Frataxin
ContentsFunction and Structural highlightsGeneral Aspects Frataxin (FXN) is a protein capable of storing, releasing and detoxifying intracellular iron. In humans, a mutation in this protein can trigger the Friedreich's ataxia, a neurodegenerative disease caused due to incapacity to form iron-sulfur groups necessary to activate the mitochondrial enzyme involved in the electron transportation chain, aconitase. It consists of a polymeric molecule that, although capable of forming larger complexes (as the 24 subunit oligomer detected by electron microscopy), exerts its activity by association of three subunits, enough to form a central channel where the ferroxidation takes place. In the following paragraphs, we describe the general features of its structure in the trimeric form as obtained by X-ray crystallography at 3Å resolution. The protein used was obtained from the Y37A yeast, which has a 40% sequence identity to the human frataxin. In the box at the right, it is possible to see its general structure in a space-fill model, in which violet, orange and light-green represent, each, a different monomer from the entire molecule. However, to cover some important aspects of the structure and function of the molecule, it is particularly useful to represent its secondary structure patterns. CyaY is a bacterial frataxin ortholog which participates in iron-sulfur assembly as an iron-dependent inhibitor of cluster formation[1]. The yeast frataxin homolog FH1 plays a role in maturation of iron-sulfur proteins[2]. General Secondary and Tertiary Structure Patterns The secondary structure is basically composed of two alpha-helices (in red) and a short helical segment (in magenta), and seven antiparallel β-sheets, in each monomer, represented here as six green setae and one short region colored yellow. Turns and coils are also represented. You can also view the monomer isolated here. From the N-terminus to the C-terminus (blue represents the N-terminus, while red the C-terminus), in the monomer, the short helical segment called alpha-helix 1 goes from Leu 68 to Gln 63, alpha-helix 2 ranges from Glu 93 to Glu 76, while alpha-helix 3 goes from Leu 171 to Ser 158 (in yellow). The sequences comprising each beta-sheet (in purple) are the following: Pro 100-Ser 105 for beta 1, Val 108-Ile 113 for beta 2, Gln 124-Gly 117 for beta 3, Ser 134-Gln 129 for beta 4, Asp 143-Gly 138 for beta 5, Trp 149-Leu 152 for beta 6 and the short region between Lys 157-Gly 155 for beta 7. Concerning the tertiary structure, each subunit has a folding pattern called α/β sandwich, in which two alpha-helices are packed against five strands of anti-parallel β-sheets. Click here to see it in the whole trimeric form.
Stabilization of the Trimeric Quaternary StructureThe trimeric structure of frataxin consists of the association of three monomers, and is mainly stabilized by the N-terminal extensions of each subunit, shown in yellow. These consist of loops, with a short helical N-terminal segment (alpha-helix 1; recall its secondary structure) highly flexible in the monomer solution, but interestingly, when in the trimeric arrangement, they play a crucial role in maintaining it. Viewing the other side of the molecule, we can notice how the N-terminal extensions, still in yellow, interact with the adjacent monomer. Taking a closer look, it is possible figure out how the N-terminal loop of the first monomer, here described as chain A, is placed with respect to chain B. Exploring even further the details, it is possible to see some residues close enough to interact. The names associated with their positions can be seen by clicking here. Click here to rotate this region. We can color the residues differently according to their hydrophilicity. In this new color scheme, polar residues are represented in pink while the hydrophobic ones appear gray. Now we are about to color all those relevant residues to specify their interactions. The package of hydrophobic residues can be seen. Here, Pro 62, Val 65 and Leu 68, shown in dark-blue, are packed against the polar uncharged aminoacids Thr 110 and Thr 118, in red (other amino acids are shown in turquoise). This interaction among the hydrophobic residues contributes to the maneintance of the loop configuration of the N-terminal region at its extremity. Another important interaction is the hydrogen bond formed between Glu 64 and Thr 118. Those are the only residues able to form hydrogen bond, since the distance separating them is within a range of approximately 3 Å (or 0.3 nm). In this image, pay special attention it the role of the carbonyl oxygen of Glu 64 involved in the hydrogen bonding. In this color scheme, carbons are grey, oxygens are red and nitrogens are blue. Now, we can devote our attention to examine what occurs at the base of the N-terminal region.Those are the residues involved in relevant interactions that contribute to the stabilization of the trimeric form. Those are their specific names (different colors of the labels simply indicates different subunits). Click here to give emphasis on them, and here to get a better spatial notion of its arrangement. If we color according to their polarities (recall: pink for charged aminoacids, and grey for aliphatic ones), it becomes evident their charged nature. Them, there is no hydrophobic packing taking place at this region. Instead, there are hydrogen bonds as the main eletrostatic interaction. Notice, again, the element composition of each amino acid: in this color scheme, again, we have C, O, and N. There are six hydrogen-bonding pairs contributing to the stabilization of the molecule. Between Lys 72 and His 74, His 74 and Glu 76 and Glu 75 and Asp 78, the pair is always formed between the carbonyls of the first and the amide groups of the second. The other type of hydrogen bonding occurs between the side chains of the amino acids involved in the pairs His 74 and Asp 79, Asp 78 and Lys 123 and Glu76 and Arg141. In any of each ways, there is always an oxygen and a nitrogen involved.
The Role of the ChannelThe channel of the trimer is formed at its 3-fold axis, in a central position. It is the strure responsible for stabilizing the atom of iron for delivery, but also acts to further stabilise the trimeric form. Stabilizing one side of the channel, there are some interactions between the loops around the central channel, which form a helical structure maintained, for instance, through hydrogen bonds between the pairs Leu 152-Gly 147 and Asn 146-Gln 154. this last one also stabilizes the ASN146 interaction with the iron atom, as described in the figure below In the structure of the channel, the side chains of the hydrophobic amino acids Leu 145, Val 150 and Leu 152 are exposed to the solvent, creating a hydrophobic lid around the channel. This hydrophobic lid isolates one of the sides of the channel core, as well as providing a hydrophobic contact surface by which other proteins can interact, hiding their hydrophobic residues from the solvent-rich environment. Other structure in the central channel of high importance is the loop formed by 125-128. This loop, stabilized by the hydrogen bonds between Asp 143-Gln 129, 127-75 and 129-126, is present in 2 different conformations. The one just described is involved in further stabilizing the Fe atom within the channel. The metal ion binds at around 4 Å from the side chains of the three Asp 143 residues (distances between residues are shown for reference). Laboratory data from X-ray crystallography suggests the Fe 2+ ion being associated with solvent molecules. In this conformation the PRO126 in pointing toward the center of the channel, further reducing contact of the iron with the environment. This create an isolation chamber for the iron inside the channel, increasing the stability for the transportation of iron.
the other conformation that the 125-128 loop can take is were it fold outside of the channel, exhibiting interactions with the residues N127-Q129-D143. in this conformations, the hydrogens interactions between N127-E75 and Q129-P126 are not present.
in the image below, it is shown the prevalence of negatively charged residues exposed (in red) in detriment of positively charged (blue)
This indicates the high metal-affinity that this conformation presents, probably to load the protein with iron. DiseaseFriedreich ataxia[3] is developed when the frataxin trimer cannot stabilize itself to perform the ferroxidase activity, with is probably dependent on conformational changes that take place when the monomers come together, being in a site around the residues H74, D78, D79, D82 and H83. There are 4 know mutations capable of creating such defects: G107V, that breaks the hydrogen bond between G107 and K123, destabilizing the region of 123-130; W131, necessary to stabilize the N-terminal extension; R141C breaks an hydrogen bound between R141-P125, which may affect the conformation of the 125-128 loop and D122Y 3D Structures of frataxin
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References
KARLBERG, Tobias et al. The structures of frataxin oligomers reveal the mechanism for the delivery and detoxification of iron. Structure, v. 14, n. 10, p. 1535-1546, 2006.
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