J. Mot. Biol. (1975) 99, 501-506
LET~.R ~o T~ En~o~ On the Conformation o f Cyclic Iron-containing Hexapeptides: The Crystal and Molecular Structure of Ferrichrysin The three-fllmensional molecular structure of the cyclic iron(III)-containing hexapeptide ferrichrysin, as elucidated from single crystal X-ray filE-faction data, is described. The molecular conformation of this biologically active compound shows similarities with the one observed for the related ferrichromo A. As has been found in similar iron(III) compounds, the iron(HI) ions arc co-ordinated b y three hydroxamate groups in an octahedral cgs-A arrangement. I n ferrichrysin all three ornithyl side chains are stabilized by intra-molecular hydrogen bonds. The fact that all ferrichromes that exhibit iron transport activities contain glycine in the same relative position in the poptide chain, suggests that the type I I fl-loop observed in both ferrichrysin and ferrichrome A is a biologically significant characteristic feature of the ferrichrome class of compounds. During the last decade a n u m b e r of naturally occurring substances, contalnln~ three hydroxamic acid groups R-CO-N(OH)- co-ordinated to a central ferric ion, have b e e n isolated, mainly from microbial sources. This paper will discuss the compounds in the ferrichrome family, for which the common basic structure is a cyclic hexapeptide (cf. Fig. 1). The three h y d r o x a m a t e groups are provided b y three acylated 8-N-hydroxyornithyl residues (Emery & Neflands, 1960) and the remaining residues are always serine, alanine or glycine (Atkin eta/., 1970). Thus, the amino acid sequence of the ferrichrome class can be written cyclo(ResS-Res2-Resl-Orn 8Orn2-0ml). ~ o r reviews on the role of these compounds in nature, we refer to the recent papers b y E m e r y (1971a) and Neflands (1966,1971). Fungal sideramlnes, such as ferrichrysin, t h a t act as growth factors for certain 0
II Orn3 -. ~C/NH'~ I'i " ~ HGI-~ylC~NH/cH It
NH /
/OH_ OH
C 2 C~--~O
Orn2 CH2~ /CH2 C~/ CH2 ~r'
O
O CH2.........
0
--C.
Ser/NH.
C
N~I
CH1,~..~
R'
C
O'..
CH2'
N
O~"
CH2
/N
O ,, / 0/
c/R IIo /
\
0
C
\R
CHZ OH /
Fzo. I. Schematic drawing of the ferrichrysin (R = CH3) and the ferrichrome A (R = CHC(CHs)CH2COOH) molecules, with the amino acid residues labelled for reference in the text. 501
502
R. NORRESTAM
E~' AL.
micro-organisms (Z~hner e~al., 1963) are said to have a role in microbial iron transport (Emery, 19715,1974; and Neilands, 1973). The biological activities of these compounds are apparently due to their outstanding ability to form strong chelate complexes with ferric iron, while the complexes with ferrous iron are relatively weak (Neilands, 1967). The stability of these complexes appears to vary with the nature of the acyl substituents on the ~-iV-hydroxyornithyl residues near the iron binding centre rather than with the amino acid sequence of the hexapeptide ring. The variations of the complex constants are probably due to differences in the accessibility of the iron(HI) ion. However, the possibility that these variations reflect differences in iron(YII) to.ordination have not been ruled out. By examining molecular models, it is easy to see that changes in the geometry of the iron(III) co-ordination affect the conformation of the whole molecule. Single crystal X-ray diffraction studies on ferrichromes have so far only been performed on a tetrahydrate of ferrichrome A (Zall~in et al., 1966). The molecular structure obtained from ferrichrome A showed a hexapeptide ring, with a configuration whose general features are in accordance with the predictions of a so-called E-structure made by Schwyzer (1959). Thus, the peptide chain of ferrichrome A can roughly be described as an antiparallel E-pleated sheet type. In idealized E-structures of cyclohexapeptides two intramolecular hydrogen bonds across the 18-membered peptide ring should be formed. From the interpretation of nuclear magnetic resonance measurements, such ideal f~-structures have been proposed for hexapeptides (Schwyzer & Ludescher, 1969) including ferrichromes in solution (Llin~s et al., 1970,1972). In the crystalline state, such a conformation exists for the trihydrate of cyclo(Gly-Gly-D-Ala-D-Ala-Gly-Gly) (Karle et al., 1970). However, in the crystalline structure of ferrichrome A, only one of the two predicted transannular hydrogen bonds of the peptide ring was found. This appears to be due to the distortion of the peptide ring caused by the co-ordination of the iron(III) ion. That an octahedral co-ordination of the iron(III) probably makes the ideal E-structure with two transannular hydrogen bonds much less energetically favourable is observed from space 6|ling molecular models. In order to elucidate some of the items discussed above a determination of the biologically highly active ferrichrysin has been carried out by X-ray diffraction techniques. Crystals of ferrichrysin, suitable for X-ray diffraction studies, were grown b y slow evaporation of an alcoholic solution. Three-dlmensional X-ray diffraction data were collected at room temperature by means of a single crystal difffractometer, using graphite-monochromatized CuK~ radiation. A total of 1822 significant reflections with Bragg-angles, 0, below 50 ° were collected. The crystal structure was deduced by application of direct methods, utilizing the variance-weighted phase-sum formula (Norrestam, 1972) followed by conventional Fourier and least-squares techniques. The absolute configuration of the ferrichrysin molecule was determinated by taking advantage of the large imaginary part of the anomalous dispersion correction for iron when using CuK~ radiation. The final refinement of the structural model yielded an unweighted linear _~ value of 0-095. The details of the structure determination are published elsewhere (Stensland & Norrestam, 1975). The resulting molecular shape is shown in Figure 2. The two seryl and the three ornithyl residues all have L-configuration, agreeing with the observations by KellerSchierlein & De~r (1963). The structural analysis showed that the crystals contained
LETTER
508
TO T H E E D I T O R
Ferrichrysin
Orn3~
~
Orn z
Gly t
\
j#
S
/
Ser 2
%
...
-...
Ser 3
-
•
............
Orn I
i
o
FIG. 2. Conformation of ferriehrysin monohydrate. The dotted lines represent probable hydrogen bonds. Dashed lines are drawn between the iron(III) ion and its 6 eo-ordinating oxygen atoms. The large open eirole denotes the iron(III) ion, small open circles the carbon atoms, solid circles the oxygen atoms, and the streaky circles the nitrogen atoms. one water molecule per ferrichrysin molecule. I t should be noted that the diffraction data collected do not permit an unambiguous determination of the hydrogen positions. Thus, the hydrogen bonds indicated in Figure 2 should only be regarded as representing a very probable hydrogen bond scheme as derived from the closest (2.8 to 3.2 .~) O ... O and 0 ... N contacts. The only transannular hydrogen bond present in this structure, as well as in t h a t of ferrichrome A (Fig. 3), is formed between the keto oxygen of the seryl residue Ser 3 and the amide nitrogen of the ornlthyl residue Orn s of the peptide main chain. The 0 ... N distance for this hydrogen bond, in ferrichrysin, is 3-15 A, while the C--O ... N angle is 134 °. As discussed above, two intramolecular transannular hydrogen bonds are predicted in true fl-struetures of hexapeptides. I n this case the second one should be formed between the keto oxygen of the ornithyl residue Orn s and the amide nitrogen of the seryl residue Set 3. The O ... N distance of 4-20 A and the C--O ... N angle of 85 ° do not indicate a second hydrogen bond. ~or comparison, the corresponding two 0 ... N distances and C--O ... N angles found in ferrichrome A were 2-98 A, 146 ° and 4-47 A, 79 °, respectively. The iron(III) ion is co-ordinated b y the six oxygens of the three 8-1V-acetyl-8-iVh y d r o x y substituents of the ornithyl residues, resulting in a distorted octahedral cis-A arrangement with Fe ... 0 distances ranging from 1-97 to 2.08 A and O ... Fe ... 0 chelate ring angles ranging from 78 to 79 °. The observed cis-A arrangement of the ligands has been found in other similar iron(III) chelating compounds like ferrichrome A, iron(HI) benzhydroxamate trihydrate (Lindner & GSttlicher, 1969) and ferrimycobaetin P (Hough & Rogers, 1974). The conformations of the three ornithyl side chains are largely dependent on the octahedral co-ordination of the iron(IH)
R. N O R R E S T A M E T AL,
504
ion. However, the ~-N-hydroxy oxygen atoms of two of the ornithyl residues, Orn 8 and Orn ~, participate in two probable hydrogen bonds (3.2 and 2.8 ~) to the amide nitrogens of Orn 1 and Orn =, respectively. This suggests t h a t the conformations of these two ornithine~ are further stabilized b y intramolecular hydrogen bonds. Three short contacts, 2-9 to 3.15 _~, are obtained between the water molecule and the two hydroxyl groups of the two seryl residues Set 2 and Ser 3 and one of the keto oxygens Ser 2 in the same ferrichrysin molecule (cf. Fig. 2). These hydrogen bonds are probably
Ferrichrorne A
Or n 5
Orn 2
Gly, j~
~..s
Ser3
Orn t
FIo. 3. Conformation of ferriohrome A as determined by Zalkin eta/, (1966). TABLE 1
Torsion angle~ Cnferrichry~¢n and ferrichrome A (in parerdhe~e~) Set 8 --166 ° (--163)
X1
177 (174) 170 (--174) 167 (60)
Set 2 --58 ° (--57)
137 (132) 179 (180) 60 (177/58)
Gly I
Orn a
Orn =
Orn 1
90 ° (82)
--155 ° (--144)
--78 ° (--76)
--124 ° (--105)
--7 (--3) --178 176 ---
--163 (--161) --176 (--176) 59
--29 (--49) --170 (--169) --63
19 (6) 170 (176) --68
(55)
(--59)
(--60)
The definitions of ~, P, oJ and X1 follow the IUPAC recommendation (1971). The labelling of the a~mino acid residues agrees with that given in Figures 1 to 3,
LETTER
TO THE
EDITOR
505
important for stabilizing the conformation of the seryl side chains, as discussed below. The molecular shape of ferrichrysin, eyclo(SerS-Ser2-Glyl-tri(~-N-acetyl-8-Nhydroxy-Orn)), described in this paper largely agrees (of. ~igs 2 and 3) with that of ferrichrome A, cyelo(Ser3-Ser2-Glyl-tri(8-N-methylghitaconyl-~-N-hydroxy-Orn)). The agreement between the conformations of the two hexapeptide chains is indicated by Table 1, which lists the most relevant torsion angles (cf. IUPAC Information, 1971) of ferrichrysin and the ferrichrome A molecules. The largest differences (up to 20 °) of the ~ and ~ angles occur for the three omit,hlnes. The rotation about the C~--C bonds, described b y the ~ angles, show the Ca-N bonds for the sequence SerS.Ser2-Glyl-Orn3-Orn2-Orn I to be cis-s~rew.trans.cis.slcrew-trans to the C--O bonds in both molecules. The values of ¢o, about 180 °, are typical for the tran8 conformation of the CO--NH group. The values of X1 show that the O~-atoms of the two serines and the C~ atoms of the three ornithines are all in the staggered position relative to N. The seryl residue Ser 2 was found to be disordered in ferrichrome A. This resulted in two different positions for the O~ atom, one of which agrees closely with the position found for the Ov atom in ferrichrysin. For the other seryl residue, Ser 8, a different staggered position of the O~ atoms is found for ferrichrysin as compared to ferrichrome A. The differences of the seryl side chain conformations in the two structures are probably due to the different number of hydrogen bonds formed to adjacent water molecules. As described above, the amide nitrogens of all three omithyl residues are involved in probable intramolecular hydrogen bonds in ferrichrysin. In ferrichrome A on the other hand, only two of these hydrogen bonds were observed, namely the trausannular hydrogen bond and the bond between the amide nitrogen of Orn 2 and the 8-Nhydroxyl oxygen of Orn 2. A hydrogen bond scheme involving all three amide nitrogens for this type of compound was also suggested from the studies by using tritiumhydrogen exchange ~cbniques on the related peptide ferrichrome, cyclo(Gly3Gly2-Glyl-tri(8.N-acetyl-$-N-hydroxy-Orn)), performed b y Emery (1967). A common feature among the ferrichromes with iron transport activity, apart from having a triomithyl entity as a part of their amino acid sequence, is that they all contain a glycyl residue (cf. ~ig. 1). In the crystalline structures of both ferrichrysin and ferrichrome A the conformation of the residues SerS-Ser2-Gly 1 agrees with a specific type of reverse turn of the peptide chain known as the fl-loop of type II. One condition for an energetically favourable type II fl-loop within peptide chains in general is, that they contain a glycyl residue in amino acid position number one of the loop (Venkatachalam, 1968). This fact suggests that the type II fl-loop, with the transannular hydrogen bond between the keto oxygen of Set 8 and the amide nitrogen of Orn 3, probably is a constant characteristic feature of this family of compounds, serving as a biological recognition property. As discussed above, studies of solution conformations of ferrichromes have suggested the presence of a second transannular hydrogen bond between the Ser 3 and Orn 3 residues. This suggestion implies that the intramolecular hydrogen bond scheme is different for ferrichromes in the crystalline state and in solution, and it also implies that the whole geometry of the main chain in solution would have to change from the energetically favourable type II fl-loop found in the solid state to a different conformation. The suggestion in this paper, that the type I I ~-loop is of biological significance for ferrichromes in general, is partly supported by the fact that the antibiotic activity
506
R. NORRESTAM
ET
AL.
of albomycin (l~aehr, 19"/1; Maehr & Pitcher, 1971), which has a n iron-binding site identical to ferrichrysin b u t a different chemical constitution a t the "~-loop region", is antagonized b y ferrichrysin. This investigation has been financially supported by the Swedish Natural Science Research Council. The authors are indebted to Professor W. Keller-Schierlein for supplying a sample of ferriehrysin. Department of Structural Chemistry Arrhenius Laboratory University of Stockholm S-104 05 Stockholm, Sweden Department of Chemistry I Agricultural College of Sweden S-750 07 Uppsala, Sweden
R. NOP~ESTAM B. STE~S~-~-D
C.-L B~rD~.N
Received 27 September 1974, and in revised form 8 September 1975 REFERENCES Atkin, C. L., Neilands, J. B. & Phaff, H. J. (1970). J. Bacteriol. 103, 722-733. Emery, T. (1967). B/ochem/~try, 6, 3858-3866. Emery, T. (I971e). Advan. En~ymoL 35, 135-185. Emery, T. (19715). Biochemistxy, 10, 1483-1488. Emery, T. (1974). Biochim. Biophys. Acta, 363, 219-225. Emery, T. & Neflands, J. B. (1960). J. Amer. Ghem. Soc. 83, 1626-1628. Hough, E. & Rogers, R. (1974). Biochem. Biophys. l~es. Commun. 57, 73-77. I U P A C Information ButtOn, A ppsnd~es on T ~ w 1Vom6ncla~re, No. 10 Abbreviations and Symbols for the Description of the Conformation of Polypeptide Chains, 1971. Karle, I. L., Gibson, J. W. & Karle, J. (1970). J. Am~r. Chem. Soc. 92, 3755-3760. Keller-Schierlein, W. & De6r, A. (1963). Helv. Chim. Ac~, 46, 1907-1920. Lindner, H. J. & GSttlicher, S. (1969). A c ~ Crys~allogr. sect. B, 25, 832-842. Llin~s, M., Klein, M. P. & Neilands, J. B. (1970). J. Mol. Biol. 52, 399-414. Llin~s, NI., Klein, 1K. P. & Neflands, J. B. (1972). J. Mol. Biol. 68, 265-284. Maehr, H. (1971). Pure Appl. Chem. 28, 603-636. Maehr, H. & Pitcher, R. (1971). J. Antibiot 24, 830-834. Neilands, J. B. (1966). Struck. Bonding, l, 59-108. Neilands, J. B. (1967). Science, 156, 1443-1447. Neilands, J. B. (1971). Struct. Bonding, 11, 145-170. Neilands, J. B. (1973). Inorg. Biochem. 1, 167-202. Norrestam, R. (1972). Acta Crysb~llogr. sect. A, 28, 303-308. Schwyzer, R. (1959). Record Chem. Progr. 20, 147. Schwyzer, R. & Ludescher, U. (1969). Helv. Ghim. Acta, 52, 2033-2040. Stensland, B. & Norrestam, R. (1975). Acta GrystaUogr. In the press. Venkatachalam, C. M. (1968). Bioloolymers, 6, 1425-1436. Za|lrln, A., Forrester, J. D. & Templeton, D . H . (1966). J. Amer. Chem.Soc. 88, 1810-1814. Z~hner, H., Keller-Schierlein, W., Hurter, R., Hess-Lcisinger, K. & De~r, A. (1963). Arch. MicrobioL 45, 119-135.
LET~.R ~o T~ En~o~ On the Conformation o f Cyclic Iron-containing Hexapeptides: The Crystal and Molecular Structure of Ferrichrysin The three-fllmensional molecular structure of the cyclic iron(III)-containing hexapeptide ferrichrysin, as elucidated from single crystal X-ray filE-faction data, is described. The molecular conformation of this biologically active compound shows similarities with the one observed for the related ferrichromo A. As has been found in similar iron(III) compounds, the iron(HI) ions arc co-ordinated b y three hydroxamate groups in an octahedral cgs-A arrangement. I n ferrichrysin all three ornithyl side chains are stabilized by intra-molecular hydrogen bonds. The fact that all ferrichromes that exhibit iron transport activities contain glycine in the same relative position in the poptide chain, suggests that the type I I fl-loop observed in both ferrichrysin and ferrichrome A is a biologically significant characteristic feature of the ferrichrome class of compounds. During the last decade a n u m b e r of naturally occurring substances, contalnln~ three hydroxamic acid groups R-CO-N(OH)- co-ordinated to a central ferric ion, have b e e n isolated, mainly from microbial sources. This paper will discuss the compounds in the ferrichrome family, for which the common basic structure is a cyclic hexapeptide (cf. Fig. 1). The three h y d r o x a m a t e groups are provided b y three acylated 8-N-hydroxyornithyl residues (Emery & Neflands, 1960) and the remaining residues are always serine, alanine or glycine (Atkin eta/., 1970). Thus, the amino acid sequence of the ferrichrome class can be written cyclo(ResS-Res2-Resl-Orn 8Orn2-0ml). ~ o r reviews on the role of these compounds in nature, we refer to the recent papers b y E m e r y (1971a) and Neflands (1966,1971). Fungal sideramlnes, such as ferrichrysin, t h a t act as growth factors for certain 0
II Orn3 -. ~C/NH'~ I'i " ~ HGI-~ylC~NH/cH It
NH /
/OH_ OH
C 2 C~--~O
Orn2 CH2~ /CH2 C~/ CH2 ~r'
O
O CH2.........
0
--C.
Ser/NH.
C
N~I
CH1,~..~
R'
C
O'..
CH2'
N
O~"
CH2
/N
O ,, / 0/
c/R IIo /
\
0
C
\R
CHZ OH /
Fzo. I. Schematic drawing of the ferrichrysin (R = CH3) and the ferrichrome A (R = CHC(CHs)CH2COOH) molecules, with the amino acid residues labelled for reference in the text. 501
502
R. NORRESTAM
E~' AL.
micro-organisms (Z~hner e~al., 1963) are said to have a role in microbial iron transport (Emery, 19715,1974; and Neilands, 1973). The biological activities of these compounds are apparently due to their outstanding ability to form strong chelate complexes with ferric iron, while the complexes with ferrous iron are relatively weak (Neilands, 1967). The stability of these complexes appears to vary with the nature of the acyl substituents on the ~-iV-hydroxyornithyl residues near the iron binding centre rather than with the amino acid sequence of the hexapeptide ring. The variations of the complex constants are probably due to differences in the accessibility of the iron(HI) ion. However, the possibility that these variations reflect differences in iron(YII) to.ordination have not been ruled out. By examining molecular models, it is easy to see that changes in the geometry of the iron(III) co-ordination affect the conformation of the whole molecule. Single crystal X-ray diffraction studies on ferrichromes have so far only been performed on a tetrahydrate of ferrichrome A (Zall~in et al., 1966). The molecular structure obtained from ferrichrome A showed a hexapeptide ring, with a configuration whose general features are in accordance with the predictions of a so-called E-structure made by Schwyzer (1959). Thus, the peptide chain of ferrichrome A can roughly be described as an antiparallel E-pleated sheet type. In idealized E-structures of cyclohexapeptides two intramolecular hydrogen bonds across the 18-membered peptide ring should be formed. From the interpretation of nuclear magnetic resonance measurements, such ideal f~-structures have been proposed for hexapeptides (Schwyzer & Ludescher, 1969) including ferrichromes in solution (Llin~s et al., 1970,1972). In the crystalline state, such a conformation exists for the trihydrate of cyclo(Gly-Gly-D-Ala-D-Ala-Gly-Gly) (Karle et al., 1970). However, in the crystalline structure of ferrichrome A, only one of the two predicted transannular hydrogen bonds of the peptide ring was found. This appears to be due to the distortion of the peptide ring caused by the co-ordination of the iron(III) ion. That an octahedral co-ordination of the iron(III) probably makes the ideal E-structure with two transannular hydrogen bonds much less energetically favourable is observed from space 6|ling molecular models. In order to elucidate some of the items discussed above a determination of the biologically highly active ferrichrysin has been carried out by X-ray diffraction techniques. Crystals of ferrichrysin, suitable for X-ray diffraction studies, were grown b y slow evaporation of an alcoholic solution. Three-dlmensional X-ray diffraction data were collected at room temperature by means of a single crystal difffractometer, using graphite-monochromatized CuK~ radiation. A total of 1822 significant reflections with Bragg-angles, 0, below 50 ° were collected. The crystal structure was deduced by application of direct methods, utilizing the variance-weighted phase-sum formula (Norrestam, 1972) followed by conventional Fourier and least-squares techniques. The absolute configuration of the ferrichrysin molecule was determinated by taking advantage of the large imaginary part of the anomalous dispersion correction for iron when using CuK~ radiation. The final refinement of the structural model yielded an unweighted linear _~ value of 0-095. The details of the structure determination are published elsewhere (Stensland & Norrestam, 1975). The resulting molecular shape is shown in Figure 2. The two seryl and the three ornithyl residues all have L-configuration, agreeing with the observations by KellerSchierlein & De~r (1963). The structural analysis showed that the crystals contained
LETTER
508
TO T H E E D I T O R
Ferrichrysin
Orn3~
~
Orn z
Gly t
\
j#
S
/
Ser 2
%
...
-...
Ser 3
-
•
............
Orn I
i
o
FIG. 2. Conformation of ferriehrysin monohydrate. The dotted lines represent probable hydrogen bonds. Dashed lines are drawn between the iron(III) ion and its 6 eo-ordinating oxygen atoms. The large open eirole denotes the iron(III) ion, small open circles the carbon atoms, solid circles the oxygen atoms, and the streaky circles the nitrogen atoms. one water molecule per ferrichrysin molecule. I t should be noted that the diffraction data collected do not permit an unambiguous determination of the hydrogen positions. Thus, the hydrogen bonds indicated in Figure 2 should only be regarded as representing a very probable hydrogen bond scheme as derived from the closest (2.8 to 3.2 .~) O ... O and 0 ... N contacts. The only transannular hydrogen bond present in this structure, as well as in t h a t of ferrichrome A (Fig. 3), is formed between the keto oxygen of the seryl residue Ser 3 and the amide nitrogen of the ornlthyl residue Orn s of the peptide main chain. The 0 ... N distance for this hydrogen bond, in ferrichrysin, is 3-15 A, while the C--O ... N angle is 134 °. As discussed above, two intramolecular transannular hydrogen bonds are predicted in true fl-struetures of hexapeptides. I n this case the second one should be formed between the keto oxygen of the ornithyl residue Orn s and the amide nitrogen of the seryl residue Set 3. The O ... N distance of 4-20 A and the C--O ... N angle of 85 ° do not indicate a second hydrogen bond. ~or comparison, the corresponding two 0 ... N distances and C--O ... N angles found in ferrichrome A were 2-98 A, 146 ° and 4-47 A, 79 °, respectively. The iron(III) ion is co-ordinated b y the six oxygens of the three 8-1V-acetyl-8-iVh y d r o x y substituents of the ornithyl residues, resulting in a distorted octahedral cis-A arrangement with Fe ... 0 distances ranging from 1-97 to 2.08 A and O ... Fe ... 0 chelate ring angles ranging from 78 to 79 °. The observed cis-A arrangement of the ligands has been found in other similar iron(III) chelating compounds like ferrichrome A, iron(HI) benzhydroxamate trihydrate (Lindner & GSttlicher, 1969) and ferrimycobaetin P (Hough & Rogers, 1974). The conformations of the three ornithyl side chains are largely dependent on the octahedral co-ordination of the iron(IH)
R. N O R R E S T A M E T AL,
504
ion. However, the ~-N-hydroxy oxygen atoms of two of the ornithyl residues, Orn 8 and Orn ~, participate in two probable hydrogen bonds (3.2 and 2.8 ~) to the amide nitrogens of Orn 1 and Orn =, respectively. This suggests t h a t the conformations of these two ornithine~ are further stabilized b y intramolecular hydrogen bonds. Three short contacts, 2-9 to 3.15 _~, are obtained between the water molecule and the two hydroxyl groups of the two seryl residues Set 2 and Ser 3 and one of the keto oxygens Ser 2 in the same ferrichrysin molecule (cf. Fig. 2). These hydrogen bonds are probably
Ferrichrorne A
Or n 5
Orn 2
Gly, j~
~..s
Ser3
Orn t
FIo. 3. Conformation of ferriohrome A as determined by Zalkin eta/, (1966). TABLE 1
Torsion angle~ Cnferrichry~¢n and ferrichrome A (in parerdhe~e~) Set 8 --166 ° (--163)
X1
177 (174) 170 (--174) 167 (60)
Set 2 --58 ° (--57)
137 (132) 179 (180) 60 (177/58)
Gly I
Orn a
Orn =
Orn 1
90 ° (82)
--155 ° (--144)
--78 ° (--76)
--124 ° (--105)
--7 (--3) --178 176 ---
--163 (--161) --176 (--176) 59
--29 (--49) --170 (--169) --63
19 (6) 170 (176) --68
(55)
(--59)
(--60)
The definitions of ~, P, oJ and X1 follow the IUPAC recommendation (1971). The labelling of the a~mino acid residues agrees with that given in Figures 1 to 3,
LETTER
TO THE
EDITOR
505
important for stabilizing the conformation of the seryl side chains, as discussed below. The molecular shape of ferrichrysin, eyclo(SerS-Ser2-Glyl-tri(~-N-acetyl-8-Nhydroxy-Orn)), described in this paper largely agrees (of. ~igs 2 and 3) with that of ferrichrome A, cyelo(Ser3-Ser2-Glyl-tri(8-N-methylghitaconyl-~-N-hydroxy-Orn)). The agreement between the conformations of the two hexapeptide chains is indicated by Table 1, which lists the most relevant torsion angles (cf. IUPAC Information, 1971) of ferrichrysin and the ferrichrome A molecules. The largest differences (up to 20 °) of the ~ and ~ angles occur for the three omit,hlnes. The rotation about the C~--C bonds, described b y the ~ angles, show the Ca-N bonds for the sequence SerS.Ser2-Glyl-Orn3-Orn2-Orn I to be cis-s~rew.trans.cis.slcrew-trans to the C--O bonds in both molecules. The values of ¢o, about 180 °, are typical for the tran8 conformation of the CO--NH group. The values of X1 show that the O~-atoms of the two serines and the C~ atoms of the three ornithines are all in the staggered position relative to N. The seryl residue Ser 2 was found to be disordered in ferrichrome A. This resulted in two different positions for the O~ atom, one of which agrees closely with the position found for the Ov atom in ferrichrysin. For the other seryl residue, Ser 8, a different staggered position of the O~ atoms is found for ferrichrysin as compared to ferrichrome A. The differences of the seryl side chain conformations in the two structures are probably due to the different number of hydrogen bonds formed to adjacent water molecules. As described above, the amide nitrogens of all three omithyl residues are involved in probable intramolecular hydrogen bonds in ferrichrysin. In ferrichrome A on the other hand, only two of these hydrogen bonds were observed, namely the trausannular hydrogen bond and the bond between the amide nitrogen of Orn 2 and the 8-Nhydroxyl oxygen of Orn 2. A hydrogen bond scheme involving all three amide nitrogens for this type of compound was also suggested from the studies by using tritiumhydrogen exchange ~cbniques on the related peptide ferrichrome, cyclo(Gly3Gly2-Glyl-tri(8.N-acetyl-$-N-hydroxy-Orn)), performed b y Emery (1967). A common feature among the ferrichromes with iron transport activity, apart from having a triomithyl entity as a part of their amino acid sequence, is that they all contain a glycyl residue (cf. ~ig. 1). In the crystalline structures of both ferrichrysin and ferrichrome A the conformation of the residues SerS-Ser2-Gly 1 agrees with a specific type of reverse turn of the peptide chain known as the fl-loop of type II. One condition for an energetically favourable type II fl-loop within peptide chains in general is, that they contain a glycyl residue in amino acid position number one of the loop (Venkatachalam, 1968). This fact suggests that the type II fl-loop, with the transannular hydrogen bond between the keto oxygen of Set 8 and the amide nitrogen of Orn 3, probably is a constant characteristic feature of this family of compounds, serving as a biological recognition property. As discussed above, studies of solution conformations of ferrichromes have suggested the presence of a second transannular hydrogen bond between the Ser 3 and Orn 3 residues. This suggestion implies that the intramolecular hydrogen bond scheme is different for ferrichromes in the crystalline state and in solution, and it also implies that the whole geometry of the main chain in solution would have to change from the energetically favourable type II fl-loop found in the solid state to a different conformation. The suggestion in this paper, that the type I I ~-loop is of biological significance for ferrichromes in general, is partly supported by the fact that the antibiotic activity
506
R. NORRESTAM
ET
AL.
of albomycin (l~aehr, 19"/1; Maehr & Pitcher, 1971), which has a n iron-binding site identical to ferrichrysin b u t a different chemical constitution a t the "~-loop region", is antagonized b y ferrichrysin. This investigation has been financially supported by the Swedish Natural Science Research Council. The authors are indebted to Professor W. Keller-Schierlein for supplying a sample of ferriehrysin. Department of Structural Chemistry Arrhenius Laboratory University of Stockholm S-104 05 Stockholm, Sweden Department of Chemistry I Agricultural College of Sweden S-750 07 Uppsala, Sweden
R. NOP~ESTAM B. STE~S~-~-D
C.-L B~rD~.N
Received 27 September 1974, and in revised form 8 September 1975 REFERENCES Atkin, C. L., Neilands, J. B. & Phaff, H. J. (1970). J. Bacteriol. 103, 722-733. Emery, T. (1967). B/ochem/~try, 6, 3858-3866. Emery, T. (I971e). Advan. En~ymoL 35, 135-185. Emery, T. (19715). Biochemistxy, 10, 1483-1488. Emery, T. (1974). Biochim. Biophys. Acta, 363, 219-225. Emery, T. & Neflands, J. B. (1960). J. Amer. Ghem. Soc. 83, 1626-1628. Hough, E. & Rogers, R. (1974). Biochem. Biophys. l~es. Commun. 57, 73-77. I U P A C Information ButtOn, A ppsnd~es on T ~ w 1Vom6ncla~re, No. 10 Abbreviations and Symbols for the Description of the Conformation of Polypeptide Chains, 1971. Karle, I. L., Gibson, J. W. & Karle, J. (1970). J. Am~r. Chem. Soc. 92, 3755-3760. Keller-Schierlein, W. & De6r, A. (1963). Helv. Chim. Ac~, 46, 1907-1920. Lindner, H. J. & GSttlicher, S. (1969). A c ~ Crys~allogr. sect. B, 25, 832-842. Llin~s, M., Klein, M. P. & Neilands, J. B. (1970). J. Mol. Biol. 52, 399-414. Llin~s, NI., Klein, 1K. P. & Neflands, J. B. (1972). J. Mol. Biol. 68, 265-284. Maehr, H. (1971). Pure Appl. Chem. 28, 603-636. Maehr, H. & Pitcher, R. (1971). J. Antibiot 24, 830-834. Neilands, J. B. (1966). Struck. Bonding, l, 59-108. Neilands, J. B. (1967). Science, 156, 1443-1447. Neilands, J. B. (1971). Struct. Bonding, 11, 145-170. Neilands, J. B. (1973). Inorg. Biochem. 1, 167-202. Norrestam, R. (1972). Acta Crysb~llogr. sect. A, 28, 303-308. Schwyzer, R. (1959). Record Chem. Progr. 20, 147. Schwyzer, R. & Ludescher, U. (1969). Helv. Ghim. Acta, 52, 2033-2040. Stensland, B. & Norrestam, R. (1975). Acta GrystaUogr. In the press. Venkatachalam, C. M. (1968). Bioloolymers, 6, 1425-1436. Za|lrln, A., Forrester, J. D. & Templeton, D . H . (1966). J. Amer. Chem.Soc. 88, 1810-1814. Z~hner, H., Keller-Schierlein, W., Hurter, R., Hess-Lcisinger, K. & De~r, A. (1963). Arch. MicrobioL 45, 119-135.
