1. {
Role of the Glutamic Acid 54
Residue in Transthyretin
Stability and
Thyroxine Binding
Masanori Miyata et. al
Reuben Sequeira
University Of Kansas
May 5, 2010

2. Presentation Overview
o Background on Transthyretin
• Mutations causing Amyloidosis
• Significance in disease study
o Purpose of this research
o Methods and techniques used
• X-­‐‑ray crystallography
• 3D transthyretin models
o Results
o Discussion
• Take home message from this research

3. Familial Amyloid Neuropathy
o Onset of disease during ages 20 – 40
o Hereditary and autosomal dominant disease→ need only
one mutation in one gene to acquire FAP
o Disease observed worldwide but first detected in
Portugal.
o E54K mutation prevalent in cases within Japan and E54G
common in the UK (1)
o Amyloid deposits in various tissues results in multiple
neuropathy

4. Transthyretin a.k.a TTR
o Transports thyroxine and retinol
o 55kDa homotetramer (127 residue monomers). Gene located on chromosome
18
o Synthesized in the liver
o Maintains normal level of retinol, Vitamin A binding protein and thyroid
hormone in blood plasma

5. Essence of this Research
o By studying the E54G and E54K mutations and their TTR products, a
comparison can be made with wt TTR and hence the importance of the
Glu54 residue in TTR.

6. Methods and Approach
Expression and Purification
• E54G/E54K plasmids prepared using site-­‐‑directed mutagenesis
• Anion-­‐‑exchange chromatography and reverse-­‐‑phase HPLC
CD (Circular dichroism) TTR stability assay
• Incubated in 0-­‐‑8M Urea at 25˚C for 96h
Tetramer-­‐‑monomer transition Assay
• 100mM KCl induced at pH 3.5-­‐‑7
Another TTR stability assay based on T4 binding
Thioflavin T binding assay after incubation with KCl
And…

7. …X-­‐‑Ray Crystallography and
Structure Determination
o Crystal formation 10days after incubation with 200 mM citrate
buffer and 3 M ammonium sulfate pH 5.3
• Steps in crystallographic study
o Crystallization
o X-­‐‑ray diffraction pahern and raw data collection
o Refinement of data
o Compute electron density map
o Build and refine 3D model

9. Results and Findings
• wt TTR and E54G TTR have similar fractions of monomers/tetramers at
indicated pH
• E54K TTR has significantly more monomer formation than wt/E54G
• stability based on urea denaturation→ wild-­‐‑type TTR (Cm=3.54)>E54G
(Cm=3.05)>E54K (Cm=2.58)

10. • Different distances
between the two 54
residues in the dimer-­‐‑
dimer interface
• Distinct surface
electrostatic potential
around residue 54

11. • Glu54 forms a hydrogen
bond with Lys15 and
His56, stabilizing
positive charge of Lys15
• In E54G, a water
molecule mediates
interaction between S52
and His56
• In E54K, H-­‐‑bond is
broken and K15/K54
causes repulsion

12. Amyloid fibril formation and stability of tetramer

13. • 54 is at the opening of the T4 binding pocket
• E54G and E54K had the highest and lowest T4 association
and dissociation rates respectively.

14. Tetramer stability and Amyloid fibril
formation in the presence of T4
• wt TTR : 12.18 monomer ratio
• monomer level of E54G TTR was 34.43
• monomer level of E54K TTR was 92.18

15. What do these findings tell us??
• wild-­‐‑type and Glu54 TTR variants have significantly different
side chain interactions and surface electrostatic potential
around residue 54
• Glu54 modulates the positive charge of Lys15 and hence
minimizes the repulsion between K15 of the two monomers
which stabilizes the tetramer
• The change of residue 54 in the TTR variants affects T4 binding
and hence decreased T4 binding increases tetramer instability

16. Closing thoughts
• Various FAP associated mutations lead to TTR tetramer
instability and hence favoring amyloidogenises
• Could another residue besides Glu54 (wt) stabilize the
tetramer?
• T4 binding studies using these residues located in the
pocket, show that binding affinity is important to tetramer
stability
• Small molecules are in the process of being developed that
can appropriately bind to this pocket with high affinity and
decrease fibril formation

17. Acknowledgements
I would like to thank HaiYan Zhao and Dr. Tang for
crystallography expertise. I would also like to thank
Shyam Mehta for giving me useful tips for this
presentation.

18. References
• Miyata et. al. (2010) Role of the Glutamic Acid 54 Residue in Transthyretin
Stability and Thyroxine Binding. Biochemistry 2010, 49, 114–123.
• Ando, Y., Araki, S., and Ando, M. (1993) Transthyretin and familial
amyloidotic polyneuropathy. Intern. Med. 32, 920–922.
• Miroy, G. J., Lai, Z., Lashuel, H. A., Peterson, S. A., Strang, C., and Kelly, J.
W. (1996) Inhibiting transthyretin amyloid fibril formation via protein
stabilization. Proc. Natl. Acad. Sci. U.S.A. 93, 15051–15056.
• Miroy, G. J., Lai, Z., Lashuel, H. A., Peterson, S. A., Strang, C., and Kelly, J.
W. (1996) Inhibiting transthyretin amyloid fibril formation via protein
stabilization. Proc. Natl. Acad. Sci. U.S.A. 93, 15051–15056.
• <hhp://www.iupui.edu/~amyloid/information.htm>
• <hhp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=tfap>
• <hhp://ghr.nlm.nih.gov/condition=transthyretinamyloidosis>

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Questions and Comments?
Thank You and Happy
Cinqo de Mayo!!