1 Dipartimento di Biochimica e Biofisica, 2 Centro di Ricerca Interdipartimentale di Scienze Computazionali e Biotecnologiche, Seconda Università di Napoli, via Costantinopoli 16, 80138 Napoli and 3 Istituto di Scienze dell'Alimentazione, CNR, via Roma 52A/C, 83100 Avellino, Italy
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Abstract |
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Keywords: consensus of methods/eIF-5A/homology modelling/model validation/prediction strategy
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Introduction |
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We applied our prediction strategy to the human eukaryotic translation initiation factor 5A (eIF-5A), a 18-kDa protein, highly conserved from yeast to mammalian cells (Park et al., 1984; Gordon et al., 1987
). eIF-5A precursor [(ec-eIF-5A(lys)] is the only cellular protein known to contain a specific lysine residue which is transformed into the unique amino acid hypusine [N
-(4-amino-2-hydroxybutyl)-lysine] by a series of post-translational reactions: (i) the transfer of the butylamine moiety from spermidine to the
-amino group of one of the lysine residues in the eIF-5A precursor protein, thus forming peptide-bound deoxyhypusine (Park et al., 1982
); (ii) the intermediate hydroxylation at C-2 of the incoming 4-aminobutyl moiety to form hypusine (Abbruzzese et al., 1985
,1986
,1988a
,b
). eIF-5A promotes the formation of the first peptide bond during the initial stage of protein synthesis (Hersey, 1991). However, the actual in vivo function of eIF-5A is to date still only partially known. eIF-5A precursors which do not contain hypusine, have little, if any, activity (Park et al., 1991
). In addition, the lysine
arginine variant is unable to stimulate methionyl-puromycin synthesis in vitro (Hersey et al., 1990; Park et al., 1991
) and is inactive in vivo (Smit-McBride et al., 1989
; Beninati et al., 1995
). Therefore, hypusine synthesis is required for the biological activity of the protein. Moreover, the ec-eIF-5A(lys) modification is correlated with cell proliferation (Abbruzzese, 1988
; Abbruzzese et al., 1988a
,b
; Beninati et al., 1990
; Schnier et al., 1991
; Caraglia et al., 1997
), and agents that block the lysinehypusine transformation (Park et al., 1984
; Abbruzzese et al., 1989
,1991
; Jakus et al., 1993
) inhibit the growth of mammalian cells (Park et al., 1993
) inducing reversible arrest at the G1-S boundary of the cell cycle (Park et al., 1981
; Cooper et al., 1982
; Abbruzzese et al., 1986
, 1988; Park, 1987
; Lalande and Hanausske-Abel, 1990
; Beninati et al., 1990
,1993
). The polyamine-dependent modification of eIF-5A has been related to the triggering of apoptosis in tumour cells (Tome and Gerner, 1997
; Tome et al., 1997
). It has also been reported that eIF-5A can accumulate at nuclear pore-associated intranuclear filaments in mammalian cells (Rosorius et al., 1999
) and interacts with the general nuclear export receptor CRM1 being transported from the nucleus to the cytoplasm (Rosorius et al., 1999
). These findings open a new scenario in which eIF-5A may also function as a nucleocytoplasmic shuttle protein of mRNAs eventually correlated with cell proliferation and apoptosis.
The knowledge of the 3D structure of human eIF-5A can help in the understanding of its function and role in the cell, in order to study the molecular interaction with other proteins, as well as to design new molecules useful to modulate its activity. Different researchers investigated the structure of this human protein, both by experimental methods (Joao et al., 1995; Klier et al., 1995
; Stiuso et al., 1999
) and predictive approaches (Gerloff et al., 1998
). However, these works are still far from giving us a 3D model of the protein. Recently, the 3D structures of two eIF-5A from archea have been solved (Kim et al., 1998
; Peat et al., 1998
). Their sequence similarity to human eIF-5A is lower than 40%, so that the comparative modelling strategy must be applied with caution, it being possible to obtain a wrong model as a consequence of a wrong sequence alignment. For this reason we chose this protein to apply our predictive scheme. This work provides an example of combining different predictive methods and experimental results, in order to develop a new prediction strategy, inclusive of an evaluation of its reliability. Finally, the 3D model of the human eIF-5A represents an essential support to further studies on its structurefunction relationships.
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Methods |
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The sequence of human eIF-5A has been taken from the SWISSPROT databases (ID code IF5A_HUMAN). Structure prediction of human eIF-5A has been based on the availability of the 3D models of the homologous protein from Methanococcus jannaschii (Kim et al., 1998), PDB code 1EIF and from Pyrobaculum aerophilum (Peat et al., 1998
), PDB code 1BKB. The search for sequence similarity within databases has been performed with the BLAST program (Altschul et al., 1997
). The alignment of eIF-5A sequences has been performed by the PILEUP program of the GCG package and by CLUSTALW (Thompson et al., 1994
). Secondary structure was predicted with the SOPMA method (Geourjon and Deleage, 1994
,1995
) which considers the consensus of different prediction methods and does not perform multiple sequence alignment. This feature is important in our approach because we need to compare independent alignments of sequences and secondary structures. PHD (Rost and Sander, 1993
) and JPRED (Cuff et al., 1998
) were also used for further comparison. The secondary structure of 3D models has been assigned with the program DSSP (Kabsch and Sander, 1983
). The programs MODELLER (Sali and Blundell, 1992) and Quanta (Molecular Simulations, Inc., San Diego, CA) were used to build 10 full-atom models of human eIF-5A according to the comparative protein modelling method. The stereochemical quality of the models was verified with the program PROCHECK (Laskowski et al., 1993
), in order to select the best model. The search for structural classification of bacterial eIF-5A was performed on SCOP (Murzin et al., 1995
) and CATH (Orengo et al., 1997
) databases. The solvent accessibility of amino acids was evaluated by the program NACCESS (Hubbard et al., 1991
) calculating the atomic accessible surface defined by rolling a probe of 1.40 Å around the van der Waals surface of the protein model. Model figures were drawn with the InsightII package (Molecular Simulations, Inc.)
In vitro: structural investigation
Protein preparation
eIF-5A precursor(Lys) was prepared by over-expression of human eIF-5A-cDNA in Escherichia coli according to the method of Smit-McBride et al. (Smit-McBride et al., 1989). The protein is homogeneous when tested by SDSPAGE showing a single band.
Protein concentration
Evaluation of protein concentration was performed spectrophotometrically by using 275 = 4100 M1 cm1. This value was calculated by the content of tyrosine and phenylalanine residues by using molar extinction coefficients of 1250 and 200 M1 cm1, respectively (Wetlaufer, 1962
). This method has been found very reliable for this purpose with a maximum error of 5% (Gill and von Hippel, 1989
). The Bradford method was also used (Bradford, 1976
).
Cysteine titration
Sulfydryl group titration of native and denatured protein was performed by the 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) method (Ellman, 1959). A large excess of DTNB is added to the protein and the increase of absorbance is monitored spectrophotometrically by using
412 = 13 600 M1 cm1 for the DTNBsulfur adduct (Ellman, 1959
). To analyse the total content of sulfydryl groups, the protein sample was denatured in 5 M guanidinium chloride (GdnHCl) for 24 h. After the addition of DTNB, the absorbance at 412 nm increased quickly, showing a monophasic transition, up to a value corresponding to four DTNBsulfur adducts. A similar protocol has been applied to the native protein. The absorbance values have been reported as the number of cysteine residues titrated.
Spectral measurement CD measurements were carried out on a Jobin Yvon Mark III spectropolarimeter equipped with a temperature-controlled cell holder, in 0.05M Tris, pH7.8 containing 0.15MKCl. Mean residue ellipticities were calculated by:
[]
= (MRWqobs)/(10lc)where [
]
is the mean residue ellipticity (deg cm2/dmol) at a particular wavelength, qobs is the observed ellipticity, MRW is the mean residue molecular weight calculated from the sequence, l is the optical path length (cm) and c is the concentration (g/ml). Cells between 0.01 and 1 cm were used in the far-UV. CD spectra were measured by computer so as to have reliable tracings even in the case of very diluted protein solutions, in addition to eliminating artefacts and obtaining good blank correction. The CD spectra were analysed in the region between 200 and 250 nm to evaluate the amount of secondary structure. A computer program with different methods of analysis (Menéndez-Arias et al., 1988) was used.
The protein samples under spectroscopic investigation were always dialysed against numerous changes of buffer, which was used as a blank for the measurements. It was reported that the eIF-5A precursor has a tendency to dimerize reversibly (Chung et al., 1991). According to these authors, the low protein concentration used in our experiments is populated by the monomeric form (dissociated state) of the protein. In fact, no concentration dependence was detected under our experimental conditions.
Chemicals and solutions Spectroscopic grade GdnHCl was from Schwarz-Mann. All chemicals were reagent grade and were purchased from BDH (British Drug Houses, UK). In denaturation experiments the protein was added to buffered solutions of GdnHCl; 0.15 M KCl was present in all solutions. The spectroscopic measurements were then followed until an apparent equilibrium was reached. pH measurements were carried out by using combination electrodes with a Radiometer pH meter.
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Results |
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The model has been compared to experimental results in order to verify the reliability of the predicted structure.
Figure 5 shows the far-UV CD spectra of the native protein. The spectrum shows a well pronounced minimum centred at approximately 205 nm, and an evident shoulder at 215216 nm. This last spectral feature suggests the presence of ß-sheet structure which is characterized by a dichroic band with a minimum at 216 nm. The relatively low intensity of the dichroic band suggests the presence of a large amount of flexible organization for the peptide backbone. The CD spectrum has been analysed by computer program (Menendez-Arias et al., 1988
) in order to calculate the secondary structure content. An RMS value of 5 was calculated for the fit of the CD spectrum. The content of secondary structure is reported in Table II
for comparison with prediction results as well as with previously published CD results. A global agreement appears between experimental and prediction results. In the Discussion, the differences will be evaluated in more detail.
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Discussion |
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The secondary structure content of the human eIF-5A, detected by far-UV CD, suggests that the protein can be classified as an all-beta protein, as confirmed by the crystallographic models from archea and their classification in the CATH (Orengo et al., 1997) and SCOP databases (Murzin et al., 1995
). As is evident from data reported in Table II
, the comparison of experimental data show that differences of secondary structure contents exist between human eIF-5A (CD spectra) and the archea homologues (crystallographic data). The most evident differences are the lower content of beta structure and the higher content of unordered structure (other) in the human protein. The same trend appears between the predicted 3D model of the human protein and the 3D experimental models of archea proteins. This means that the archea structures, used as templates for the homology modelling, have been correctly modified in the modelling procedure, and the final model is in good agreement with the expected differences between the human and the archea proteins. The few differences in the percentage content of secondary structure between CD data and the model can be ascribed, at least in part, to the presence of an unordered segment in the N-terminal region of the human sequence, without correspondence in the template proteins, and therefore not folded by the homology modelling. Secondary structure predictions assign coil conformation to this segment, but it is possible that it partially assumes helical conformation, thus explaining the presence of an alpha helix detected by the CD spectrum, although to a very low extent. The secondary structure predictions, on the contrary, show higher differences to the experimental results, thus confirming that the homology modelling approach, applied in suitable conditions, represents the best method for protein structure prediction.
Comparisons of detailed structural features of the modelled protein to experimental results also confirms the reliability of the model. Cysteine titration by DTNB and labelling by fluorophore constitute a complex set of information. In fact, experimental results indicate that: (i) all four cysteines are titrated when the protein is in the presence of a high denaturant concentration; (ii) only one cysteine is quickly titrated when the protein is in native conditions; (iii) the other two cysteines are titrated after a longer incubation time; and (iv) the fourth cysteine is not titratable. The first cysteine titrated should be Cys72, as obtained by tryptic mapping data. Moreover, as previously published (Stiuso et al., 1999), the N-(iodoacetylaminoethyl)-5-naphtylamine-1-sulfonic acid (IAEDANS) labelling of cysteine side chains, followed by tryptic mapping, revealed that Cys72 is evidently marked and the spectral features of the labelled protein suggest that the IAEDANS bounded is in an apolar environment. These results can be explained by accurate evaluation of the cysteine environment in the predicted model. In Figure 4
the cysteine residues have been highlighted. Cys128 appears well exposed on the surface of the protein and the analysis of the model with specific software confirms that 69% of the amino acid surface is accessible to the solvent, whereas Cys72 has an intermediate exposure (9%). Cys21 and Cys37 are buried, their solvent accessibility being 0.8 and 0.4%, respectively. It could be expected that it can be much easier to titrate and label the most exposed Cys128 than Cys72. However, the apolar environment detected for the IAEDANS fluorophore seems not to be suitable for the high exposure of Cys128 because the fluorophore, after the labelling of such a side chain, might be very exposed to the solvent. Cys72 is partially buried and this can justify the apolar environment of the IAEDANS. The preference for Cys72 instead of Cys128 may be the consequence of other environmental conditions, like the presence of specific functional groups which facilitate or prevent the labelling.
It was previously shown (Stiuso et al., 1999) that human eIF-5A consists of two different domains which seem to react differently to the presence of denaturant. The fluorescence of tyrosine side chains, mainly present in the C-terminal domain (Figure 4C
), show a structural transition at low denaturant concentration (mid-point at approximately 1 M GdnHCl) whereas the IAEDANS-labelled protein, i.e. the protein with derivatized cysteines, mainly present in the N-terminal domain (Figure 4B
) seems to be more stable, its fluorescence being subjected to a transition at a higher GdnHCl concentration (mid-point at 2 M). This information suggested that the N-terminal domain is more stable than the C-terminal domain. This statement is in good agreement with our structural model, in which two distinct domains are evident and the N-terminal domain has a lower amount of unordered structure, which can mean a more compact and rigid backbone structure.
In the past, secondary structure predictions allowed one to hypothesize the nature of the packing of the two domains (Klier et al., 1995). However, such a hypothesis appears to be strongly affected by the excessive alpha helix prediction, evidently overestimated as compared to both our CD spectra analysis and CD published results of the same authors (Joao et al., 1995
; Klier et al., 1995
). In a more recent predictive paper (Gerloff et al., 1998
) a lower content of helix was predicted by using the newest available methods. In the absence of homologue structures, the fold recognition approach was applied and mainly beta folds were suggested, like the open barrel and the immunoglobulin-like beta sandwich. Finally, as a further improvement, our prediction gives a 3D model of the human eIF-5A, based on the comparative modelling approach applied after a careful adjustment of the sequence alignment. This structural model of eIF-5A can be useful for designing ligands able to inhibit the lysine
hypusine modification, thus acting as new antiproliferative agents raised against eIF-5A in order to potentiate the apoptosis induced by IFN
in cancer cells. In fact, we have demonstrated that the addition of IFN
to human epidermoid cancer KB cells induces apoptosis (Caraglia et al., 1999
) and reduces hypusine synthesis (Caraglia et al., 1995,1999
). Such effects are both antagonized when IFN
-treated KB cells are exposed to EGF for 12 h (Caraglia et al., 2000
). Therefore, on the basis of these results, hypusine synthesis could be part of an anti-apoptotic response raised by EGF IFN
-treated cells. The knowledge of the eIF-5A structure could allow a pharmacological screening on a computational basis in order to identify pharmacological substances able to bind the hypusine containing site of eIF-5A and inhibit its function. Therefore, eIF-5A, on the basis of its intrinsic biochemical and structural properties, could represent an useful target in combined approaches for the inhibition of tumour cell proliferation (Caraglia et al., 2000
).
In conclusion, the comparison of experimental data and structural features highlighted on the predicted model allows us to validate both the homology modelling strategy, based on an accurate refinement of the sequence alignment, and the predicted model. This will be useful for further investigations in pharmacological studies aimed at modulating the eIF-5A function.
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Notes |
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Acknowledgments |
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References |
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Received February 19, 2001; revised July 27, 2001; accepted August 9, 2001.