Protein Engineering, Vol. 12, No. 11, 895-897,
November 1999
© 1999 Oxford University Press
Short Communication |
Homology modeling of the multicopper oxidase Fet3 gives new insights in the mechanism of iron transport in yeast
Dip. Scienze Biochimiche `A.Rossi-Fanelli' and Centro di Biologia Molecolare del CNR, Università di Roma `La Sapienza', P.le A. Moro 5, 00185 Roma, Italy and 2 Dip. Biologia, Terza Università di Roma, V.le Marconi 446, 00146 Roma, Italy
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Abstract |
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Fet3, the multicopper oxidase of yeast, oxidizes extracellular ferrous iron which is then transported into the cell through the permease Ftr1. A three-dimensional model structure of Fet3 has been derived by homology modeling. Fet3 consists of three cupredoxin domains joined by a trinuclear copper cluster which is connected to the blue copper site located in the third domain. Close to this site, which is the primary electron acceptor from the substrate, residues for a potential iron binding site could be identified. The surface disposition of negatively charged residues suggests that Fet3 can translocate Fe3+ to the permease Ftr1 through a pathway under electrostatic guidance.
Keywords: cupredoxin/ferroxidase activity/Fet3/homology modeling
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Iron homeostasis in eukaryotes as distantly related as yeast and humans is controlled by multiprotein systems which include a multicopper oxidase with ferroxidase activity, such as Fet3 and ceruloplasmin (Askwith and Kaplan, 1998a
). The molecular details of iron handling by these two enzymes and the mechanism of translocation to the actual iron transporter(s) are still unclear, although in yeast the iron permease Ftr1 has been identified and the metabolic links with Fet3 have been outlined (Stearman et al., 1996).
Multicopper oxidases couple the one-electron oxidation of substrate(s) to full reduction of molecular oxygen to water by employing a functional unit formed by three types of copper binding sites with different spectroscopic and functional properties (Solomon et al., 1996). Type 1 blue copper (T1) is the primary electron acceptor from the substrate, while a trinuclear cluster formed by type 2 copper and binuclear type 3 copper (T2/T3) is the oxygen binding and reduction site. Fet3 shows high sequence similarity to laccase and ascorbate oxidase, but is endowed with ferroxidase activity as ceruloplasmin. This strengthens the importance of Fet3 as a model system for the study of the mechanisms underlying the functional role of ceruloplasmin in iron homeostasis. An experimentally determined structure for Fet3 is not currently available; however, the high structural similarity displayed even at low sequence identity by the cupredoxin fold, the Greek key ß-barrel of multicopper oxidases (Murphy et al., 1997), prompted the calculation of a three-dimensional homology model for Fet3 using the ascorbate oxidase monomer as template, in an attempt to shed light on the structural determinants required for ferroxidase activity.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Comparative modeling utilized Homology, Modeller and Discover in the InsightII suite (InsightII User Guide 1997, MSI San Diego, CA). Model consistency was assessed with ProsaII (Sippl, 1993
) and Profile_3D (Lüthy et al., 1992). Procheck (Laskowsky et al., 1993) and restrained energy minimization were used for portions of the model which required manual refinement. Multiple and pairwise sequence alignments between the structural template ascorbate oxidase (PDB code 1AOZ) and Saccharomyces cerevisiae Fet3 (Swissprot code fet3_yeast) and its homologues were calculated with Clustal W (Higgins and Sharp, 1989) and GAP (GCG package), respectively. The initial sequence alignment between 1AOZ and fet3_yeast was iteratively modified to optimize the ProsaII energy profile of the corresponding model obtained by Modeller until no further improvement could be achieved. The final alignment accounts for 29% sequence identity between 1AOZ and fet3_yeast. The electrostatic potential field around the protein was modeled with the program DelPhi and displayed with GRASP (Nicholls et al., 1991). Secondary structure prediction was performed through the PHD server (Rost and Sander, 1994). Model coordinates are available upon request to the authors. |
Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Figure 1
shows the sequence alignment and the superposition of the C
traces of the Fet3 model and of the structural template ascorbate oxidase. The core three-domain structure of Fet3 includes residues 22–529 with each domain exhibiting the typical eight-stranded cupredoxin ß-barrel, leaving out the signal sequence (residues 1–21) which is removed from the mature protein, and a stretch of 30 amino acids before the putative transmembrane and cytoplasmic regions (residues 560–636). The copper sites are arranged as in the other three-domain multicopper oxidases. The T1 site is located in domain 3 toward the C-terminus with the canonical axial Met ligand replaced by L494 in a geometry typical of a high redox potential copper site (Solomon et al., 1996). The T2/T3 cluster lies at the interface between the first and last domain with His ligands supplied symmetrically by domains 1 and 3. Two His residues flank the type 1 copper Cys ligand within the motif His–Cys–His, forming the Y-shaped 13 Å-long pathway for intramolecular electron transfer from reduced T1 copper to the trinuclear site, which is an absolute requirement for the catalytic mechanism of the blue oxidases (Messerschmidt and Huber, 1990).
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An iron binding site, involving residues E272, E935, H940 and D1025, has been identified in the crystal structure of human ceruloplasmin near T1 copper in domain 6 (Lindley et al., 1997
). Two potential iron ligands, E185 and Y354, are found close to T1 copper of Fet3 in positions homologous to E272 and H940 of human ceruloplasmin. E185 and Y354 together with D409 could form the iron binding site of Fet3, comprising also the T1 ligand H489 (Figure 1B
). All these residues are found within conserved regions in the sequences of S.cerevisiae Fet5, C.albicans Fet3 and Schizosaccharomyces pombe Fio1. A similar arrangement of the substrate binding site is predicted also in ascorbate oxidase, where W163 and W362 (structurally homologous to E185 and Y354) and the T1 ligand H512 would be involved in L-ascorbate binding, according to docking experiments (Messerschmidt et al., 1992).
A constellation of negatively charged residues, highlighted in red in Figure 1B, suggests a possible pathway for Fe3+ translocation from Fet3 to the permease Ftr1. D278 and D279 would provide an electrostatic guidance for Fe3+ released from the active site toward the negatively charged region defined by D312, D315, D319 and D320. Analysis of the sequence of Ftr1 predicts the presence of an extracellular highly positively charged loop (residues 108–146) just before the transmembrane segment containing the REGLE motif essential for iron uptake (Stearman et al., 1996). The distribution of positively charged residues in this loop is such that it would guarantee the necessary electrostatic complementarity with the above-mentioned region of Fet3, if a complex were to form between the proteins.
This model explains how Fet3 organizes its extracellular catalytic domain and is compatible with experimental data reported so far for Fet3. The proximity of two polypeptide stretches (residues 164–178 and 306–321) to the T1 site accounts for the inhibitory effect of antibodies generated against them on the catalytic activity (De Silva et al., 1995). Two acidic residues predicted to be responsible for ferroxidase activity (Murphy et al., 1997) are buried (E227) or point outside the contact region (E230), easily explaining why their mutation was ineffective (Askwith and Kaplan, 1998b). The model offers a new framework for a de novo rational design of the site-directed mutagenesis and of other experimental approaches aimed at clarifying the mechanism of action of Fet3.
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Acknowledgments |
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This work was partially supported by MURST funds and by CNR grant 97.02338.12.
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Notes |
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1 To whom correspondence should be addressed
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Received June 9, 1999; revised July 28, 1999; accepted July 28, 1999.
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