Structural study of human FKBP38 and its interaction with calmodulin by NMR and computational methods [Elektronische Ressource] / von Mitcheell Maestre Martínez

Sujets

• Biologie

Informations

• Publié par: martin-luther-universitat_halle-wittenberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 31
• Langue: English
• Poids de l'ouvrage: 15 Mo

Extrait

Structural study of human FKBP38
and its interaction with calmodulin by
NMR and computational methods





Dissertation



zur Erlangung des akademisches Grades
Doctor rerum naturalium (Dr. rer. nat.)


vorgelegt an der Naturwissenschaftlichen Fakultät I der
Martin-Luther-Universität Halle-Wittenberg



von
Mitcheell Maestre Martínez
Geb. am 29. April 1974 in Havanna, Kuba



Halle/Saale, Februar 2008




Gutachter: 1. PD. Dr. Christian Lücke
Max-Planck-Forschungsstelle für Enzymologie der Proteinfaltung,
Halle/Saale

2. Prof. Dr. Milton T. Stubbs
Martin-Luther-Universität Halle-Wittenberg

3. Prof. Dr. Thomas Peters
Universität zu Lübeck


Verteidigungsdatum: 13.02.2008
urn:nbn:de:gbv:3-000014742
[http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000014742]Index

1. Introduction ………………………………………………………….…………...……. 1
1.1. PPIases ……………………………………………...….….….…………………… 1
1.1.1. Human FKBPs ……………………………………………………………... 3
1.1.2. The human FKBP38 ……………………………………………………….. 4
1.1.3. Three-dimensional structures of FKBPs …………………………………… 6
1.1.3.1. The prototypic FKBP12 ………………………………………..……. 6
1.1.3.2. FKBPs with TPR domains: FKBP51, FKBP52 and AtFKBP42 …….. 8
1.2. Calcium and calcium-binding proteins ……………………………………………. 11
1.2.1. Calmodulin ……………………………………………………..………….. 12
1.2.2. Calmodulin binding to target proteins ………………………………..……. 14
1.3. Objectives ………………………………………………………………….……… 19

2. Materials and Methods ………………………………………………………………… 20
2.1. Materials …………………………………………………………………………... 20
2.1.1. Chemicals and materials …………………………………………………… 20
2.1.2. Enzymes …………………………. 21
2.1.3. Plasmids and templates …………………………………………………….. 21
2.1.4. PCR primers ……………………………………………………………….. 21
2.1.5. Escherichia coli cells ………………………………………………………. 22
2.1.6. Proteins and peptides ………………………………………………………. 22
2.1.7. Chromatography columns ………………………. 22
2.1.8. Standards …………………………………………………………………... 22
2.1.9. Kits …………………………. 22
2.1.10. Buffers, media and stock solutions ………………………………………… 22
2.1.11. Equipment ………………………………………………………………….. 23
2.2. Methods …………………………………………………………………………… 23
2.2.1. Molecular biology methods ………………………………………………... 23
2.2.1.1. Polymerase chain reaction …………………………………………… 23
2.2.1.2. Agarose gel electrophoresis ………………….. 24
2.2.1.3. DNA quantification ………………………………. 24
2.2.1.4. Enzymatic modification of DNA …………………………………….. 24
2.2.1.5. Plasmid mini-preparation ……………………………………………. 25
2.2.1.6. Transformation into competent Escherichia coli cells ………………. 25
I 2.2.1.7. Culturing of Escherichia coli cells …………………………………... 25
2.2.2. Preparative methods ……………………………………………………….. 25
2.2.2.1. Overexpression tests …………………………………………………. 25
35-1532.2.2.2. Expression of recombinant FKBP38 …………………………… 26
2.2.2.3. Lysis of Escherichia coli cells ……………………………………….. 26
35-1532.2.2.4. Purification of FKBP38 ………………………………………… 26
2.2.3. Analytical methods ………………………………………………………… 26
2.2.3.1. SDS-PAGE …………………………………………………………... 26
2.2.3.2. Protein quantification ………………………………………………... 27
2.2.3.3. Internet-based programs ………………………….. 27
2.2.4. NMR spectroscopy ………………………………………………………… 27
35-153
2.2.4.1. Structural study of FKBP38 …………………………………….. 27
2.2.4.1.1. Sample preparation …………………………………………….. 27
2.2.4.1.2. NMR experiments ……………………………………………… 28
2.2.4.1.3. Resonance assignment …………………………………………. 28
2.2.4.1.4. Structure calculation and refinement …………….. 29
35-153 2+ 2+2.2.4.2. Study of the interactions of FKBP38 with Ca and Mg ……... 30
2.2.4.3. Study of the interactions between FKBP38 and CaM ……………….. 30
2.2.4.3.1. Resonance assignments of apo-CaM, holo-CaM and
290-313FKBP38 ……………………………………………………... 30
2.2.4.3.2. Chemical shift perturbation experiments ………………………. 31
2.2.4.3.3. Docking calculations …………………………………………... 32
2.2.5. Molecular dynamics simulations …………………………………………... 34
35-1532.2.6. Crystal structure analysis of FKBP38 ………………………………… 36

3. Results and discussion ………………………………………………………………… 38
1 15 35-1533.1. NMR assignment of the H and N resonances of FKBP38 ………………... 38
35-153
3.2. Three-dimensional structure of FKBP38 ……………………………………. 42
35-153 2+ 2+
3.3. Interaction of FKBP38 with Ca and Mg …………………………………. 47
35-1533.4. Interaction of FKBP38 with calmodulin …………………………………….. 52
3.4.1. NMR assignment of the backbone amide resonances of apo- and holo-
calmodulin …………………………………………………………………… 52
35-1533.4.2. Interaction of FKBP38 with apo-calmodulin ………………………… 57
35-153
3.4.3. Interaction of FKBP38 with holo-calmodulin ………………………... 62
35-153
3.4.4. Comparison of the interactions of FKBP38 with apo- and holo-
IIcalmodulin …………………………………………………………………… 64
35-153
3.4.5. Three-dimensional structures of the FKBP38 /CaM complexes ……… 66
35-1533.4.5.1. Three-dimensional structure of the FKBP38 /apo-CaM complex 66
35-1533.4.5.2. Three-dimensional structure of the FKBP38 /holo-CaM complex 70
35-
3.4.5.3. Comparison of the three-dimensional structures of the FKBP38
153/CaM complexes ……………………………………………………... 74
290-3133.5. Interaction of FKBP38 with holo-calmodulin ……………………………… 75
290-313
3.5.1. Three-dimensional structure of the FKBP38 /holo-CaM complex …... 79
3.6. Three-dimensional models of the overall FKBP38/CaM complexes …………….. 82
3.6.1. Comparison of the overall FKBP38/CaM complexes with other known
CaM complexes ……………………………………………………………… 84

4. Summary ………………………………………………………………………………. 86

5. References ……………………………………………………………………………... 89

6. Figure Index …………………………………………………………………………… 99

7. Abbreviations ………………………………………………………………………….. 103

8. Appendix ………………………………………… 105
8.1. Acknowledgments ………………………………………………………………... 105
8.2. Publications ……………………………………………………………………….. 106
8.3. Curriculum vitae …………………………………. 107

Eidesstattliche Erklärung ………………………………………………………………….. 108



III1. Introduction

The human FK506-binding protein 38 (FKBP38) is a constitutively inactive peptidyl prolyl
2+cis/trans isomerase (PPIase) that is activated by calmodulin (CaM) and calcium (Ca ).
Furthermore, this protein plays a key role in Bcl-2 related apoptotic pathways (Edlich et al.,
2005; Edlich et al., 2006). Because of all these singular properties, the molecular structure of
FKBP38 and the characterization of its interaction with CaM are of major interest.

1.1. PPIases

Proteins play a fundamental role in virtually every biological process, displaying a multitude of
functions such as the catalysis of biochemical reactions, the transmission of biological messages
in signal transduction pathways, and the trafficking of a wide variety of chemical substances
across cell membranes. All proteins are synthesized in the ribosome as linear polypeptide chains.
In order to become biologically active, the polypeptide chain has to fold into a unique native
three-dimensional structure. Moreover, the failure of proteins to fold correctly and efficiently is
associated with the malfunction of biological systems. A variety of diseases such as cystic
fibrosis and Alzheimer’s disease are the result of protein misfolding (Chaudhuri and Paul, 2006;
Cohen and Kelly, 2003).

Although the information for correct folding is encoded by the amino acid sequence for most
proteins (Anfinsen, 1973), living organisms are additionally equipped with an efficient folding
machinery, consisting of chaperones (Bukau et al., 2006; Hartl, 1996), protein disulfide
isomerases (Ellgaard and Ruddock, 2005) and peptide bond isomerases (Fischer, 1994; Fischer
and Aumüller, 2003).

Peptide bond isomerases assist the cis/trans interconversion of peptide bonds, which possess
partial double bond character due to the delocalization of the lone electron pair of the nitrogen
atom across the entire amide group. Peptide bonds therefore can only adopt two planar
conformations (cis or trans), which interconvert slowly in comparison with the other torsion
angles that define the protein conformation. There are two classes of peptide bond isomerases: (i)
the secondary amide peptide bond isomerases (APIases) (Schiene-Fischer et al., 2002) and (ii)
the major class of the peptidyl prolyl cis/trans isomerases (PPIases, Enzyme class 5.2.1.8.)
(Fischer et al., 1989) which assists the interconversion of peptide bonds where proline is in the
C-terminal position.

1 PPIases are ubiquitous in life. The subfamilies of this enzyme class (i) are unrelated to each other
in their amino acid sequences, (ii) have distinct substrate specificities, and (iii) prove to be
sensitive to different inhibitors. A classification of these enzymes according to their ligand-
specificity and sequence similarities allows the identification of three PPIases families: the
FK506-binding proteins (FKBPs), the cyclophilins (Cyps) and the parvulins. The members of the
first two families are characterized by their ability to bind the low-