Crystal and molecular structure of the dipeptide l -Tyr-l -Lys

Crystal and molecular structure of the dipeptide L-Tyr-L-Lys L. Urpi, M. Coil and J. A. Subirana Unitat de Quimica Macromolecular del CSIC, Escola T~cnica Superior d'Enginyers Industrials, Diagonal 647, 08028-Barcelona, Spain (Received 16 April 1987; revised 16 July 1987) We have determined the structure of the dipeptide L-Tyr-L-Lys by X-ray diffraction. The molecule is found as a zwitterion with a protonated e-amino group of lysine and a negatively charged terminal carboxylate group. The terminal amino group is found in its neutral form. It acts both as an H-bond donor and acceptor. The dipeptide backbone is folded and the side chains have the most stable conformation which is commonly found in both amino acids. The crystal structure is stabilized by intermolecular hydrogen bonding. No stacking of the tyrosine aromatic ring is observed. Keywords: Aromatic~aromaticinteractions;crystal structure; conformation;L-Tyrosyl-L-lysine;X-ray studies

Introduction We have undertaken this study in order to better understand the interaction of aromatic residues and basic side chains in peptides and proteins. Furthermore, the structure of small basic peptides gives detailed information on the conformation of such residues and is a useful complementary approach in the study of D N A protein interactions. In fact the interaction of tyrosine side chains with DNA has been well documented ~'2. It has also been recently shown 3'4 that aromatic rings inside proteins do not show any tendency to stack, rather they tend to interact in a perpendicular manner. In agreement with these observations, the results we have obtained do not show any stacking interactions among the aromatic rings.

Experimental The compound was obtained from Bachem AG and crystallized, without further purification, by vapour diffusion of an aqueous M PD solution of the dipeptide in an atmosphere of 2-methyl-2,4-pentanodiol (MPD). Triclinic plate-like triangular crystals formed after one week. A crystal 0.62 × 0.36 × 0.I mm was mounted in a capillary. X-ray data were collected using an Enraf-Nonius CAD-4 diffractometer with MoK~t radiation (2= 0.71069A) and graphite monochromator. The cell parameters were determined from 25 reflections (4 < 0 < 9). The crystallographic data are listed in Table 1; 1372 independent reflections (0 < 25 °) were recorded, of which 1068 with I > 1.5a(l) were considered as observed. The data were collected at room temperature with the w-scan technique, three reflections were measured every 50 reflections as an intensity control and no significant differences were observed. The h k I range was = +5, +_1O, + 11. Lp correction was applied. Absorption was ignored. The structure was determined by direct methods using the M I T H R I L computer program package 5. A Debye curve with scattering factors for the tyrosine ring group 0141-8130/88/010055-05503.00 1988 Butterworth & Co. (Publishers) Ltd

was computed in order to normalize the structure factors. Previous attempts to solve the structure using a Wilson normalization curve failed. Non-hydrogen atoms were refined anisotropically by full-matrix least squares using the SHELX 76 computer program 6. The scattering factors f, f ' and f " for all atoms were obtained from the International Tables for X-ray Crystallography 7. All the H atoms were visible in difference Fourier maps. Those which are bonded to the O atom of the phenol ring of the tyrosine and to N atoms (all H which can form the H bonding net) were placed in the positions found in the difference Fourier map and refined isotropically. Their coordinates, bond distances and angles are presented in Tables 2 and 3, with the other atoms. The remaining H atoms were included at calculated positions and refined isotropicaUy with geometrical constraints. The final isotropic temperature factor for calculated H atoms bonded to CA1 and CA2 was 0.0449 (64), for those bonded to CB2, CG2, CD2 and CE2 was 0.0702 (40), for those bonded to CD1, CD'I, CE1 and CE'I was 0.0784 (64) and for those bonded to CB1 was 0.0681 (85)A 2. The refinement converged to a final R = 0.032, Rw = 0.034. The weighting scheme used was (a2(F)+ 0.001317F2) - 1. Maximum and minimum heights in the

Table 1 Crystallographic data

Molecular formula: C15H2aNaO 4 Molecular weight: 309.36 Space group: PI a = 5.021 (4) A b-- 8.911 (1)A c = 9.430 (2) A -- 74.74 (1)° fl = 83.75 (3) ° 7 = 75.10 (2) ° V = 393 (7) A3 Z= 1 Calculated density: 1.31 rag m- a # = 0.103mm -l

Int. J. Biol. Macromol., 1988, Vol 10, February

55

Structure of the dipeptide L-Tyr-L-Lys: L. Urpi et al. Table2 Fractional atomic coordinates with e.s.d.s in parentheses and equivalent isotropic thermal parameters (A2)

N1 HN1 H'N1 CA1 CB1 CG1 CD1 CD'I CE1 CE'I CZ1 OH1 HOHI C1 Ol N2 HN2 CA2 CB2 CG2 CD2 CE2 NZ2 HNZ2 H'NZ H"NZ C2 O12 022

x~

Wa

z~

aeq o

0.4794 (12) 0.5435 (88) 0.2960 (114) 0.5728 (10) 0.4775 (11) 0.5603 (11) 0.8060 (12) 0.3854 (11) 0.8764 (12) 0.4575 (12) 0.7012 (11) 0.7740 (10) 0.6532 (113) 0.4601 (I0) 0.2089 (9) 0.6384 (9) 0.8168 (86) 0.5517 (9) 0.4571 (10) 0.6894 (10) 0.5992 (10) 0.8338 (11) p.7385 (0) 0.8683(111) 0.6425 (80) 0.6119 (99) 0.3384 (10) 0.3995 (9) 0.1329 (10)

0.2028 (6) 0.2485 (51) 0.2284 (56) 0.2687 (6) 0.1888 (6) 0.2476 (6) 0.1781 (7) 0.3748 (7) 0.2318 (7) 0.4283 (7) 0.3568 (6) 0.4059 (6) 0.5038 (69) 0.4485 (6) 0.5049 (5) 0.5391 (5) 0.4968 (40) 0.7131 (5) 0.7957 (6) 0.7647 (6) 0.8406 (6) 0.8134 (7) 0.8693 (0) 0.8647 (60) 0.9908 (51) 0.7987 (57) 0.7590 (5) 0.6842 (5) 0.8691 (5)

0.3616 (5) 0.4259 (54) 0.3702 (54) 0.2082 (5) 0.1038 (5) 0.9435 (5) 0.8811 (6) 0.8517 (6) 0.7328 (6) 0.7042 (5) 0.6447 (5) 0.4979 (4) 0.4368(64) 0.1678 (5) 0.1599 (5) 0.1493 (4) 0.1584 (39) 0.1248 (5) 0.9688 (5) 0.8542 (5) 0.6984 (5) 0.5873 (5) 0.4352 (0) 0.3663(64) 0.4086 (44) 0.4279 (53) 0.2460 (5) 0.3779 (4) 0.2114 (4)

4.52 4.98 5.63 3.53 4.09 3.64 5.00 5.19 5.32 5.17 4.17 5.59 7.36 3.05 4.05 2.88 2.70 2.94 3.27 3.89 3.71 4.46 3.86 6.36 4.08 5.68 3.33 4.32 4.69

a Beq = 8/3• 2Z, Z~ U0a*a*a,aj final difference Fourier synthesis were: 0.13 and - 0 . 1 4 e A -3. A Vax 750 computer has been used for all the calculations.

Results Conformation of the peptide chain The molecular structure and atomic numbering are shown in Figure 1 and the final positional and thermal parameters are listed in Table 2. Bond lengths and bond angles are given in Table 3 and conformational angles (IUPAC-IUB Commission on Biochemical Nomenclature) s are presented in Table 4. The bond lengths and angles have the expected values. The conformational angles show that the dipeptide backbone is extended at the centre, from the CA1 to the CA2 atoms (oJ is trans), but the ends are not extended (~k1, 42 and ~ are not trans). This is most clearly seen in the diagram of the molecule presented in Figure 1.

~k1 angle. This torsion angle (111.3 °) is very different from those reported in the literature for dipeptides of the type L-Tyr-L-A where A is Pro 9, Phe 1°, Gly ~ , Va112 or Glu ~3. The ~k~ values of such structures are between 150 and 166 ° in agreement with conformational energy calculations for dipeptides which show that Tyr and Phe have the most stable conformation when the main chain is extended with ~9_ 150 ° (Ref. 10). The reason for this deviation of ~ki from the expected value may be due to the fact that the N-terminal group is neutral in this structure, whereas most of the peptides of the type L-Tyr-L-A have a protonated N-terminal group. Only in the dipeptide

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Int. J. Biol. Macromol., 1988, Vol. 10, February

Ac-L-Phe-L-Tyr the value of ~kl (134.7 °) approaches the value found by us in L-Tyr-L-Lys.

09angle. It indicates the normal trans conformation of the peptidic bond but with a considerable deviation from planarity. This feature is also found in the dipeptide structures discussed in the previous paragraph, although all these peptides have a positive value of 09. ~P2 angle. The considerable deviation of this angle from 180° contributes most significantly to the folded conformation of the dipeptide backbone. According to Suresh and Vijayan 1¢ this angle is sufficient to describe the backbone conformation ofa dipeptide with deblocked N and C terminal groups. This is not exactly true in our structure, since the hypothesis made by these authors that if1, oJ and ~,~ are around 180° and ~k~ is close to 0 ° is not adequate. Only o~ has the expected value. In the dipeptide L-Pro-L-Lys acetate (unpublished results) we have found a very similar (63.0°) value for this angle. No other lysine dipeptide structures are available for comparison. ~'2 angle. This dihedral angle is very different from those found in the literature for charged L-lysine amino acid salts ls'~6 or small peptides like Ac-Gly-L-Lys Me ester ~7. But the last comparison with a blocked dipeptide is not very meaningful because the present structure has a charged terminal carboxylate. On the other hand, there are some dipeptide structures lacking the amino acid Llysine and which have a charged C-terminal carboxylic group t4 which show a great spread of values in the range of - 7 3 to + 71 ° with many examples of negative values and without any near 48 °, the value found in the present structure. The diversity of ~k~ values observed in the Cterminal group of dipeptides indicates that the carboxylate acquires the most favourable orientation in order to make H bonds with the H donor groups. O(O12-C2-CA2-CB2) angle. This angle is determined by the ~k~ angle. From Figure 1 it can be seen that the 0(O12-C2-CA2-CB2) angle is trans. This conformation is unusual in long polypeptide chains. The situation found here is a typical small peptide effect. Furthermore the tendency to optimize the side chain interactions and to maximize the number of H-bonds in the crystal may favour the conformation we have found in this dipeptide. This tendency may also have an influence on the unexpected ~k~ value.

Conformation of the side chains As it can be seen in Figure 1 the side chains of tyrosine and lysine are in a parallel arrangement. The aromatic ring of tyrosine is planar (the biggest deviation from the plane defined by the six carbon atoms is 0.003 A). The oxygen atom OH1 shows a small deviation from this plane of - 0.026 A. The tyrosine side chain torsion angles g~, Z2'1 and g 2'2 agree fairly well with those found in the dipeptide Ac-L-Phe-L-Tyr (Ref. 10) (Z~=180.4 ° and Z2'1=78.6°). This is one of the three most stable conformations for the side chain of aromatic amino acids in dipeptides ( g ] = - 6 0 , 60 or 180 °) as reviewed by Benedetti et al. is. The side chain of lysine also shows one of the most stable conformations: g-(z~), t(z2), t(g~), t(Z~), in agreement with most structures found in the literature16'l 8.

Structure of the dipeptide L-Tyr-L-Lys: L. Urpi et al. Table 3

Bond distances (A) and angles (o) with e.s.d.s, in parentheses

N1--CA1 CA1-C1 CA1-CB1 C1-O1 C1-N2 N2-CA2 CA2-CB2 CA2-C2 C2-O12 C2-O22

1.484 1.514 1.533 1.234 1.322 1.461 1.528 1.541 1.276 1.232

(5) (5) (5) (4) (4) (4) (4) (5) (4) (4)

CBI-CG1 CG1-CD1 CG1--CD'I CD1-CE1 CD'I-CE'I CE1-CZ1 CE'I-CZ1 CZ1-OH1

1.513 1.376 1.394 1.392 1.386 1.369 1.362 1.376

(5) (6) (5) (6) (6) (6) (6) (5)

CB2-CG2 CG2-CD2 CD2-CE2 CE2-NZ2 NZ2-HNZ2 NZ2-H'NZ2 NZ2-H"NZ2

1.524 1.511 1.504 1.478 0.870 1.041 1.021

(4) (5) (5) (5) (59) (43) (50)

NI-HN1 N1-H'N1

0.937 (50) 0.890 (53)

OHI-HOHI N2-HN2

1.000 (57) 0.882 (41)

N1-CA1-C1 N1-CA1-CB1 CB1--CA1-C1 CA1-C1-N2 CA1-C1--O1 OI-C1-N2 C1-N2~A2 N2-CA2--C2 N2-CA2-CB2 C2-CA2-CB2 CA2-C2--O22 CA2--C2-O12 O22--C2-O 12 CA1-CB1-CG1 CB1-CG1--CD1 CB1-CG1--CD'I CD'I-CG1--CD1 CGI--CD1-CE1 CG1-CD'I-CE'I CD1-CE1-CZ1 CD'l-CE'142Z1 CE'I--CZ1-CE1 CE1-CZ1--OH1 CE'I-CZ1-OH1 CA2-CB2-CG2 CB2-CG2-CD2 CG2-CD2--CE2 CD2-CE2-NZ2 HNZ2-NZ2-H'NZ2 HNZ2-NZ2-H"NZ2 H'NZ2-NZ2-H"NZ2 HNZ2-NZ2-CE2 H'NZ2-NZ2-CE2 H"NZ2-NZ2-CE2 HN1-NI-H'N1 HN1-N1-CAI H'N1-N1-CA1 HOH1-OH1-CZ1 HN2-N2-CA2 HN2-N2-C1

N1

O1

022

109.1 (3) 109.7 (3) 111.5 (3) 117.5 (3) 119.8 (3) 122.6 (3) 122.4 (3) 110.2 (3) 112.4 (3) 113.8 (3) 119.1 (3) 116.0 (3) 124.7 (3) 114.4 (3) 122.3 (3) 120.6 (4) 117.0 (3) 121.5 (4) 121.2 (4) 120.4 (4) 120.7 (4) 119.2 (4) 119.2 (4) 121.6 (4) 111.5 (3) 112.7 (3) 111.8 (3) 111.8 (3) 101.1 (3.9) 109.3 (4.3) 112.9 (3.3) 115.4 (3.4) 112.2 (2.2) 106.0 (2.6) 107.0 (4.3) 109.8 (2.6) 110.0 (3.2) 118.5 (3.4) 115.9 (2.1) 121.5 (2.1)

O12

Figure 1 ORTEP stereo drawingz3 of the molecular structure and atom numbering. Thermal ellipsoids of 50% of probability are shown

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57

Structure of the dipeptide L-Tyr-~-Lys: L. Urpi et al. Table 4 Conformational angles (°) N1-CA1-C1-N2: CA I--C1-N2-CA2: C1-N2-CA2-C2: N2-CA2-C2-O 12: N2-CA2-C24~)22:

{k~ ~o ~b2 ~ ~

N1-CA1-CB1-CG1 : CA1-CB1-CG1-CD1 : CB1-CG1-CD1-CE1 : CG1-CD1-CE1-CZ1 : CD1-CE1-CZ1-OHI :

Z] . . . . . X~ . . . . . Z3 . . . . .

N2-CA2-CB2~G2: CA2-CB2-CG2-CD2: CB2-CG2q2D2-CE2: CG2-CD2~CE2-NZ2:

X~ Z~ X23 Z~

..... ..... ..... ..... ..... -

111.3 173.7 52.7 47.6 136.5 180.0 89.4 178.8

.....

o.o

Z~ . . . . . - 179.0 ..... ..... .... ....

-61.8 178.2 178.1 172.7

0--~---~7 ---------~ ..~,

the x-axis of the unit cell. This H - b o n d is of the $ 5 - - , 4 type according to the classification of Suresh and Vijayan 14 and it is responsible for the growth of the crystal in one direction. This organization is reminiscent of a//-pleated sheet, but the orientation of the side chains is anomalous in the peptide we have studied, since both lie on the same side of the peptide plane. In some amino acids salts 14'2° and small peptides 2~ a head-to-tail interaction involving the N - and C-terminal groups has been described, but in our case this organization is not found. Since the N-terminal is uncharged and acts mainly as an acceptor such an organization may be difficult. The N-terminal acts also as a donor, so that H-N1 forms a weak hydrogen bond with the phenolic oxygen. The other amino hydrogen (H'-N 1) does not form any H-bond. In fact it is the only potential donor that is not involved in the H-bonding network. The presence of polar and charged groups in the side chains may also be responsible for preventing a head-to-tail pattern. In the carboxylate 0 2 2 forms only one hydrogen bond and O12 forms two. This fact correlates with the shorter O22-C2 bond length (1.232 (4) A) when compared with O12-C2 (1.276 (4)A). Finally it is interesting to note that the charged e-amino group of lysine forms three hydrogen bonds with three different molecules.

Discussion

Figure 2 An ORTEP drawing 23 of the crystal structure of L-Tyr-t-Lys. The orientation of the crystallographic axes is indicated at the bottom of the figure. All the dipeptide molecules which are H-bonded to the central molecule (in black) are shown. The broken lines indicate hydrogen bonds; those involving the peptide group are represented as continuous lines

Hydrogen bonding and crystal packing Since we have crystallized the zwitterionic form of the dipeptide, the N-terminal amino group remains neutral, whereas the side chain amino group is charged, as expected from the relative p K of both groupsa 9. Thus the N-terminal group acts as a hydrogen bond acceptor, coming from a positively charged e-amino group of a lysine side chain in a neighbouring molecule. In fact the structure is fully stabilized by a complex network of intermolecular hydrogen bonds, as shown in Figure 2. The hydrogen bond lengths and angles have values in the standard range, except for the bond between the Nterminal amino group and the phenolic oxygen, which is slightly longer than expected (3.183 (6) A) (Table 5). The peptide groups form an infinitely hydrogen bonded chain, with the H bond direction (indicated by continuous lines in Figure 2) approximately parallel to

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Int. J. Biol. Macromol., 1988, Vol. 10, February

In the results section we have carried out a detailed comparison of the structure we have determined with other related dipeptides. The structure has an unusual main chain conformation, which m a y be due to the optimization of the extensive network of hydrogen bonds which is most clearly seen in Figure 2. As a result of this conformation, the lysine and tyrosine side chains lie on the same side of the peptide backbone and even show Van der Waals contacts, as it is clearly apparent in Figure 3. This organization is in contrast with what is found in the usual //-sheet conformation, where consecutive side chains lie on opposite sides of the main chain. Nevertheless, the peptide group has its normal planar conformation and forms an extended chain of hydrogen bonds similar to those found in//-sheets, as shown in

Figure 2. In spite of this unusual conformation, it appears of interest to consider the interactions of the tyrosine aromatic ring, as well as the conformation of the lysine

Table 5 Hydrogen bond lengths (A) and angles (°) H D-HA

D-A

N1-H4DH1 (a) N2-H-O1 (b) NZ2-H4922 (c) NZ2-H'-N1 (d) NZ2-H"-O12 (e) OH1-H-O12 (e)

3.183 2.812 2.732 2.851 2.833 2.748

(6) (4) (4) (5) (4) (4)

H-A

D

A

2.297 (49) 1.990 (41) 1.864 (61) 1.811 (45) 1.823 (51) 1.773 (58)

157.6(3.7) 154.5(3.0) 176.0(4.9) 179.3 (3.2) 169.2 (4.2) 163.8 (5.1)

Symmetry code: (a) x,y,z+l; (b) x+l,y,z; (c) x + l , y , z - 1 ; (d) x , y + l , z - 1 ; (e) x,y,z- 1

Structure of the dipeptide t.-Tyr-L-Lys: L. Urpi et al. and E. Molins for the facilities offered to us. This work has been supported by a CAICYT grant (2679/83). Lourdcs Urpi acknowledges with thanks support received from the Comit6 Conjunto Hispano-norteamericano (grant CCB-8402039) and Miquel Coll, a predoctoral fellowship from the CSIC.

C=•,•

X+l,y-l,Z C~

References 1

X-l,y,z x,y-l,Z Figure 3 This ORTEP drawing2a shows the nearest neighbours of the tyrosine aromatic fingo The regions which have Van de Waals contacts with the central tyrosine ring (molecule x, y, z) are shown in black. Those above and below the plane of the paper are not shown. The crystallographic coordinates of each molecule are indicated. It can be seen that the aromatic ring is surrounded by miscellaneous atoms, no ring stacking occurs

2 3 4 5 6 7 8

side chain, and compare them with those found in proteins. Our structure does not show any stacking of the aromatic rings in agreement with what is found in proteins 3'4. This is most clearly seen in Figure 3, where it is apparent that the phenyl plane lies between two lysine side chains and has many other Van der Waals interactions. It is likely that for stacking to occur it is necessary to have a system with several aromatic rings, as it is found in DNA. The lysine side chain is in the usual all-trans extended conformation; only the first bond, between the main chain and the side chain is in the usual g- conformation. These are the standard features found in oligopeptide crystals 17't9. However it is striking that this side chain conformation is only seldom found (once in 12 cases) in high resolution studies of protein side chain conformations 22. More data should be available in order to allow an explanation for this discrepancy.

9 10 11 12 13 14 15 16 17 18 19 20 21

Acknowledgements

22

Diffraction data were collected in the Institut Jaume Almera (CSIC) and we are most thankful to C. Miravitlles

23

H6i6ne,C. and Lancelot, G. Prog. Biophys. Molec. Biol. 1982, 39,1 Mayer, R., Toulme, F., Montenay-Garestier, T. and Helene, C. J. Biol. Chem. 1979, 254, 75 Burley,S. K. and Petsko, G. A. Science 1985, 229, 23 Singh, J. and Thornton, J. M. FEBS Lett. 1985, 191, 1 Gilmore, C. J. 'MITHRIL. A Computer Program for the Automatic Solution of Crystal Structures from X-ray Data', University of Glasgow, Scotland, 1983 Sheldrick,G. M. 'SHELX 76. Program for Crystal Structure Determination', University of Cambridge, England, 1976 International Tables for X-Ray Crystallography, Kynoch Press, Birmingham, 1974, Vol. IV IUPAC-IUB Commission on Biochemical Nomenclature Biochemistry 1970, 9, 3471 Precigoux,G., G-eoffre, S., Hospital, M. and Leroy, F. Acta Crystallogr. 1982, B38, 2172 Cotrait, M., Barrans, Y. and Leroy, F. Acta Crystallogr. 1982, B38, 1626 Carson, W. M. and Hackert, M. L. Acta Crystallogr. 1978,ILM, 1275 Ramakrislman, B., Seshadri, T. P. and Viswamitra, M. A. Acta Crystallogr. 1984, C40, 1248 Pandit, J., Seshadri, T. P. and Viswamitra, M. A. Acta Crystallogr. 1984, C40, 169 Suresh,C. G. and Vijayan, M. Int. J. Pept. Protein Res. 1985,26, 311 Capasso, S., Mattia, C. A., MazzareUa, U and Zagari, A. Acta Crystallogr. 1983, C39, 281 Bhat,T. N. and Vijayan, M. Acta Crystallogr. 1976, B32, 891 Salunke, D. M. and Vijayan, M. Acta Crystallogr. 1982,B38, 287 Benedetti,E., Moreili, G., Nemethy, G. and Scheraga, H. A. Int. J. Pept. Protein Res. 1983, 22, 1 Tanford, C. 'Physical Chemistry of Macromolecules', J. Wiley, New York, 1961, p. 556 Suresh,C. G. and Vijayan, M. Int. J. Pept. Protein Res. 1983,22, 617 Coil, M., Subirana, J. A., Solans, X., Font-Altaba, M. and Mayer, R. Int. J. Pept. Protein Res. 1987, 29, 708 Smith, J. L., Hendrickson, W. A., Honzatko, R. B. and Sheriff,S. Biochemistry 1986, 25, 5018 Johnson, C. K. ORTEP. Report ORNL-3794, Oak-Ridge National Laboratory, Tennesse, 1965

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