private study only. The thesis may not be reproduced elsewhere without
the permission of the Author.
•
and anion substituted human lactoferrin
A thesis presented in partial fulfllment of the requirements for the degree of Doctor of Philosophy in Chemistry
at Massey University
Clyde Alexander Smith
1992
Abstract
Lactoferrin, isolated from human colostrum, is an 80 kDa glycoprotein capable of binding two ferric ions concomitantly with two carbonate anions. The protein consists of a single polypeptide chain folded into two similar halves, each of which bind one iron and one carbonate. It is a member of the transferrin family of proteins, which includes serum transferrin, an iron transport protein found in the blood, ovotransferrin from avian and reptilian egg-white and melanotransferrin expressed by human melanoma cells. These proteins have important roles in the transport of iron around the body and the iron-binding function of lactoferrin has implications for the health of breast-fed infants.
Two lactoferrin complexes with copper substituted for both iron atoms and the other substituted with copper and an oxalate anion for one of the carbonate anions, have been prepared and crystallised, and the structures of both determined by X-ray crystallography to
2.1
A
resolution. Whereas in diferric lactoferrin, both anions are coordinated in a bidentate fashion to the two iron atoms, in the dicupric complex the carbonate in the N-terminal site is monodentate, resulting in a 5-coordinate copper geometry with an elongated apical ligand(:;2.7
A).
In the second complex, oxalate replaces carbonate in the C-terminal half only.Analysis of the structure indicates that the extent of closure of the lobes has an important role in determining the stereochemistry at the metal binding site.
In addition to the structural studies, the binding of a variety of other metal ions, including other transition metals, aluminium, the lanthanides and thorium have been investigated spectroscopically. All of these metal ions form 2:1 complexes with lactoferrin but with varying degrees of stability. Some binding constants for a number of the lanthanides have been estimated, Irrstly to provide a comparison with results obtained for serum transferrin and secondly to investigate the relationship between the size of the metal ion and the strength of binding. In the course of the investigation of the binding of the lanthanide ions, it was established that cerium interacts with lactoferrin to give a complex which slowly developes a brown colour after standing for a few days. This can be attributed to the slow oxidation of Ce(lll) to Ce(lV).
These results, when compared with earlier structural analyses on lactoferrin, show that the protein is very flexible and can accommodate other metals without altering it's overall
structure to any great extent. The predicted binding of other metal ions, including vanadium and the lanthanides, is described based on a combination of the crystallographic and
physicochemical studies.
Acknowledgemen ts
I wish to thank my supervisors, Ted Baker and Andrew Brodie for their advise, assistance and encouragement throughout the course of this work. I would also like to thank Bryan Anderson for his help in data collection and with the computing, Heather Baker for her help with protein purification and some of the spectroscopic work, and the other members of the Crystallography Group (past and present). Ted and Heather must also be thanked for teaching us how to put up wallpaper.
I should also like to thank Eric Ainscough and Gill Norris for their valued contributions and helpful discussions, and Catherine Day for critically reading some of this work.
Thanks are also due to the following:
Dr. Peter Lindley for supplying the coordinates of diferric rabbit serum transferrin and the iron-saturated N-lobe half-molecule of rabbit serum transferrin. I would also like to thank him for arranging a visit to his laboratory in 1991.
Drs. Derek Knighton and Jeff Plowman at the Dairy Research Institute for allowing me to use the CD instrument, and for their generous help with running the spectra.
All the mothers who kindly donated breastmilk for use in this study. Thanks also to the staff at the Palmerston North, Wanganui, Otaki and Dannevirke hospitals for arranging for the collection of the samples.
Massey University and the Chemistry and Biochemistry Department for the award of a Graduate Assistantship and the position of Assistant Lecturer.
And last, but certainly not least my family for putting up with me and my work for the past four years. Special thanks to Claire for her support and encouragement, and for helping with the fmal stages of this thesis.
Table of contents
Abstract
Acknowledgements Table of contents List of Figures List of Tables List of Plates Abbreviations
Chapter 1:
Chapter 2:
Chapter 3 :
Chapter 4:
Chapter 5:
A review of the Literature
1 . 1 Historical and Functional aspects of the transferrins 1 .2 Physical and Structural characteristics of the transferrins 1 .3 Concluding remarks
Introduction
2. 1 The interaction of metals with the transferrins 2.2 The interaction of anions with the transferrins 2.3 Questions to be addressed by this thesis
Crystallographic studies
3 . 1 Experimental proceedures
3.2 Results A: Dicupric-dicarbonato-lactoferrin 3.3 Results B: Dicupric-carbonato-oxalato-lactoferrin 3 .4 Discussion
Spectroscopic Studies 4. 1 Introduction
4.2 Experimental proceedures
4.3 Results A: Transition metals and aluminium 4.4 Discussion A: Transition metals
4.5 Results B: i-block metals 4.6 Discussion B: i-block metals 4.7 Concluding remarks
Conclusions 5. 1 Metal binding 5.2 Anion binding
page
11
iv v vii x
xu Xlll
1 4 24
25 27 33
39 67 123 146
1 64 174 1 89 204 217 232 244
246 249
References
Appendices
5.3 C032- or HC03 -; implications for the mechanism of metal binding and release
5.4 Additional metal-anion-Iactoferrin complexes 5.5 Implications for future research
I Metal and anion binding II X -ray data processing
III Structure refinement statistics
N Hydrogen bonding in the helices, �-sheets and turns in dicupric lactoferrin
V Hydrogen bonding at the metal and anion sites in CU2Lf
..
•
252 255 255
258
280 285 294 3 1 6 328
Figyre 1.1
1.2 1.3 1.4
1.5 1.6 1.7 1.8 2.1 3.1 3.2 3.3 3.4
3.5 3.6 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14 3.15
3.16 3.17 3.18 3.19 3.20 3.21 3.22
List of figures
Amino acid sequences of human serum transferrin, chicken ovotransferrin and human lactoferrin.
Ca plot of diferric lactoferrin.
Schematic representation of the polypeptide chain in diferric lactoferrin Folding patterns of diferric lactoferrin (N-Iobe) and the sulphate binding protein (SBP)
The N-Iobe iron and carbonate binding sites of diferric lactoferrin.
The N-Iobes of diferric lactoferrin and apolactoferrin.
Four possible conformations of a single lobe of lactoferrin.
A possible evolutionary scheme for the transferrins.
The interlocking sites model and the structure of a general synergistic anion Electronic absorption spectrum of CU20xLf and CU2Lf.
Percentage of unique reflections as a function of resolution for the diffractometer data sets of CU2Lf and CU20xLf
The percentage of unique reflections in each resolution shell in the final data set for CU2Lf.
The percentage of unique reflections in each resolution shell in the [mal data set for CU20xLf
The course of the PROLSQ refinement of CU2Lf.
The course of the PROLSQ refinement of Cu2oxLf.
en plot of dicupric lactoferrin.
Luzzati plot of resolution against the R factor for the final model of CU2Lf.
Real space correlation coefficient against residue number for CU2Lf.
Ramachandran plot of the <I> and 'II angles for CU2Lf.
Schematic representation of the secondary structure elements in CU2Lf.
The classical y-turns at residues 299 and 642.
Average mainchain B against residue number for CU2Lf.
Wilson plot for CU2Lf.
Correlation of mainchain B with nns deviations in mainchain atoms between CU2Lf and Fe2Lf.
Solvent B values as a function of the distance from the copper atoms Common solvent B values against difference in position.
The solvent structure in the N- and C-Iobe binding clefts of CU2Lf.
The first 2Fo-Fc electron density in the N-and C-Iobe binding sites of CU2Lf.
Coordination geometry in the N- and C-Iobe binding sites of CU2Lf.
The hydrogen bonding interactions of Arg210 and Lys564.
nns deviation in Ca positions against residue number.
Page
6 11 12
13 14 18 19 24 31 42 53 55
56 61 65 67 68 69 72 74 83 84 85
88 92 96 97 102 106 110 116
3.23 Luzzati plot for the final model of CU20xLf. 123 3.24 Correlation of B values with rms deviations between CU20xLf and Fe2Lf,
and between CU20xLf and CU2Lf. 124
3.25 Ramachandran plot of the mainchain torsion angles for CU20xLf. 126 3.26 Average mainchain B against residue number for Cu20xLf. 127 3.27 The fIrst 2F o-F c electron density in the N- and C-Iobe sites of CU20xLf. 132 3.28 Coordination geometry in the N- and C-Iobe binding sites of CU20xLf. 133
3.29 Modes of carbonate coordination. 150
3.30 Modelling a lanthanide ion in the N-Iobe of CU2Lf. 155
3.31 Modes of oxalate coordination. 160
4.1 Visible absorption spectra of transition metal complexes of lactoferrin. 165
4.2 ESR spectrum of diferric lactoferrin. 167
4.3 ESR spectrum of dicupric serum transferrin. 167
4.4 ESR spectrum of divanadyl(IV) serum transferrin. 168
4.5 ESR spectrum of gadolinium(Ill) serum transferrin. 169 4.6 Fluorescence titrations curves of lactoferrin and transferrin with Fe3+ 170
4.7 Iron binding scheme for serum transferrin 172
4.8 Elution profile of apolactoferrin from a Sephadex CM-50 column 178
4.9 Preparation scheme for CU2Lf and CU20xLf 184
4.10 Electronic absorption spectra of Fe2U 189
4.11 Electronic absorption spectra of CU2Lf and CU20xLf 190 4.12 UV difference spectra of apoLf upon the addition of Fe3+, Cu2+, V (Ill),
V(IV) and V (V) 191
4.13 Titration curves for Fe, Cu, V(m) and V(IV) addition to apoLf 192
4.14 Titration cw;ves for V(V) addition to apoLf 193
4.15 UV difference spectrum of aluminium lactoferrin 193
4.16 Titration curves for A13+ and AI(NT A) addition to apoLf 194 4.17 Fluorescence titration curves for Fe3+ and Cu2+ addition to apoLf 194 4.18 Fluorescence titration curves for Fe3+ addition to apoTf and apoMTf 195
4.19 CD spectrum of apoLf 197
4.20 CD spectrum of Fe2Lf 197
4.21 CD spectrum of CU2Lf 198
4.22 CD spectrum of Mn2U 198
4.23 CD spectrum of C02Lf 198
4.24 Electronic absorption difference spectrum of CU2Lf 199 4.25 a: ESR specta of (V02+hLf under argon and exposed to air 200 4.25 b & c: ESR specta of V(Ill)-Lf under argon and exposed to air 201
4.26 ESR specta of CU2Lf and CU20xLf 203
4.27 4.28 4.29 4.30 4.30 4.31 4.32 4.33 4.34 4.35 4.36 4.37
4.38 4.39 4.40 4.41 4.42 4.43 4.44 4.45 4.46 4.47 4.48
Crystal field diagrams for Cu2+
N- and C-Iobe copper coordination geometries
Molecular energy transitions involved in tryptophan fluorescence
a & b: Modelling of V02+ in the N-Iobe binding site of Fe2Lf
c & d: Modelling of v()2+ in the N-Iobe binding site of CU2Lf
Electronic absorption spectrum of Ce(IIIhLf and Ce(IVhLf pH mediated cerium release from Ce(IVhLf
U V difference spectra of apoLf upon addition of Pr3+ and Gd3+
Titration curves for Pr3+ and Gd3+ addition to apoLf Fluorescence titration curves for Ce(III) addition to apoLf UV difference spectra of apoLf upon addition of Eu3+ and Yb3+
Titration curves for La3+, Nd3+, Sm3+, Eu3+, Tb3+, Ho3+ and Yb3+
addition to apoLf
LogK2 as a function of the ionic radius for several Ln3+-Lf complexes Titration curves for Ce(lll) addition to apoLf
205 207 2 1 1 2 15 2 15 2 17 2 19 220 220 221 222
222 226 227
Titration curves for Th(IV) addition to apoLf 228
U V difference spectra for apoLf upon the addition of Nd(NT A)x and Yb(NT A)x 229 Plot of M: as a function of the NT A concentration 229 ESR spectrum of a 1: 1 gadolinium-lactoferrin complex 230 ESR spectra of gadolinium-Iactoferrin complexes with different ratios of Gd:Lf 231 Crystal field splitting diagram for Mn(Il) and Mn(III) complexes 233 Plot of M:l and .1Etot for various lanthanide complexes of lactoferrin 236
The structure of HBED and diMeHBED 237
LogK as a function of ionic radius for 4 chelating ligands and human lactoferrin 239
List of tables
Table Page
1.1 The occurence of lactoferrin in various human bodily secretions 2
1.2 The molecular masses of the transferrins 4
1.3 Primary structure relationships between some members of the transferrins 5 1.4 Selected metal binding fragments of the transferrins 7 1.5 Preliminary crystallographic data for several transferrins 10 1.6 Residues involved in metal and anion binding in the transferrins 15
2.1 Metal ions which bind to the transferrins 29
2.2 Synergistic anions 31
3.1 Cell dimensions for CU2Lf, CU20xLf and Fe2oxLf. 44
3.2 Diffractometer data collection for CU2Lf. 46
3.3 Diffractometer data collection for CU2oxLf. 46
3.4 Scale factors for CU2Lf. 50
3.5 Scale factors for CU2oxLf. 50
3.6 Diffractometer data collection summary for CU2Lf and CU2oxLf. 52 3.7 Photographic data collection summary for CU2Lf and CU2oxLf. 57 3.8 Statistics on final data sets for CU2Lf and CU2oxLf. 57 3.9 Geometrical constraints used in the refinement of CU2Lf and CU2oxLf. 59
3.10 Refmement statistics for CU2Lf and CU2oxLf. 64
3.11 Secondary structure in CU2Lf. 76
3.12 Hydrogen bonding interactions in CU2Lf. 79
3.13 A & B: Characteristics of the two 'Y-turns in human lactoferrin. 82 3.14 A & B: Temperature factors.for the flexible regions in Cu2Lf. 87 3.14 C: Correlation of average B values with rms deviation in mainchain atom
positions between CU2Lf and Fe2Lf. 89
3.15 A: Summary of the solvent structure in CU2Lf. 90
3.15 B: Solvent hydrogen bonding. 91
3.16 A: Common solvent molecules. 95
3.16 B: Hydrogen bonding involving common solvent molecules. 96 3.17 A & B: Bond distances and angles in the copper coordination sphere of CU2Lf. 108 3.18 Hydrogen bonding interactions in the vicinity of the metal and anion
sites in CU2Lf. 109
3.19 Superposition of CU2Lf on Fe2Lf. 113
3.20 A & B: Largest rms displacements in atom positions between CU2Lf and Fe2Lf. 115
3.21 Domain relationships between CU2Lf and Fe2Lf. 118
3.22 A: Average rms displacements in Cn and mainchain atomic positions between
CU2Lf and Fe2Lf in the metal and anion binding sites. 120 3.22 B: rms differences in the positions of the metal and anion binding ligand. 121
3.23 Temperature factors for CU20xLf 126 3.24 Bond distances in the copper coordination sphere of CU20xLf. 134 3.25 Bond angles in the copper coordination sphere of CU20xLf. 134 3.26 Hydrogen bonding interactions in the metal and anion sites in CU20xLf. 137
3.27 Domain relationships between CU20xLf and Fe2Lf. 140
3.28 Domain relationships between CU20xLf and CU2Lf. 141
3.29 Differences between atom positions in the binding sites of
CU20xLf and CU2Lf 142
4.1 Visible absorption maxima for some metallo-transferrin complexes 165 4.2 Characteristics of the CD spectra of metallo-Iactoferrin complexes 196 4.3 Distances of the tryptophan residues from the metal atoms in CU2Lf and Fe2Lf 209 4.4 Relative fluorescence of some metal-Iactoferrin complexes 211 4.5 Electronic absorption spectral data for coloured metallo-Iactoferrin complexes 218 4.6 Spectrophotmetric titrations of human lactoferrin with various metals 224 4.7 Data derived from binding experiments of selected lanthanides to lactoferrin 226 4.6 Characteristics of the ESR spectrum of 1: 1 gadolinium lactoferrin 231
List of Plates
Plate �
3.1 Crystals of CU2Lf and CU20xLf. 40
3.2 Zero-layer precession photographs of the hkO and Old reciprocal lattice
zones for CU2Lf and CU20xLf. 43
3.3 Richardson-type schematic representations of the polypeptide chains of
the N- and C-lobes of CU2Lf. 75
3.4 Superposition of the N-lobe of CU2Lf onto the C-lobe of CU2Lf. 75
3.5 Representative regions of electron density. 81
3.6 Solvent in CU2Lf and some water molecules in their electron density. 94 3.7 Stereo views of the 2F o-F e electron density associated with the glycan
chains in the N- and C-lobes of CU2Lf. 100
3.8 Stereo views of the 2F o-F e electron density in the N-lobe binding sites of CU2Lf. 104 3.9 Stereo views of the 2F o-F e electron density in the C-lobe binding sites of CU2Lf. 105 3.10 The hydrogen bonding patterns around the bicarbonate anions in the N-
and C-lobe sites of CU2Lf.
3.1 1 Fe2Lf - CU2Lf difference electron density map (F(Fe2Lt) - F(CU2Lt). 112 3. 12 Superposition of the N- and C-lobes of CU2Lf onto the corresponding
lobes of Fe2Lf. 114
3. 13 Superposition of the N- and C-lobe metal binding sites of Cu2Lf onto the
corresponding sites in Fe2Lf 119
3. 14 Stereo views of the 2Fo-Fe electron density associated with the glycan
chains in the N- and C-lobes of CU20xLf. 130
3.15 CU20xLf - CU2Lf difference electron density map (F(CU2oxLf) - F(CU2Lf)
in the C-lobe binding site only 130
3. 16 Stereo views of the 2Fo-Fe electron density in the N-lobe binding sites
of CU20xLf. 135
3. 17 Stereo views of the 2Fo-Fe electron density in the C-lobe binding sites
Of CU20xLf. 136
3.18 The hydrogen bonding patterns around the bicarbonate anions in the N-
and C-lobe sites of CU20xLf. 138
3. 19 CU20xLf - Fe2Lf difference electron density maps. 139 3.20 Superposition of CU2Lf and CU20xLf on to Fe2Lf - metal binding sites. 143 3.21 Superposition of Arg465 of CU20xLf on to Arg465 of CU2Lf. 144 3.22 Superposition of Fe2Lf onto CU2Lf - the metal and anion sites. 153
Ab breviations
lactoferrin Tf serum transferrin
ovotransferrin MTf melanotransferrin
Lf OTf
mLf
bLfdOTf bTf msTf rat Tf
bovine lactoferrin apoLf metal-free human lactoferrin mouse lactoferrin cOTf chicken ovotransferrin duck ovotransferrin hTf human transferrin bovine transferrin rTf rabbit transferrin Tobacco Homworm transferrin XTf Xenopus transferrin rat serum transferrin rTiN N-Iobe of rabbit transferrin LfN isolated N-Iobe of human lactoferrin (from recombinant DNA)
apoTf metal-free human serum transferrin Fe2Lf diferric human lactoferrin apobTf metal-free bovine transferrin apoOTf metal-free hen ovotransferrin
diferric human transferrin diferric bovine transferrin CU2 Tf dicupric serum transferrin
Cil20xLf dicupric-( oxalato-carbonato) lactoferrin
diferric hen ovotransferrin diferric rabbit transferrin dicupric lactoferrin
Mn2Lf dimanganese(ID) lactoferrin C02Lf dicobalt(ill) lactoferrin Ce(llIhLf dicerium(ill) lactoferrin Ce(IVhLf dicerium(IV) lactoferrin EDTA ethylenediaminetetraacetic acid EDDA ethylenediaminediacetic acid NTA nitrilotriacetic acid
J3
-meJ3
-mercaptoethanold.d. water distilled deionised water SDS sodium dodecylsulphate UV Nis electronic absorption spectroscopy ESR electron spin resonance XANES X-ray-absorption near-edge-structure CD circular dichroism UV Ultraviolet
EXAFS extended-X -ray-absorption-fine-structure ESEEM electron spin echo envelope modulation
