Student Project 2 for UMass Chemistry 423 Spring 2015

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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

Ribbon diagram of the transthyretin (TTR) monomer with its beta-sheets

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Encoded by Human Transthyretin gene, transthyretin (TTR) is a protein composed of identical 127-aa sandwich subunits (shown in purple). Its main function is to transport retinol [1] and thyroxine (T4) [2] throughout the body. Interestingly, transthyretin’s name is coming from its function: transports thyroxine and retinol. 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 [3]. In order to transport T4, four TTR proteins must bind together to form a four-protein unit (homotetramer). In addition, TTR also carries retinol [4] (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 [5]. 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)[6] [7] .

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. [8]






















Overall Structure

Overall Structure of Human Transthyretin

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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[9]. 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 (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 and on the bottom of the cleft. There is a short (meshed for better spatial resolution), that is folded back relative to strand A, which is involved in the dimer–dimer contact[10].

A total of 13 buried water molecules are found per dimer. Five are located at the monomer–monomer . 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 at position 12 and 55. Several amyloidogenic mutations are linked to these residues, with Leu55Pro being concluded as the most aggressive [11].

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[12].




















Binding Interactions

3kgt, Ligand Protein Binding Interactions

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The inner sheets of two dimers (AB and CD) of TTR interface – strands A, D, G, and H – form , also known as T4 channel, with two binding sites, which we will be referring to as and . 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.

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 with Genistein hydroxyl group. The nonpolar residues of Leu17, Leu110, Lys15, and Ala108 stabilize ligand-protein binding through .

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 [13]. 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.






Additional Features

3kgt, 2 Chain Structure of Transthyretin

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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 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.

The binding and transport of thyroxine and retinol requires this tetramer. Retinol, the vitamin A alcohol, requires , RBP4 [14]. This is the RBP complex with . The are RBP complexed with Transthyretin. is bound with the RBP, clearly seen as non-yellow. Thyroxine bound to TTR is shown .



















Quiz Question 1

pdbcode, Insert caption here

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The substitution of a 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.





















Quiz Question 2

pdbcode, Insert caption here

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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[15]. What Kinetic value would you expect to change for this mutant from Wild Type TTR? ( bound to Genistein, polar groups in grey, non polar groups in purple.)






















See Also


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

  1. Monaco HL. The transthyretin-retinol-binding protein complex. Biochim Biophys Acta. 2000 Oct 18;1482(1-2):65-72. PMID:11058748
  2. PMID: PMC4126162
  3. Cookson EJ, Hall MR, Glover J. The transport of plasma thyroxine in white storks (Ciconia ciconia) and the association of high levels of plasma transthyretin (thyroxine-binding prealbumin) with moult. J Endocrinol. 1988 Apr;117(1):75-84. PMID:3128623
  4. Monaco HL. The transthyretin-retinol-binding protein complex. Biochim Biophys Acta. 2000 Oct 18;1482(1-2):65-72. PMID:11058748
  5. Koo EH, Lansbury PT Jr, Kelly JW. Amyloid diseases: abnormal protein aggregation in neurodegeneration. Proc Natl Acad Sci U S A. 1999 Aug 31;96(18):9989-90. PMID:10468546
  6. Connelly S, Choi S, Johnson SM, Kelly JW, Wilson IA. Structure-based design of kinetic stabilizers that ameliorate the transthyretin amyloidoses. Curr Opin Struct Biol. 2010 Feb;20(1):54-62. doi: 10.1016/j.sbi.2009.12.009. Epub, 2010 Feb 3. PMID:20133122 doi:http://dx.doi.org/10.1016/j.sbi.2009.12.009
  7. Gustavsson A, Jahr H, Tobiassen R, Jacobson DR, Sletten K, Westermark P. Amyloid fibril composition and transthyretin gene structure in senile systemic amyloidosis. Lab Invest. 1995 Nov;73(5):703-8. PMID:7474944
  8. Faria TQ, Almeida ZL, Cruz PF, Jesus CS, Castanheira P, Brito RM. A look into amyloid formation by transthyretin: aggregation pathway and a novel kinetic model. Phys Chem Chem Phys. 2015 Mar 4;17(11):7255-63. doi: 10.1039/c4cp04549a. PMID:25694367 doi:http://dx.doi.org/10.1039/c4cp04549a
  9. Foss TR, Wiseman RL, Kelly JW. The pathway by which the tetrameric protein transthyretin dissociates. Biochemistry. 2005 Nov 29;44(47):15525-33. PMID:16300401 doi:http://dx.doi.org/10.1021/bi051608t
  10. Haupt M, Blakeley MP, Fisher SJ, Mason SA, Cooper JB, Mitchell EP, Forsyth VT. Binding site asymmetry in human transthyretin: insights from a joint neutron and X-ray crystallographic analysis using perdeuterated protein. IUCrJ. 2014 Oct 21;1(Pt 6):429-38. doi: 10.1107/S2052252514021113. eCollection, 2014 Nov 1. PMID:25485123 doi:http://dx.doi.org/10.1107/S2052252514021113
  11. Sousa MM, Fernandes R, Palha JA, Taboada A, Vieira P, Saraiva MJ. Evidence for early cytotoxic aggregates in transgenic mice for human transthyretin Leu55Pro. Am J Pathol. 2002 Nov;161(5):1935-48. PMID:12414539 doi:http://dx.doi.org/10.1016/S0002-9440(10)64469-0
  12. Green NS, Foss TR, Kelly JW. Genistein, a natural product from soy, is a potent inhibitor of transthyretin amyloidosis. Proc Natl Acad Sci U S A. 2005 Oct 11;102(41):14545-50. Epub 2005 Sep 29. PMID:16195386 doi:http://dx.doi.org/10.1073/pnas.0501609102
  13. Trivella DB, Bleicher L, Palmieri Lde C, Wiggers HJ, Montanari CA, Kelly JW, Lima LM, Foguel D, Polikarpov I. Conformational differences between the wild type and V30M mutant transthyretin modulate its binding to genistein: implications to tetramer stability and ligand-binding. J Struct Biol. 2010 Jun;170(3):522-31. Epub 2010 Mar 6. PMID:20211733 doi:10.1016/j.jsb.2010.03.002
  14. Coward P, Conn M, Tang J, Xiong F, Menjares A, Reagan JD. Application of an allosteric model to describe the interactions among retinol binding protein 4, transthyretin, and small molecule retinol binding protein 4 ligands. Anal Biochem. 2009 Jan 15;384(2):312-20. doi: 10.1016/j.ab.2008.09.051. Epub 2008, Oct 12. PMID:18952041 doi:http://dx.doi.org/10.1016/j.ab.2008.09.051
  15. Trivella DB, Bleicher L, Palmieri Lde C, Wiggers HJ, Montanari CA, Kelly JW, Lima LM, Foguel D, Polikarpov I. Conformational differences between the wild type and V30M mutant transthyretin modulate its binding to genistein: implications to tetramer stability and ligand-binding. J Struct Biol. 2010 Jun;170(3):522-31. Epub 2010 Mar 6. PMID:20211733 doi:10.1016/j.jsb.2010.03.002

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