{

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

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

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

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

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.

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…

…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

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)

•  Different  distances  between  the  two  54  residues  in  the  dimer-­‐‑dimer  interface

•  Distinct  surface  electrostatic  potential  around  residue  54

•  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

Amyloid  fibril  formation  and  stability  of  tetramer

•  54  is  at  the  opening  of  the  T4  binding  pocket

•  E54G  and  E54K  had  the  highest    and  lowest  T4  association and  dissociation  rates  respectively.

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  

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

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

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.

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>

{ Questions  and  Comments?

Thank  You  and  Happy  Cinqo  de  Mayo!!