Sandbox Reserved 431

Introduction

Rickets is a disease caused by a vitamin D deficiency. Vitamin D can be obtained from ultra violet radiation and from various food sources. Cytochrome P450 enzymes are involved in the first step to regulate and process vitamin D in the human body.

Overall Structure

Cytochrome P450 exists as an . Each dimeric unit contains 12 α-helices (labeled A-L) along with β-sheets, which are mostly located on one side of the molecule. Helices F and G from each of the two units form the dimeric interface of cytochrome P450, and are also involved in the formation of the active site. This dimeric interface of the protein is stabilized by between the G helix of one the units with the F helix of the second unit, and vice versa. Two molecules of 2-hydroxypropyl-β-cyclodextrin, which is used to dissolve vitamin D3, are also found near the dimer interface.

Binding Interactions

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 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. 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.

Additional Features

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.

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

• Cytochrome P450
• Drug Metabolism by CYP450 Enzymes
• 3w0y
• 3w0c

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