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=='''Phosphatase Inhibitor complexes: pdb 1nny'''==
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=='''Vitamin D activation by cytochrome P450, Rickets (3c6g)<ref>PMID: 18511070 </ref>'''==
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<Structure load='1nny' size='500' frame='true' align='right' caption='PTP1B inhibitor' scene='Insert optional scene name here' />
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by Isabel Hand, Elizabeth Humble, Kati Johnson, Samantha Kriksceonaitis, and Matthew Tiller
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===Introduction===
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[[Student Projects for UMass Chemistry 423 Spring 2016]]
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<scene name='Sandbox_Reserved_431/Monomer/1'>Monomer</scene>
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Diabetes is a quickly growing disease that is affecting more and more people every day. Diabetes is caused when the body either does not produce enough insulin, or has a resistance to insulin. Insulin resistance occurs when an increase in the concentration of insulin in the cells leads to a decrease in the cell’s uptake of insulin.
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==Introduction==
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Rickets is a disease resulting from prolonged vitamin D deficiency. As vitamin D is vital for the absorption of phosphorus and calcium, a deficiency would cause weakening of bones. Rickets also causes muscle weakness, skeletal deformities, dental problems, inhibition of growth, and muscle spasms. In some cases, rickets can be inherited due to mutations in genes responsible for coding for human CYP2R1.
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When insulin binds to a normal insulin receptor, three specific tyrosine residues are phosphorylated and this serves as the first step in insulin signaling. The insulin receptor is bound to the cell by a beta strand that extends through the membrane into the interior of the cell. The insulin binds to the two different binding sites on the receptor and is used to move the glucose in the blood stream to different tissues in order to use as energy.
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In the human body, CYP2R1, a member of the cytochrome P450 family, is responsible for the first steps of the conversion of vitamin D into a bioavailable form within the liver. CYP2R1 is also known as Vitamin D 25-hydroxylase as it hydroxylates vitamin D3 into calcidiol, the bioavailable form of the vitamin, which would then be converted to calcitriol via the enzyme 25-hydroxyvitamin D3 1-alpha-hydroxylase, as seen in figure 1.
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In a person with type 2 diabetes, the cells are unable to uptake glucose due to decreased insulin receptor signaling. This decrease in signaling can be caused by the protein tyrosine phosphatase (PTP1B). PTP1B is responsible for the dephosphorylation of the insulin receptor, and therefore responsible for the down regulation of insulin signaling and the cause of diabetes.
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[[Image:Action_Of_CYP2R1.jpg]]
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As explained below in the section labelled 'Binding Interactions,' recent efforts have been made to develop new drugs that would inhibit the effects of the PTP1B protein. A small, potent and selective inhibitor was developed that competitively and reversibly binds to the binding sites on the insulin receptor and selectively inhibits PTP1B. This PTP1B inhibitor increases the half-life of the phosphorylated insulin receptor, which in turn enhances the effects of insulin in patients with type 2 diabetes.
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Fig. 1, the conversion of vitamin D3 into calcidiol via Vitamin D 25-hydroxylase
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<ref>Szczepankiewicz BG, Liu G, Hajduk PJ, Abad-Zapatero C, Pei Z, Xin Z, Lubben TH, Trevillyan JM, Stashko MA, Ballaron SJ, Liang H, Huang F, Hutchins CW, Fesik SW, Jirousek MR. Discovery of a potent, selective protein tyrosine phosphatase 1B inhibitor using a linked-fragment strategy. J Am Chem Soc. 2003 Apr 9;125(14):4087-96. PMID:12670229 doi:10.1021/ja0296733</ref>
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<scene name='48/483888/Human_p450/1'>Human P450 Cytochrome</scene> is shown with amino acids in teal and the heme center shown as a space filling model, with the nitrogen shown in blue, carbon in gray, oxygen in red, and the iron center shown in orange
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<StructureSection load='3c6g' size='350' side='right' caption='Structure of Human Cytochrome p450' scene=''>
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==Overall Structure==
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Cytochrome P450 is an <scene name='48/483888/Cytochrome_p450_dimer/1'>asymmetric dimer</scene>, which means the protein is made of two subunits that are structurally very similar to one another, but not identical. Each dimeric subunit contain 12 α-helices (labeled A-L) along with some β-sheets that are localized to one side of the molecule. Helices F and G from each of the units form the dimer interface of cytochrome P450, and are involved in the formation of the active site. This dimeric interface of the protein is stabilized by <scene name='48/483888/Cytochrome_p450_interface/1'>hydrogen bonding interactions</scene> between residues from the G helix of one the units with residues located on the the F helix of the second unit, and vice versa. Two molecules of 2-hydroxypropyl-β-cyclodextrin are also found near the dimer interface. The cyclodextrins are believed to help further stabilize the protein, and also shield the hydrophobic part of the F-G helix transition loop from the solvent by <scene name='48/483888/Cytochrome_p450_cyclodextrins/1'>encapsulating the Phe240 residue within its cavity</scene>. Cytochrome P450 has an apparent mass of ~120 kDa.
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==Binding Interactions==
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CYP2R1 binds vitamin D3 at an extended binding site that orients the bound molecule to bring its side chain close to the heme and allow for hydroxylation. The binding site is located at the channel between the G and I helices and the B' helix/B-C loop. The active site has non-polar residues which allows for nonpolar interactions with D3. In the <scene name='48/483888/Spacefilled_binding_site/1'>space filling representation </scene> you can see the residues that interact to bind Vitamin D3 and the channel between. Once bound, the D3 molecule is submerged into the protein, with only its 3-OH group showing.
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The B' helix is one of the substrate recognition sites and has a flexible C terminus which unwinds outward to allow entrance of the substrate into the active site channel. Due to the stabilizing interactions of B' with the F-G loop, binding of the substrate causes the protein to adopt a closed conformation which closes the access channel.
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<Structure load='1nny' size='500' frame='true' align='left' caption='PTP1B catalytic loop' scene='Insert optional scene name here' />
 
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<br><br>
 
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===Overall Structure===
 
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The secondary structure of the monomer includes 8 <font color='magenta'>alpha </font>
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==Additional Features==
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<scene name='Sandbox_Reserved_431/Alpha_helix/3'>helices</scene> and 10 beta strands, 8 <font color='deepskyblue'>beta strands</font> make up the main <scene name='Sandbox_Reserved_431/Beta_sheet/3'>beta sheet</scene>. The beta sheet adopts a highly twisted configuration.
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The <font color = 'mediumspringgreen'>C-terminal </font> <scene name='Sandbox_Reserved_431/C_terminus/1'>35 residues</scene> are predominantly hydrophobic in nature and function in targeting the enzyme to the cytoplasmic face of membranes of the endoplasmic reticulum (ER).
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A structural feature that is highly conserved among PTPs is the catalytic, or <font color = 'limegreen'>PTP </font> <scene name='Sandbox_Reserved_431/Catalytic_site/1'>loop</scene>. The phosphate recognition site is created from a loop that is located at the amino-terminus of an alpha helix. PTP loop is made up of the following residues (I/V)HCXAGXGR(S/T)G that includes the <scene name='Sandbox_Reserved_431/Catalytic_site/2'>active site</scene> <font color = 'red'>Cys215 </font>. <ref> PMID:12829250 </ref> Another conserved loop, the recognition loop, plays an important role in substrate recognition. The pTyr loop contains Tyr 46 (tyrosine), which defines the depth of the cleft and contributes to the specificity for phosphotyrosine-containing substrates. The <scene name='Sandbox_Reserved_431/Tyr_46_and_val_49/7'>residues</scene> <font color = 'yellow'> Tyr 46 </font> and <font color = 'darkorange'> Val 49 </font> assist the substrate's insertion into catalytic site.
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<scene name='Sandbox_Reserved_431/Ser216/1'>Ser</scene> <font color ='navy'> 216 </font> of the PTP loop forms a hydrogen bond with the the recognition loop, stabilizing the active site cleft. A third conserved loop is the <font color = 'blue'> WPD </font>
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<scene name='Sandbox_Reserved_431/Wpd_loop/1'>loop</scene>. For this specific inhibitor of the enzyme, once the substrate binds there is no conformational change in the structure of the loop.<sup>[1]</sup>
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For other inhibitors of this enzyme, once the substrate binds to the binding site, there is a conformational change in which the WPD loop closes around the side chain of the pTyr residue of the substrate. This causes Phe 282 to stack against the phenyl side chain of of the substrate pTyr, stabilizing the closed loop.<sup>[1]</sup> This conformation positions the <font color = 'teal'>Asp </font> <scene name='Sandbox_Reserved_431/Asp_181/2'>181</scene> to function as a general acid in the initial part of the phosphorylation transfer step.
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PTP1B represents an example of the concept of an induced fit, which means that the binding of the substrate induces a conformational change that creates a form of the enzyme that is catalytic.
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<br> <br><br><br>
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===Binding Interactions===
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Cytochrome P450 has a central iron-bound heme, which, combined with its structural conformation, allows for hydroxylation with the attached substrate. Cytochrome P450 has specific vitamin D 25-hydroxylase activity, which does not function properly when a person has rickets. Rickets is caused by a lack of sufficient vitamin D in their system, which is often caused by a vitamin D-25 hydroxylation defect. Leu99Pro is an evolutionarily conserved mutation in the beta helix which contributes to the hydroxylation defect. Leu99 does not inhibit substrate binding; however, Leu99Pro disturbs hydrogen binding around the heme and interferes with the helix steric properties, causing protein instability. When Leu99 does not have the proline mutation, its carboxyl group forms hydrogen bonds with Arg445, which are both located around the central heme and allows for hydroxylation of vitamin D.
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PTP1B is thought to primarily be responsible for the dephosphorylation of the insulin receptor and, therefore, acts to downregulate insulin signaling. Inhibiting PTP1B is linked to improved insulin response and activation of the insulin pathway. PTP1B deficient mice showed a resistance to diet induced diabetes.
 
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<Structure load='1nny' size='500' frame='true' align='right' caption='Enzyme active site' scene='Insert optional scene name here' />
 
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Overall the <scene name='Sandbox_Reserved_431/Working_ligand/1'>ligand</scene> is a competitive inhibitor which reversibly binds to two sites simultaneously and has a high selectivity for PTP1B over other phosphatases. This ligand was engineered by a research group from Abbot labs and the steps they took in its design revolve around many important binding interactions. The group designed the drug in <scene name='Sandbox_Reserved_431/Ligand_design/1'>three different stages</scene>. In order to identify this inhibiting ligand, the research group of interest first used a computer to scan a library of 10,000 organic compounds based on there NMR spectra, searching for those who have an affinity for the enzymes active site. The best match produced by the screen was diaryloxamic acid which the group then modified to <font color = 'dodgerblue'>naphthyloxamic acid</font> , reasoning that an inhibitor that took up more space would be a more potent inhibitor. When the group mixed this molecule with enzyme, NMR data showed a chemical shift in <scene name='Sandbox_Reserved_431/1st_binding/4'>Val49, Gly220, and Gly 218</scene>, part of the active site of the enzyme. The observed kinetics indicated competitive and reversible inhibition.
 
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After their initial success, the group then reasoned that since the active domain of PTP1B is somewhat conserved by many other phosphatases and extremely similar in some ( TCPTP), incorporation of binding to a second and nonactive site would dramatically increase selectivity. Based off of naphthyloxamic acid, the group then created what they called <font color = 'darkorange'>compound 12</font> hoping that it would satisfy these conditions. Mixing the new ligand and enzyme then screening with NMR N-15 and C-13 resonance yielded data showing that in addition to binding to Val49, Gly220, and Gly 218, the new ligand bound to additional active sites, those being <scene name='Sandbox_Reserved_431/Active_site_2/5'>Gln262 and Arg221</scene> The new ligand also forms hydrogen bonds to the backbone of residues <font color = 'darkorange'> Ser216-Gly220</font> as well as forming a hydrophobic interaction with Tyr46. The group improved the site two ligand creating <font color = 'magenta'>compound 23</font> compound 23, which primarily adds interactions with <scene name='Sandbox_Reserved_431/Active_site_2/6'>Met258, Arg24, and Arg254</scene>. It is not yet know whether <font color = 'magenta'> Arg 24, Arg 254,</font> or both residues are interacting with the ligand, but it is believed that they are either forming hydrogen bonds to the ligand or forming a salt bridge. The groups final ligand shows excellent potency for PTP1B while also showing high seletivity against other phosphatases.
 
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===Additional Features===
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<scene name='48/483888/Hemegroup/1'>Leu99 and Arg445</scene>
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<Structure load='1nny' size='500' frame='true' align='right' caption='Dephosphorylation reaction' scene='Insert optional scene name here' />
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</StructureSection>
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'''Dephosphorylation of the Tyrosine-Phosphate Residue'''
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==Quiz Question 1==
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A phosphorylated residue enters the active site of the protein, facilitated by the recognition loop residues <scene name='Sandbox_Reserved_431/Tyr_46_and_val_49/5'>Tyr 46 and Val 49</scene>. The base of the entrance through which it is brought is known as the PTP loop. The phosphotyrosine entering is amphipathic, thus requiring a non-polar pocket for its phenol ring while the polar end is positioned in the catalytic site. Once inside, the substrate causes a conformational change in the WPD loop, causing the substrate to be held in place for a nucleophilic attack. Also, the residue, <scene name='Sandbox_Reserved_431/Asp_181/1'>Asp 181</scene>, is shifted, so that it can act as an acid. The first step of the reaction involves Asp 181 attaching a hydrogen atom to the oxygen of tyrosine, thus neutralizing it and allowing it to diffuse from the site. Binding in the PTP loop occurs, so the connection between Arg 221 and the phosphate of the substrate is maximized. The WPD loop now has a very stable conformation, as binding has increased with multiple surrounding residues, <scene name='Sandbox_Reserved_431/Pro_180_trp_179_and_phe_182/2'>Pro 180, Trp 179, and Phe 182</scene>. The tyrosine residue is now positioned in close proximity to the <scene name='Sandbox_Reserved_431/Initial_view/2'>sulfur of Cys 215</scene>, allowing Cys 215 to remove the phosphate as an intermediate step in the reaction. Now, the phosphate binds to the <scene name='Sandbox_Reserved_431/Cys_215_and_asp_181/2'>Cys 215</scene>, forming the cysteinyl-phosphate intermediate. This intermediate will then be dephosphorylated again, creating a water phosphate complex. Afterwards, the enzyme will return to its resting conformation and will be able to accept another tyrosine for dephosphorylation.[3]
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1A) Why is Proline so poorly suited for inclusion in Alpha Helices?
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A) It cannot hydrogen bond due to the position of its amide
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B) The residue is incapable of forming the correct phi and psi angles in a helix
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C) The steric hindrance of its sidechain
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D) A and C
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E) All of the above
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===Credits===
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1B) This protein being a dimer, has symmetry between its two large sections, from <scene name='48/483888/Gandfhelices/1'>this orientation</scene> where most of the molecule has been cut away for simplicity, you can see where one half (in green) comes within close proximity of the other half (in blue). These 2 pairs of helices help hold the dimer together via electrostatic interactions. If the black residue is Arg, and the white residue is Asp, what is most likely to be on the opposite helix
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(Arg match, Asp match)
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A) Asp, His
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B) Gly, Val
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C) Met, Lys
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Introduction - Jill Carlson
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==See Also==
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*[[Cytochrome P450]]
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*[[Drug Metabolism by CYP450 Enzymes]]
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*[[3w0y]]
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*[[3w0c]]
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Overall Structure - Polina Berdnikova
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==Credits==
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Drug Binding Site - Brett Clinton
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Introduction - Sami Kriksceonaitis
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Additional Features - James Hamblin
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Overall Structure - Kati Johnson
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===References===
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Binding Interactions - Isabel Hand
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Additional Features - Elizabeth Humble
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Quiz Question 1 - Matthew Tiller
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==References==
<references/>
<references/>
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3. Edwards, K., T. Davis, D. Marcey, J. Kurihara, D. Yamamoto. 2001. Comparative Analysis of the Band 4.1/ezrin-related Protein Tyrosine Phosphatase Pez from Two Drosophila Species: Implication for Structure and Function. Gene 275: 195-205.
 

Current revision


This Sandbox is Reserved from January 19, 2016, through August 31, 2016 for use for Proteopedia Team Projects by the class Chemistry 423 Biochemistry for Chemists taught by Lynmarie K Thompson at University of Massachusetts Amherst, USA. This reservation includes Sandbox Reserved 425 through Sandbox Reserved 439.


Contents

Vitamin D activation by cytochrome P450, Rickets (3c6g)[1]

by Isabel Hand, Elizabeth Humble, Kati Johnson, Samantha Kriksceonaitis, and Matthew Tiller

Student Projects for UMass Chemistry 423 Spring 2016

Introduction

Rickets is a disease resulting from prolonged vitamin D deficiency. As vitamin D is vital for the absorption of phosphorus and calcium, a deficiency would cause weakening of bones. Rickets also causes muscle weakness, skeletal deformities, dental problems, inhibition of growth, and muscle spasms. In some cases, rickets can be inherited due to mutations in genes responsible for coding for human CYP2R1.

In the human body, CYP2R1, a member of the cytochrome P450 family, is responsible for the first steps of the conversion of vitamin D into a bioavailable form within the liver. CYP2R1 is also known as Vitamin D 25-hydroxylase as it hydroxylates vitamin D3 into calcidiol, the bioavailable form of the vitamin, which would then be converted to calcitriol via the enzyme 25-hydroxyvitamin D3 1-alpha-hydroxylase, as seen in figure 1.

Image:Action_Of_CYP2R1.jpg

Fig. 1, the conversion of vitamin D3 into calcidiol via Vitamin D 25-hydroxylase

is shown with amino acids in teal and the heme center shown as a space filling model, with the nitrogen shown in blue, carbon in gray, oxygen in red, and the iron center shown in orange

Structure of Human Cytochrome p450

Drag the structure with the mouse to rotate

Quiz Question 1

1A) Why is Proline so poorly suited for inclusion in Alpha Helices? 

    A) It cannot hydrogen bond due to the position of its amide
    B) The residue is incapable of forming the correct phi and psi angles in a helix
    C) The steric hindrance of its sidechain
    D) A and C
    E) All of the above

1B) This protein being a dimer, has symmetry between its two large sections, from where most of the molecule has been cut away for simplicity, you can see where one half (in green) comes within close proximity of the other half (in blue). These 2 pairs of helices help hold the dimer together via electrostatic interactions. If the black residue is Arg, and the white residue is Asp, what is most likely to be on the opposite helix 

       (Arg match, Asp match)
    A) Asp, His
    B) Gly, Val
    C) Met, Lys

See Also

Credits

Introduction - Sami Kriksceonaitis

Overall Structure - Kati Johnson

Binding Interactions - Isabel Hand

Additional Features - Elizabeth Humble

Quiz Question 1 - Matthew Tiller

References

  1. ↑ Strushkevich N, Usanov SA, Plotnikov AN, Jones G, Park HW. Structural analysis of CYP2R1 in complex with vitamin D3. J Mol Biol. 2008 Jun 27;380(1):95-106. Epub 2008 Apr 8. PMID:18511070 doi:10.1016/j.jmb.2008.03.065
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