Crystal structure of the amino acid-vitamin complex lysine pantothenate

Biochimica et Biophysica Acta, 798 (1984) 175-179

175

Elsevier BBA 21704

CRYSTAL S T R U C T U R E OF T H E A M I N O A C I D - V I T A M I N C O M P L E X L Y S I N E P A N T O T H E N A T E * DINAKAR M. SALUNKE and MAMANNAMANA VIJAYAN Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India)

(Received July 20th, 1983) (Revised manuscript received December 15th, 1983)

Key words: Amino acid-vitamin complex; Lysine-pantothenate," Crystal structure

L-Lysine D-pantothenate, a 1 : 1 amino acid-vitamin complex, crystallizes in the monoclinic space group P2 t with a = 8.883(2), b = 16.218(5), c = 10.024(2) A, and fl = 106.6(2) °. The structure has been solved by direct methods and refined to an R value of 0.053 for 1868 observed reflections. The zwitterionic positively charged lysine molecules in the structure assume the sterically most favourable conformation with an all.trans side chain trans to the a-carboxylate group. The pantotbenate anion has a somewhat folded conformation stabilised by an intramolecular bifurcated hydrogen bond. The unlike molecules aggregate into separate alternating layers. The molecules in the lysine layers form a head-to-taft sequence parallel to the a-axis. The interactions which hold the adjacent layers together include those between the side chain amino group of lysine and the carboxylate group in the pantothenate anion. The geometry of these interactions is such that each carboxylate group is sandwiched between two amino groups in a periodic arrangement of alternating carboxylate and amino groups.

intoduction We have been pursuing a programme of the preparation and the X-ray analysis of crystalline complexes involving amino acids and short peptides, among themselves as well as with other molecules, in an attempt to elucidate the atomic details of the non-covalent interactions that are important in the structure and action of proteins [1]. These studies have led to the geometrical definition of some biologically significant specific in* This work is part XI of 'X-ray Studies on Crystalline Complexes InvolvingAmino Acids and Peptides'. Supplementary data to this article are deposited with, and can be obtained from, Elsevier Science Publishers, B.V., BBA Data Deposition, P.O. Box 1345, 1000 BH Amsterdam, The Netherlands. References should be made to BBA/DD/227/21704/798 (1984) 175. The supplementaryinformation includes: the list of observed and calculated structure factors, the anisotropic thermal parameters of the individual non-hydrogen atoms and hydrogen parameters. 0304-4165/84/$03.00 © 1984 Elsevier Science Publishers B.V.

teractions [2,3] and have been suggested to be of probable relevance to chemical evolution [4]. The complexes already analysed in this laboratory include two involving the vitamin L-ascorbic acid [5,6]. Here we report the crystal structure of a complex of another vitamin, namely, D-pantothenic acid, with the amino acid L-lysine.

Experimental Sample. Free pantothenic acid was prepared by the addition in molar proportion of oxalic acid to an aqueous solution of a hemicalcium salt of pantothenic acid obtained commercially from Sigma Chemical Company, U.S.A., and the consequent precipitation of calcium oxalate. L-Lysine in molar proportion was then added to t h e supernatant. This solution on slow evaporation yielded the crystals of the complex. Data collection and structure analysis. The unit

176 TABLE 1 CRYSTAL DATA Compound Molecular formula Molecular weight Crystal system Space group

L-lysine D-pantothenate C6H15N20~-. C9H16NO5

t/

b c

/3 V Z Density (measured) Density (calculated)

365.4 monoclinic P2~ 5.883(2) A 16.218(5) ,~ 10.024(2) 106.6(2)° 916.5 ~t 3 2 1.326(8) mg.m -3 1.324 mg-m -a 8.674 cm- 1 (for CuK a-radiation)

TABLE II POSITIONAL PARAMETERS ( x 104) AND EQUIVALENT ISOTROPIC TEMPERATURE FACTORS [22] OF NONHYDROGEN ATOMS Estimated standard deviations are given in parentheses. Atom

X

N(1) O(1) 0(2) C(1) C(2) C(3) C(4) C(5) C(6) N(7) O(11) 0(12) C(13) C(14) C(15) N(16) C(17) 0(18) C(19) 0(20) C(21) C(22) C(23) C(24) 0(25)

- 1955 1892 4347 2362 390 917 -313 597 - 264 691 5144 7 764 5681 3630 4371 5740 4941 2904 6662 8943 5878 3659 5455 7 949 7572

(4) (4) (3) (4) (4) (5) (5) (6) (6) (4) (4) (4) (5) (5) (5) (4) (4) (3) (4) (3) (4) (5) (7) (5) (5)

Y

Z

Beq

7133 (0) 6936(2) 7499 (2) 7227 (2) 7 304 (2) 6702 (2) 6879(2) 6260 (2) 6436 (2) 5 808 (2) 5254(2) 4419 (2) 4624(2) 4088 (2) 3303 (2) 3469 (2) 3378 (2) 3116(2) 3619 (2) 3701 (2) 4436 (2) 4275 (2) 5109(2) 4672 (2) 5433 (2)

5410 (2) 4528 (2) 6420 (2) 5 706 (2) 6434 (2) 7658 (3) 8788 (3) 9972 (3) 11219 (3) 12325 (2) 12146 (2) 11602 (3) 11544 (3) 10716 (3) 10135 (3) 9156 (2) 7785 (3) 7174 (2) 6963 (3) 7944 (2) 6143 (3) 4920 (3) 7100 (4) 5 546 (2) 4803 (2)

1.7 (1) ,~2 2.6 (1) 2.8 (1) 1.6 (1) 1.5 (1) 1.9 (1) 2.1 (1) 2.8 (1) 2.7 (1) 2.0 (1) 2.7(1) 3.4 (1) 2.0(1) 2.6 (1) 2.1 (1) 2.2 (1) 1.6 (1) 2.3(1) 1.6 (1) 2,6 (1) 1.9 (1) 2.7 (1) 3.1 (1) 2.4 (1) 3.1 (1)

cell d i m e n s i o n s a n d the space group were det e r m i n e d from oscillation a n d W e i s s e n b e r g p h o t o graphs. T h e former were s u b s e q u e n t l y refined on a four circle c o m p u t e r c o n t r o l l e d C A D - 4 diffractometer. T h e crystal d a t a are listed in T a b l e I. 1914 reflections were m e a s u r e d u p t o a Bragg angle of 75 ° using g r a p h i t e m o n o c h r o m a t e d C u K aradiation. Of these, 1868 reflections (with I > 2o ( I ) ) were used for structure d e t e r m i n a t i o n a n d refinement. The d a t a were corrected for Lorentz a n d p o l a r i s a t i o n factors. T h e structure was solved by direct m e t h o d s using M U L T A N [7] a n d refined b y the b l o c k - d i a g o n a l least squares p r o c e d u r e using a m o d i f i e d version of the p r o g r a m m e originaly w r i t t e n b y R. Shiano. T h e h e a v y a t o m s a n d the h y d r o g e n a t o m s were given a n i s o t r o p o c a n d isotropic t e m p e r a t u r e factors, respectively. T h e weighting function used in the final cycles of r e f i n e m e n t h a d the f o r m l / ( a + b [ F 0 [ + c i F o [ 2 ) . T h e c o n s t a n t s a, b a n d c were c h o s e n from an analysis of ( l i F o [ - [Fcl[2), in different ranges of I Fo] a n d h a d values 1.380, - 0 . 1 5 4 a n d 0.027, respectively. The scattering factors for the n o n - h y d r o g e n a t o m s a n d the h y d r o g e n a t o m s were taken from Refs. 8 a n d 9, respectively. The final p a r a m e t e r s of the n o n - h y d r o g e n a t o m s are listed in T a b l e II. Results and discussion

T h e lysine molecule in the structure is a zwitter i o n c a r r y i n g a net positive charge with two positively c h a r g e d a m i n o groups a n d a negatively c h a r g e d c a r b o x y l a t e group. T h e p a n t o t h e n a t e ion, o n the other hand, carries a negative charge with a d e p r o t o n a t e d c a r b o x y l group. Molecular dimensions

T h e b o n d lengths a n d angles in the structure, s h o w n in Fig. 1, are normal. T h e torsion angles which describe the c o n f o r m a t i o n of the lysine m o l e c u l e are as in Ref. 10: xo1 = -10.3(3) °

•=168.3(2) °

Xj = -79.0(3) X3 = -175.1(2)

X 2 = -175.1(2)

X' = -179.9(2)

T h e molecule assumes the sterically most favourable c o n f o r m a t i o n with an all-trans side chain trans

177 o (1)

o(12)

~ 0"-~ "~

0~2)

119.ot3)C(13 ) 1'27l(4}O111)

.,w

x,,, ,o

~ .~

TABLE IV

~ ,~,

w , # C ( 2 )i-~9lta) los.ml ~ P d ~" " 1 ' J

#"

C(3) ,16.3 (l)

,ov~(l)C(4)

C(5) ,,3.1(3)

,,o.,

~ 7C(6) (3)

C(1/-,) .~.7 (31

m z (l}C(15)

N(16) ,is.Io)

1is 9(l) C(17),2s~ (* O(18)

0(20)(3.__.~ "41s L C(19) ~,~e"~~"(~)~ C(22)

N ,7

HYDROGEN BOND PARAMETERS D-H...A

D...A

H-D...A

N(1)-HI(N1)... 0025) b N(1)-H2(N1)... 0(2) b N(1)-H3(N1)... O(18) f N(7)-HI(N7)... 0011)" N(7)-H2(N7)... 001) ~ N(7)-H3(N7)... O(12) b N(16)-HI(N16)... O(12)" N(16)-HI(N16)... 0(20) a O(20)-H1(O20)... O(18) a O(25)-H1(O25)... 0011) c

2.820 (3) A 2.714 (3) 2.956 (3) 2.823 (4) 2.789 (3) 2.801 (4) 2.545 (4) 2.853 (4) 2.821 (3) 2.654 (3)

10 (3)° 1 (3) 4 (3) 5 (3) 5 (3) 28 (2) 49 (3) 56 (3) 12 (4) 12 (4)

(a) x,y, z; (b) x - l , y , z ; (c) x,y,z-1; (d) x +l,y, z; (e)x,y, z + l ; ( f ) - x , y + l / 2 , z+l.

l~0.e (3) C(21) 10a.~ (l)

C(19)-- C(21)-- C(24) =105.5(2) C(22)'C(21)--C(23) ,111.8(3)

~amC(24) li 0(25)

Fig. 1. Bond lengths (A) and bond angles (o) involvong non-hydrogen atoms. Estimated standard deviations are given in parentheses.

to the carboxylate group [11] as in the crystal structure of L-lysine hydrochloride [12] and L-lysine L-aspartate [13]. The torsion angles in the pantothenate ion are listed in Table III. Each of these torsion angles. except perhaps C(15)-N(16)-C(17)-C(19), has more than one stericaUy allowed value. Different combi-

TABLE III TORSION ANGLES THAT DEFINE THE CONFORMATION OF PANTOTHENATE ION IN L-LYSINE D-PANTOTHENATE AND CaBr SALT OF D-PANTOTHENIC ACID

nations of them lead to a large n u m b e r of possible conformations for the pantothenate ion. Although some of them are likely to b e c o m e disallowed when all the torsion angles are considered simultaneously, the total n u m b e r of sterically allowed conformations is likely to be still large (a detailed treatment of the molecular conformation of pantothenic acid using semiempirical energy minimisation methods is described in the following paper [14]). The conformation of the pantothenate ion has been characterised earlier only in the crystal structure of a calcium bromide salt of D-pantothenic acid [15]. The conformation observed in the present structure is very different from that observed in the calcium bromide salt, as can be seen from Fig. 2 and Table IV. The c o n f o r m a t i o n in the metal complex is such as to facilitate tridentate chelation, with O(12), O(18) and 0(25) coordinating to the calcium ion. A n interesting feature of the conformation of the vitamin in the present structure is the occurrence of a bifurcated intramolecular hydrogen b o n d involving N(16) as the donor, and O(12) and 0(20) as the acceptors.

Torsion angle

Lysine salt

CaBr salt

Crystal structure and hydrogen bonding

0(11)-C(13)-C(14)-C(15)

- 174.4 (3) ° - 61.6 (3) - 106.8 (3) 177.9 (3) - 107.9 (3) 171.8 (2) - 177.5 (2)

- 168.4 (4)° 69.0 (5) - 113.0 (5) 180.0 (4) - 93.4 (5) - 65.7 (6) 76.8 (6)

The crystal structure o f L-lysine D-pantothenate, shown in Fig. 3, is stabilised by ionic interactions and hydrogen bonds. The parameters of the hydrogen bonds including those of the intramolecular h y d r o g e n b o n d referred to earlier, are given in Table IV. As in the crystal structures of most of the

C(13)-C(14)-C(15)-N(16) C(14)-C(15)-N(16)-C(17) C(15)-N(16)-C(17)-C(19) N(16)-C(17)-C(19)-C(21) C(17)-C(19)-C(21)-C(24) C(19)-C(21)-C(24)-O(25)

178

(a)

(b) Fig. 2. The conformation of the pantothenate ion in (a) L-lysine D-pantothenate and (b) calcium bromide salt of D-pantothenic acid.

complexes, analysed in this laboratory [1,5,13,1619] the unlike molecules aggregate into separate alternating layers in the present structure also. The layers in the a c plane are stacked along the b-axis. The stabilisation of the lysine layer involves two intermolecular hydrogen bonds, one between the

side chain amino group and O(1) of the c~carboxylate group, and the other between the c~amino group and 0(2) of the c~-carboxylate group. The latter gives rise to a head-to-tail sequence [20] . parallel to the a-axis. The pantothenate layer is also stabilised by two intermolecular hydrogen bonds, one between the carboxylate and the terminal hydroxyl groups, and the other between the central hydroxyl and the amide carbonyl groups. The interactions between the layers of unlike molecules involve four hydrogen bonds. The c~amino group of lysine is the donor in two of them, with the terminal hydroxyl oxygen atom and the amide carbonyl oxygen atom of the pantothenate ion as the acceptors. The remaining two involve interactions between the side chain amino group of lysine, as the donor, and the oxygen atom of the carboxylate groups of the pantothenate ion, as acceptors. In the context of protein-vitamin association, the interactions between the positively charged side chain amino group of lysine and the negatively charged carboxylate group of pantothenate merit particular attention. The geometry of these interactions, observed in the crystal structure, is illustrated in Fig. 4. The carboxylate group of each pantothenate ion is sandwiched between two ad-

'x

008)CQ7)

C1~99~ C1721 C(2 e.)

.

23)

[11)

'~,

(1)

\

C(6)

C(3)

O(1J

Cl6~ Ell,)

•

" @ ' "

,

~

C(5 J " - .

1

."

~

".0(12) CI21)

/ /

(15)

0(11)

0(12)l~) C (13)

0(2)

t

/

J

Fig. 3. Crystal structure as viewed down the a-axis. The broken lines indicate hydrogen bonds. N(1) and 0(20) are hydrogenbonded to 0(2) and O(18), respectively, of the molecule related by an a-translation.

/

I

Fig. 4. Interactions involving the side-chain amino group of lysine and the carboxylate group of the pantothenate ion.

179

jacent side chain amino groups of lysine, in a periodic arrangement in which the amino and the carboxylate groups alternate along the a-axis. This arrangement is strikingly similar to that involving the side chain amino group of the lysyl residue and the acetate group, observed in the crystal structure of N-acetylglycyl L-lysine methyl ester acetate [21].

Acknowledgement We thank the Department of Science and Technology, India, for financial support.

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