View source for Student Project 2 for UMass Chemistry 423 Spring 2015

=='''Human Transthyretin (TTR) complexed with genistein '''==
by Arash Manafirad, Mahdiyeh Yazdani, Allison Coutu, Jack Caudwell, Christopher Borcoche, Thanh Nguyen, Sonny Nguyen

[[Student Projects for UMass Chemistry 423 Spring 2015]]

==Introduction==
<Structure load='3kgt' size='300' frame='true' align='right' caption='Ribbon diagram of the transthyretin (TTR) monomer with its beta-sheets' />


Encoded by Human Transthyretin gene, transthyretin (TTR) is a protein composed of identical 127-aa <scene name='48/483883/Betasheets/1'>Betasheet</scene> sandwich subunits (shown in purple). Its main function is to transport retinol <ref>PMID: 11058748 </ref>  and thyroxine (T4) <ref>PMID: PMC4126162 </ref>  throughout the body. Interestingly, transthyretin’s name is coming from its function: '''trans'''ports '''thy'''roxine and '''retin'''ol. Mainly, TTR is produced by the liver, although it is also produced in smaller amounts in the choroid plexus and retinal pigment epithelium. The concentration of TTR in human plasma and cerebrospinal fluid is 0.2-0.3 mg mL-1 and 0.02 mg mL-1 respectively.

T4 is one of two major hormones produced by the thyroid gland which help control the regulation of metabolism and thus the rate at which the body uses energy. Along with two other proteins (thyroxine-binding globulin and albumin), TTR is responsible for carrying T4 in the bloodstream <ref>PMID: 3128623</ref>. In order to transport T4, four TTR proteins must bind together to form a four-protein unit (homotetramer). In addition, TTR also carries retinol <ref>PMID: 11058748 </ref>  (one of the major forms of vitamin A) in the blood. In this case, retinol-binding proteins (RBP) should bind to TTR (in its tetramer form).

Inappropriate folding in proteins cause a disease named amyloidosis <ref>PMID: 10468546</ref>. Amyloids (misfolded proteins) become insoluble, lose their normal function and deposit in different organs and tissues. TTR is one of the proteins that can unfold and aggregate into amyloid ﬁbrils. TTR amyloidoses include central nervous system selective amyloidoses (CNSA), familial amyloid cardiomyopathy (FAC), familial amyloid polyneuropathy (FAP), and senile systemic amyloidosis (SSA)<ref>PMID: 20133122</ref> <ref>PMID: 7474944</ref> .

In SSA fibrils are derived from wild-type TTR which cause deposition of amyloid in the heart leading to congestive heart failure. FAP, FAC and CNSA are caused by different TTR variants. These include deposition of amyloid fibrils in peripheral nerves, heart, and leptomeninges and subarachnoid vessels, respectively. It is also worthwhile to mention that different mutations of TTR have been found which many of them are amyloidogenic. <ref>PMID: 25694367</ref>


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==Overall Structure==
<Structure load='3kgt' size='300' frame='true' align='right' caption='Overall Structure of Human Transthyretin' scene='Sandbox_Reserved_430/>
Human transthyretin (TTR) is a 55 kDa homotetramer (or more precisely, a dimer of dimers).  The monomer contains a sandwich-like tertiary structure with two four-stranded β-sheets<ref>PMID:16300401</ref>. The monomer–monomer interface is defined by six backbone hydrogen bonds. The intermolecular contacts formed by the dimer–dimer interface result in the formation of a spacious channel (40 A ̊ long). The dimer–dimer contact is mediated by only eight backbone hydrogen bonds. The <scene name='48/483883/Channel/1'>channel</scene> (with the all 4 ''Ser'' residues shown as dotted form) is about 10 A ̊ wide at the outer rim and narrows in the centre to about 4 A ̊ . This narrowing is defined by the alignment of <scene name='48/483883/Ser117a/1'>Ser117A</scene> and <scene name='48/483883/Ser117b/1'>Ser117B</scene> on the bottom of the cleft. There is a short <scene name='48/483883/Small_b-sheet/1'>β􏰆-strand</scene> (meshed for better spatial resolution), that is folded back relative to strand '''A''', which is involved in the dimer–dimer contact<ref>PMID:25485123</ref>.

A total of 13 buried water molecules are found per dimer. Five are located at the monomer–monomer <scene name='48/483883/Interface/1'>interface</scene>. Four more are located in each of the monomers. One water molecule is situated at the junction of strands A and D, making hydrogen bonds to oxygen of these <scene name='48/483883/Water-trap/1'>Leu residues </scene> at position 12 and 55. Several amyloidogenic mutations are linked to these residues, with Leu55Pro being concluded as the most aggressive <ref>PMID:12414539 </ref>.

As noted, The main function of tranthyretin is to transport retinol and thyroxine throughout the body. To transport retinol, transthyretin must form a tetramer and then bind to retinol binding patch.  Even tough both polar and nonpolar interactions are involved in this binding event. However, several hydrophobic residues such as Val20, Leu17, Val121, Leu110 and Thr119 involved in hydrophobic contacts that further stabilize the tetramer. Furthermore, the substitution of a hydrophilic for a hydrophobic side chains in the regions of contact can cause a decrease or even loss in retinol-binding affinity.  This reveals the importance of hydrophobic interactions and the high degree of complementarity between the binding of retinol-binding protein and transthyretin<ref>PMID:16195386</ref>.
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==Binding Interactions==

<Structure load='3kgt' size='300' frame='true' align='right' caption='3kgt, Ligand Protein Binding Interactions' scene='' />
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The inner sheets of two dimers (AB and CD) of TTR interface – strands A, D, G, and H – form <scene name='48/483883/Pocket/1'>a central hydrophobic pocket</scene>, also known as T4 channel, with two binding sites, which we will be referring to as <scene name='48/483883/Ac/1'>AC</scene> and <scene name='48/483883/Bd-green/2'>BD</scene>. T4 channel binding sites are governed by negative cooperativity, in which the binding of ligand to one site reduces the ligand binding affinity of the other. In fact, genistein molecule bind BD sites with higher affinity in compare to that of AC sites, making BD sites the high affinity binding sites and AC the low affinity binding sites.
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There are two type of protein-ligand binding involves in this ligand-protein complex including hydrogen bond and hydrophobic interaction . Genistein is positioned in the manner that its phenyl group is buried within the hydrophobic pocket and its hydroxyl group is accessible for hydrogen bonding. The side chain residues of Lys15 and Ser17 located at the entrance and bottom of the binding sites, are  <scene name='48/483883/Hydrogen_bonding/7'>hydrogen bond</scene> with Genistein hydroxyl group.  The nonpolar residues of Leu17, Leu110, Lys15, and Ala108 stabilize ligand-protein binding through <scene name='48/483883/Hydrophobic/2'>hydrophobic interaction</scene>. 
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At site BD, genistein bond tightly with residues Lys15 and Ser117 side chains. Within the two hydrogen bonding residues, LYS15 can also hydrogen bond to water molecules at the entrance of the BD channel <ref>PMID:20211733</ref>. This acts to further increase the stability of the ligand-protein surface. At the second binding site, AC, one of the SER117 side chains, is turned away from the ligand, in the direction of the BD site. This will weakened the ligand: protein binding in the AC site. From the given information, the LYS15 and SER117 forms two important bonding between the ligand and protein.

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==Additional Features==
<Structure load='3kgt' size='300' frame='true' align='right' caption='3kgt, 2 Chain Structure of Transthyretin' scene='Insert optional scene name here' />
This starting model is Transthyretin complexed with Genistein. The binding of substrate to Transthyretin requires four TTR proteins to be bound to each other simultaneously. This <scene name='48/483883/Homotetramer_of_ttr/1'>complex</scene> is the tetramer of TTR. As mentioned earlier, it is a homo-tetramer, this is because each of the four protein constituents are identical. The Transthyretin units are yellow, pink, green, and grey.
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The binding and transport of thyroxine and retinol requires this tetramer. Retinol, the vitamin A alcohol, requires <scene name='48/483883/Human_retinol-binding_protein4/1'>Human Retinol-Binding Protein 4</scene>, RBP4 <ref>PMID:18952041</ref>. This is the RBP complex with <scene name='48/483883/Retinol-binding_protein/1'>retinol</scene>. The <scene name='48/483883/Homotetramer_of_ttr/1'>magenta and cyan units</scene> are RBP complexed with Transthyretin. <scene name='48/483883/Homotetramer_of_ttr/2'>Retinoic acid</scene> is bound with the RBP, clearly seen as non-yellow. Thyroxine bound to TTR is shown <scene name='48/483883/Ttr_complex_with_thyroxine/1'>here</scene>.
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==Quiz Question 1==
<Structure load='3kgt' size='300' frame='true' align='right' caption='pdbcode, Insert caption here' scene='Insert optional scene name here' />The substitution of a <scene name='48/483883/Quiz_1/5'>transthyretin</scene> hydrophobic side chain (maroon) for a hydrophilic side chain (blue) that is in the contact region between retinol binding protein and transthyretin results in:

A.  An increase in binding affinity and increase in hydrophobic interactions.  

B.  A decrease or even complete loss of binding affinity. 

C.  No change in affinity, both polar and nonpolar interactions bind retinol-binding protein and transthyretin.

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==Quiz Question 2==
<Structure load='3kgt' size='300' frame='true' align='right' caption='pdbcode, Insert caption here' scene='Insert optional scene name here' /> Genistein is a molecule extracted from Soy. Genistein binding to TTR has shown to stabilize the tetramer.

a) How could a A108Q point mutation affect the binding of Genistein?


b) The V30M point mutation of TTR has shown to affect it's binding affinity to Genistein<ref>PMID:20211733</ref>. What Kinetic value would you expect to change for this mutant from Wild Type TTR? (<scene name='48/483883/Ttr/2'>TTR</scene> bound to Genistein, polar groups in grey, non polar groups in purple.)


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==See Also==
*[[1dvq]]
*[[1dvx]]
*[[1bmz]]
*[[4pm1]]
*[[1bm7]]
*[[1e4h]]
*[[3cft]]



==Credits==

Introduction - Mahdieh Yazdani

Overall Structure - Arash Manafirad

Drug Binding Site - Sonny Nguyen, Thanh Nguyen

Additional Features - Christopher Borcoche

Quiz Question 1 - Allison Coutu

Quiz Question 2 - Jack Caudwell

==References==
<references/>