to a
co-crystal structure between Spo0F and Spo0B, revealed the residues within the vicinity of the
active site, including loop regions and the interface between α-helix 1 and α-helix 5, are involved in crucial hydrophobic contacts that are important for protein recognition [90, 93].
NMR studies of Spo0F in the unphosphorylated and the phosphorylation-mimic BeF3− -bound forms have revealed changes involved in activation via phosphorylation [80]. These
studies are believed to support a pre-existing equilibrium model, where in the
unphos-phorylated form, Spo0F samples and fluxes between the inactive and active
conforma-tions [132, 133]. Upon phosphorylation, or BeF3−-induced activation, the equilibrium is pushed from the inactive to active conformation. NMR studies of the backbone
dy-namics of inactive form of Spo0F revealed residues that experience motions from the
picosecond-to-millisecond time-scales. Interestingly, residues that exhibit slow motions on
the microsecond-to-millisecond time-scale correlate to regions of Spo0F that were shown to
be important for protein:protein interactions in the alanine scanning mutagenesis studies.
Additionally, Spo0F has been characterized in terms of metal-binding. The most
vi-sual of these studies include x-ray crystal structures the Y13S mutant of Spo0F bound to
Mn2+ and Ca2+ [73, 134]. Both structures reveal a single divalent metal bound in the active site Asp pocket, primarily in an octahedral coordination geometry. There are
using fluorescence in a Spo0F Y13W mutant. These studies revealed Spo0F, like CheY
and DivK, bind various metal ions with different affinities. Titration of Mg2+ followed by
15N-HSQC NMR revealed characteristics that correlate well with the Ca2+ and Mn2+Y13S
Spo0F crystal structures. Mg2+-induced perturbations were observed near the active site Asp pocket with a Kd of 20 ± 5 mM. However, titration of higher Mg2+ concentrations
resulted in additional unsaturated binding curves in the region of the α4/β5/α5 surface and the β4-α4 loop. These effects are observed at concentrations above the physiological 1mM concentration of Mg2+, and it was suggested that this region may be a binding site for a different metal cation.
The studies presented in this chapter report on a comprehensive analysis of the effects
of various divalent metals on the structure and function of B. subtilis Spo0F. The results
obtained using the complementary techniques of µESI-MS and NMR suggest that certain divalent metals, such as Ca2+, Mg2+ and Mn2+, primarily display 1:1 protein:metal binding profiles and allow for phosphotransfer between KinA and Spo0F, while other metals, such
as Cu2+, display unique multiple-metal binding profiles and do not support phosphotrans-fer. A potential mechanism for in vitro divalent metal-mediated phosphotransfer and its
implications inB. subtilis and a related organism,P. penetrans, are discussed.
2.4
Methods
Unlabeled and15N labeled Spo0F samples were expressed and purified as previously de-scribed [90, 130, 135]. 15N labeled Spo0F samples used for NMR data collection contained approximately 1 mM protein, were approximately 99% pure, and were dialyzed into a buffer
of 25 mM Tris pH 6.9, 50 mM KCl, and 0.02% NaN3. Metal titrations were performed
us-ing chloride salts and monitored by means of 1H-15N HSQC experiments [136, 137]. NMR experiments were run at 300 K on either a Bruker DRX 500 equipped with 3 radiofrequency
channels and a triple axis pulsed field gradient triple resonance probe or on a Varian
field gradient triple resonance probe. Data were referenced to previously published
chem-ical shifts for B. subtilis Spo0F [55], processed with NMRPipe [138] and analyzed with
NMRView [139].
Concentration ranges of metal ions during the titrations were as follows: Ca2+: 0− 151 mM; Cu2+: 0−1.169 mM; Mg2+: 0−147 mM; Mn2+: 0−433µM. Diamagnetic metal concentration ranges were chosen to be similar to the protein:metal ratios a previous Mg2+ titration [130]. Titrations of diamagnetic metals were analyzed by measuring changes in
1H
N-15N backbone chemical shifts as a function of metal concentration using the minimum
chemical shift difference method [140, 141]:
∆δmin= [∆δ(1HN)2+ 0.1×∆δ(15N)2]1/2 (2.1)
Titrations of paramagnetic metals were analyzed by measuring1H-15N chemical shift peak
line width broadening as a function of metal concentration and normalized based on the
individual intensities for each peak in the base spectrum with no metal. The following trends
in chemical shift changes and line broadening were considered when plotting trends onto the
structure of Spo0F: Ca2+,∆δmin>0.23 ppm; Mg2+, ∆δmin >0.20 ppm; Mn2+ and Cu2+, residues that experience the most significant decreases in slope and intensity as a function
of metal concentration. The colors and symbols used in the graphical plots are numbered
respective of sequence position, with the exception of residues in the paramagnetic titrations
that display relatively little change in intensity or line broadening, which are shown with
blue dotted lines. Distances of residues with reference to the Asp pocket were measured
between the HNatom of the residue of interest and the Cγatom of D11 from Spo0F (PDB:
1NAT) [142]. Graphical analysis was performed using MATLAB (The MathWorks, Inc,
Natick, MA) and structural images were prepared withPyMOL (DeLano Scientific).
TheP. penetrans Spo0F homology model was created usingMODELLER6v2 [143] using
Table 2.1: Detectable protein-metal complexes in µESI-MS metal concentration-based titration experiments
Metal Group Detected protein-metal complex
K+ 1 Apo form is the main detected complex; observable weak one metal-bound complex at high concentrations of metal
Ca2+ 2 Primarily apo and one metal-bound complexes detected; weak two and three metal-bound com-plexes detected
Cu2+ 3 One, two and three metal-bound complexes detectable with relatively high affinity compared to
the other divalent metals
Mg2+ 2 Primarily apo and one metal-bound complexes detected; weak two and three metal-bound
com-plexes detected
Mn2+ 2 Primarily apo and one metal-bound complexes detected; somewhat weak two and weak three metal-bound complexes detected, which are more intense than that for Ca2+and Mg2+
Zn2+ 3 One, two and three metal-bound complexes detectable with relatively high affinity compared to the other divalent metals, but not as strong as that for Cu2+
Fe3+ 1 Apo form is the main detected complex; observable weak one metal-bound complex at high
concentrations of metal
Mn3+ 1 Apo form is the main detected complex; observable weak one metal-bound complex at high
concentrations of metal
2.5
Results
2.5.1 µESI-MS
µESI-MS is a technique that allows for the study of noncovalent protein−ligand com-plexes [144–146]. This technique was used as a quick analysis of the relative binding affinities
and stoichiometries of Spo0F metal binding as a function of ion concentration and pH. The
overall goal of these experiments was to identify and group Spo0F metal binding trends in
terms of the identity of the metal. Metal concentration-basedµESI-MS experiments reveal three groups of metal binding trends for Spo0F (Table 2.1). Raw profiles for the Mg2+, Ca2+, Mn2+ and Cu2+ titrations are shown in Figure 2.1.
The monovalent K+ and trivalent Mn3+ metals were found to not specifically bind to Spo0F to any significant degree. This result was not surprising since response regulators
0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100
13800 14000 14200 14400 14600 14800 15000 15200 13800 14000 14200 14400 14600 14800 15000 15200 13800 14000 14200 14400 14600 14800 15000 15200 13800 14000 14200 14400 14600 14800 15000 15200
0µM Mg2+ 125µM Mg2+ 250µM Mg2+ 375µM Mg2+
apo apo apo apo
1Mg2+ 1Mg2+
1Mg2+
2Mg2+
a b c d
* * * * 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100
13800 14000 14200 14400 14600 14800 15000 15200 13800 14000 14200 14400 14600 14800 15000 15200 13800 14000 14200 14400 14600 14800 15000 15200 13800 14000 14200 14400 14600 14800 15000 15200
0µM Ca2+ 125µM Ca2+ 250µM Ca2+ 375µM Ca2+
apo apo apo apo
1Ca2+
1Ca2+
1Ca2+
2Ca2+ 2Ca2+
e f g h
* * * * 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100
13800 14000 14200 14400 14600 14800 15000 15200 13800 14000 14200 14400 14600 14800 15000 15200 13800 14000 14200 14400 14600 14800 15000 15200 13800 14000 14200 14400 14600 14800 15000 15200
0µM Cu2+ 125µM Cu2+ 250µM Cu2+ 375µM Cu2+
apo apo apo
apo 1Cu2+
2Cu2+
3Cu2+ 1Cu2+
m n o p
* * * * 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100
13800 14000 14200 14400 14600 14800 15000 15200 13800 14000 14200 14400 14600 14800 15000 15200 13800 14000 14200 14400 14600 14800 15000 15200 13800 14000 14200 14400 14600 14800 15000 15200
0µM Mn2+ 125µM Mn2+ 250µM Mn2+ 375µM Mn2+
apo apo apo apo
1Mn2+
1Mn2+ 2Mn2+
i j k l
*
* * *
Relative Ion Abundance
Relative Ion Abundance
Relative Ion Abundance
Relative Ion Abundance
MR MR MR MR
MR MR MR MR
MR MR MR MR
MR MR MR MR
cavanagh_fig5
Figure 2.1: Spo0F-metal complexes analyzed usingµESI-MS. Metal concentration-based titrations of Spo0F (6µM) in the presence of increasing concentrations of (a-d) Mg2+, (e-h)
Ca2+, (i-l) Mn2+ and (m-p) Cu2+. The majority of protein complexes observed are from
15N-Spo0F, while low relative ion abundance peaks are observed for 14N-Spo0F, which are
