Thạc Sĩ 3D domain swapping: Structural characterizations of domain-swapped dimer proteins FVE and rhodocetin

TABLE OF CONTENTS



CHAPTER 1: Introduction 1
3D DOMAIN SWAPPING 2
Background of 3D domain swapping 2
3D domain swapping definition 4
Helpful definitions 6
History of 3D domain swapping Diphtheria toxin 6
More than one domain swapping 7
Examples of 3D domain swapping 9
Single mutation induce 3D domain swapping 11
Design of 3D domain-swapped molecule 19
Human cystatin C dimerizes through 3D domain swapping 22
Hinge loop role in 3D domain swapping 26
PROTEIN X-RAY CRYSTALLOGRAPHY 28
Protein crystallization 29
Crystal systems and symmetry 29
X-Ray diffraction and Bragg's Law 30
Ewald construction 31
The structure factor 31
Fourier transform and phase problem 33
Model building 35
Refinement 35 AIM AND SCOPE OF THE THESIS 36
CHAPTER 2: Structural characterizations of fungal immunomodulatory
protein: Fve
37
MATERIALS AND METHODS 39
Protein purification 39
Protein crystallization and data collection 40
Structure solution and refinement 40
RESULTS AND DISCUSSION 43
Overall fold 43
Topology of FNIII fold in Fve 48
Determinants of the Ig-like fold 51
Structure-function relationships 56
Dimerization by 3D domain swapping 57
CHAPTER 3: Structural characterizations of venom of the Malayan pit
viper: Rhodocetin
67
MATERIALS AND METHODS 69
Protein purification 69
Protein crystallization and data collection 69
Structure solution and refinement 70
RESULTS AND DISCUSSION 72
Overall fold 72
Structure comparison with C-type lectin 76
Structure-function relationships 80 Dimerization by 3-D domain swapping 84
CHAPTER 4: Conclusion 88
Bibliography 93
Appendix 112

















SUMMARY

Fve, a major fruiting body protein from Flammulina velutipes, a mushroom possessing
immunomodulatory activity, stimulates lymphocyte mitogenesis, suppresses systemic
anaphylaxis reactions and edema, enhances transcription of IL-2, IFN-γ and TNF-α, and
hemagglutinates RBCs. It appears to be a lectin with specificity for complex cell surface
carbohydrates. Fve is a non-covalently linked homodimer containing no Cys, His and Met.
It shares sequence similarity only to the other Fungal Immunomodulatory Proteins (FIPs)
LZ-8, Gts, Vvo and Vvl, all of unknown structure. The 1.7 Å structure of Fve solved by
Single Anomalous Diffraction of NaBr-soaked crystals is novel: each monomer consists
of an N-terminal α-helix followed by a fibronectin III (FNIII) fold. The FNIII fold is the
first instance of “pseudo-h-type” topology – a transition between the seven β-stranded s-
type and the eight β-stranded h-type topologies. The structure suggests that dimerization,
critical for the activity of FIPs, occurs by 3-D domain swapping of the N-terminal helices
and is stabilized predominantly by hydrophobic interactions. The structure of Fve is the
first in this lectin family, and the first of an FNIII domain-containing protein of fungal
origin.
Rhodocetin is a unique heterodimer consisting of α and β subunits of 133 and 129
residues respectively. The molecule, purified from the crude venom of the Malayan pit
viper, Calloselasma rhodostoma, functions as an inhibitor of collagen induced platelet
aggregation. Rhodocetin has been shown to have activity only when present as a dimer.
The dimer is formed without an inter-subunit disulfide bridge as observed with all the other Ca
2+
- dependent lectin-like proteins (CLPs). The 1.9 Å resolution structure of
rhodocetin is determined by molecular replacement. The structure reveals the inter-
subunit interface which has compensatory interactions for forming the dimer in the
absence of the disulfide bridge. This is the first structure of a CLP without a disulfide
connecting the subunits and thus represents a novel molecule which can help to
understand a new set of protein-protein interactions. Further, unlike other CLPs,
rhodocetin does not require metal ions for its functional activity. However, like other
CLPs, rhodocetin also forms the heterodimer by domain swapping, in which the central
looped region is swapped.