Computer Modeling of Carbohydrate Molecules - American Chemical

Chapter 14

Downloaded by UNIV OF TEXAS AT DALLAS on July 11, 2016 | http://pubs.acs.org Publication Date: July 6, 1990 | doi: 10.1021/bk-1990-0430.ch014

Molecular Modeling Studies on Unbranched Complex Carbohydrates Application to a Linkage Region Fragment from Connective Tissue Proteoglycans N. Rama Krishna, Bo-Young Choe, and Stephen C. Harvey Departments of Biochemistry and Physics, and the Comprehensive Cancer Center, University of Alabama, Birmingham, AL 35294 A general molecular modeling methodology particularly suitable for unbranched complex carbohydrates was described. This methodology employed molecular dynam ics (MD) and energy minimization (EM) procedures together with inter-residue spatial constraints across the linkages derived from 2D-NOESY spectroscopy. The f i r s t step in this methodology is the generation of a wide variety of starting conformations that span the (Ф,ψ) space for each linkage. In the present study, for each linkage, nine starting conformations that span the (Ф,ψ) space in a resonable manner were con structed using the torsion angles Ф and ψ correspond ing to the gauche+, gauche-, and trans configurations across each of the two bonds constituting the linkage. These conformations were subjected to a combined MD/EM refinement using the NOESY derived constraints as pseudoenergy functions. Families of conformations for the whole molecule were then constructed from the structures derived for each linkage. This procedure was demonstrated on a fragment from the carbohydrateprotein linkage region of connective tissue proteogly cans. Connective tissue proteoglycans are predominantly composed of glycosaminoglycans, some oligosaccharides and a core protein (1). The glycosaminoglycans are covalently attached to the core protein through a unique linkage region composed of a short oligosaccha ride. During the course of our investigation on the various oligo saccharides derived from the proteoglycans, we have developed a general molecular modeling methodology based on NMR and molecular dynamics (MD) and energy minimization (EM). While the methodology can be adapted for any complex carbohydrate, unbranched oligosac charides particularly lend themselves to a simplified analysis by this methodology since the conformation for the whole oligosaecha0097-6156/90/043O-O227$06.00A) © 1990 American Chemical Society

French and Brady; Computer Modeling of Carbohydrate Molecules ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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COMPUTER MODELING OF CARBOHYDRATE MOLECULES


r i d e can be c o n s t r u c t e d from the c o n f o r m a t i o n s of the i n d i v i d u a l modules composed o f two r e s i d u e s l i n k e d t o g e t h e r ( a d i s a c c h a r i d e or a monosaccharide l i n k e d t o an amino a c i d ) . I m p l i c i t i n t h i s modular a n a l y s i s approach i s the assumption t h a t i n t e r a c t i o n s between nonn e i g h b o u r i n g r e s i d u e s are n e g l i g i b l e . Such a s i t u a t i o n i s commonly r e a l i z e d a t the o l i g o s a c c h a r i d e l e v e l i n c o n n e c t i v e t i s s u e p r o t e o g l y c a n s where 1->3 and 1 — > l i n k a g e s are f r e q u e n t l y encountered. We w i l l i l l u s t r a t e our methodology u s i n g Ο-β-D-galactopyranosyl - ( 1 - > ^ ) - 0 - 3 - D - x y l o p y r a n o s y l - ( 1 - > 0 ) - L - s e r i n e (GXS), a fragment from the c a r b o h y d r a t e - p r o t e i n l i n k a g e r e g i o n of x y l o s e / s e r i n e l i n k e d proteoglycans. Methodology: The combined use of MD, EM and c o n s t r a i n t s d e r i v e d from n u c l e a r Overhauser e f f e c t s (NOEs) t o r e f i n e model s t r u c t u r e s f o r b i o l o g i c a l m o l e c u l e s i s now w e l l e s t a b l i s h e d (2-7 ). The p r o t o c o l used i n our methodology i s shown i n F i g u r e 1. Because of the l a c k of a g e n e r a l s o l u t i o n t o t r e a t m u l t i p l e minima i n the (φ,ψ) space ( 8 , 9 ) , i t i s e s s e n t i a l t o c o n s t r u c t s e v e r a l s t a r t i n g s t r u c t u r e s so t h a t they can span the a v a i l a b l e c o n f o r m a t i o n a l space i n a r e a s o n a b l e manner ( 8 ) . Hence Stage 1 of the p r o t o c o l i n v o l v e s the c o n s t r u c t i o n of s e v e r a l s t a r t i n g s t r u c t u r e s t h a t s a t i s f y t h i s r e q u i r e m e n t . Our i n i t i a l a t t e m p t s t o g e n e r a t e such s t r u c t u r e s by s u b j e c t i n g an a r b i t r a r y c o n f o r m a t i o n of GXS t o a h i g h - t e m p e r a t u r e (1000 K) MD s i m u l a t i o n ( a 50 ps MD s i m u l a t i o n w i t h o u t c o n s t r a i n t s , where random atomic v e l o c i t i e s c o r r e s p o n d i n g t o 1000 Κ were r e p e a t e d l y a s s i g n e d every 5 ps) showed t h a t t h i s MD s i m u l a t i o n c o u l d not overcome the b a r r i e r s a c r o s s the i n t e r r e s i d u e l i n k a g e s , and as a r e s u l t the c o n f o r m a t i o n s tended t o l o c a l i z e around t h r e e p o i n t s i n the (φ,ψ) space r a t h e r than sample the a v a i l a b l e space. Ha e t a l have chosen, i n t h e i r s i m u l a t i o n of m a l t o s e ( 8 ) , s t a r t i n g c o n f o r m a t i o n s d e f i n e d on a 20° g r i d i n the (φ,ψ) space and t h e s e were s u b s e q u e n t l y energy m i n i mized. I n the p r e s e n t s t u d y , we chose a t o t a l of n i n e s t a r t i n g con f o r m a t i o n s over the (φ,ψ) space f o r each i n t e r r e s i d u e l i n k a g e ( i . e . , 81 s t a r t i n g c o n f o r m a t i o n s f o r GXS, and i n g e n e r a l 9 start i n g c o n f o r m a t i o n s f o r an o l i g o s a c c h a r i d e w i t h " η l i n k a g e s ) and s u b j e c t e d each o f these t o h i g h - t e m p e r a t u r e MD e v o l u t i o n (1000 K, 5 p s ) . The s t a r t i n g c o n f o r m a t i o n s were d e f i n e d by a s s i g n i n g the t h r e e t o r s i o n a l a n g l e v a l u e s c o r r e s p o n d i n g t o gauche+, gauche- and t r a n s c o n f i g u r a t i o n s a c r o s s the C-0 and 0-C bonds d e f i n i n g the l i n k a g e . Our c h o i c e of 9 s t a r t i n g c o n f o r m a t i o n s f o r each l i n k a g e was d i c t a t e d by our d e s i r e t o l i m i t the c o m p u t a t i o n s t o a t r a c t a b l e num b e r , w h i l e a s s u r i n g t h a t enough c o n f o r m a t i o n s have been s e l e c t e d t o span the (φ,ψ) space i n a r e a s o n a b l e manner. C l e a r l y , the p r o t o c o l shown i n F i g u r e 1 can be used f o r any a r b i t r a r y number of s t a r t i n g c o n f o r m a t i o n s s p a n n i n g the (φ,ψ) space. Our p r o t o c o l and the a d i a b a t i c mapping procedure d e s c r i b e d by Ha e t a l (8) have two f e a t u r e s i n common. They are both based on m o l e c u l a r mechanics approach, and they both s t a r t w i t h s e v e r a l s t r u c t u r e s s c a t t e r e d a t r e g u l a r i n t e r v a l s over the (φ,ψ) space. But the o b j e c t i v e s of the approaches are f u n d a m e n t a l l y d i f f e r e n t . Whereas the a d i a b a t i c mapping procedure i s i n t e n d e d t o f u l l y c h a r a c t e r i z e the c o n f o r m a t i o n a l energy s u r f a c e of d i s a c c h a r i d e s , our 11

f



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STAGE 1

Unbranched Complex Carbohydrates

STAGE2

STAGE3

STAGE4

STRUCTURES

STRUCTURES

STRUCTURES

N STARTING STRUCTURES

229

STAGE5 F!NA] ÎAL STRUCTURES

Figure 1: The protocol used i n the molecular modeling studies on complex carbohydrates. For unbranched carbohy drates, this protocol i s applied to each interresidue l i n kage at a time and uses Ν starting conformations (Stage 1) and arrives at Ν f i n a l structures (Stage 5 ) . In the cur rent investigation, we chose 9 s t a r t i n g conformations as described i n the text. The Stage 5 structures generated for each linkage serve as modules f o r the construction of the structures f o r the entire molecule. These are subjected to a f i n a l step of energy minimization.



230


p r o c e d u r e i s aimed a t f i n d i n g a l l c o n f o r m a t i o n s t h a t a r e c o n s i s t e n t w i t h a s e t o f e x p e r i m e n t a l l y determined d i s t a n c e c o n s t r a i n t s . I n o r d e r t o r e l a t e t h e modeled s t r u c t u r e s t o e x p e r i m e n t a l observables during t h i s high-temperature MD s i m u l a t i o n , s p a t i a l c o n s t r a i n t s between hydrogen atoms a c r o s s the l i n k a g e were i n c l u d e d i n t h e form o f a pseudoenergy f u n c t i o n ( v i d e i n f r a ) . The r e s u l t i n g s t r u c t u r e s (Stage 2) were annealed by an a d d i t i o n a l 5 ps MD s i m u l a t i o n a t 300 Κ w i t h NOE c o n s t r a i n t s t o a r r i v e a t t h e s e t o f s t r u c t u r e s i n "Stage 3". These s t r u c t u r e s were s u b j e c t e d t o 200 s t e p s of energy m i n i m i z a t i o n u s i n g t h e c o n j u g a t e g r a d i e n t method. The NOE c o n s t r a i n t s were r e t a i n e d d u r i n g t h i s s t e p t o produce models c o m p a t i b l e w i t h NOE d a t a . The r e s u l t i n g s e t o f n i n e s t r u c t u r e s (Stage 4) were then s u b j e c t e d t o 200 s t e p s o f energy m i n i m i z a t i o n w i t h o u t NOE c o n s t r a i n t s . T h i s l a s t s t e p r e l i e v e s the s t r a i n s i n t h e s t r u c t u r e s i n t r o d u c e d by t h e NOE pseudoenergy f u n c t i o n , and p r o duces models (Stage 5) w i t h a c c e p t a b l e s t e r e o c h e m i s t r y i n terms o f bond l e n g t h s and bond a n g l e s . The parameters o f t h e p o t e n t i a l energy f u n c t i o n used i n o u r c a l c u l a t i o n s on GXS were the same as those used i n an e a r l i e r s t u d y of c y c l o d e x t r i n (10), which were d e r i v e d from GROMOS (11,12) w i t h s l i g h t m o d i f i c a t i o n , as f o l l o w s . The p a r t i a l c h a r g e s f o r a s i n g l e sugar u n i t i n c l u d i n g t h e hydrogens were c a l c u l a t e d (10) u s i n g t h e G a u s s i a n 80 (UCSF) program w i t h minimal b a s i s s e t and w i t h o u t g e o m e t r i c a l o p t i m i z a t i o n . The atomic c h a r g e s f o r t h e s e r i n e r e s i due were those o f GROMOS (11). The amino group o f s e r i n e was assumed t o be d e p r o t o n a t e d and t h e c a r b o x y l was assumed t o be p r o t o n a t e d t o remove t h e p o s i t i v e and n e g a t i v e c h a r g e s s o a s t o make the charge d i s t r i b u t i o n c o m p a t i b l e w i t h t h a t i n t h e n a t i v e c o r e protein. T a b l e I c o n t a i n s a l i s t o f p a r t i a l c h a r g e s f o r t h e GXS m o l e c u l e used i n t h e computer modeling s t u d i e s . A d i s t a n t depen dent d i e l e c t r i c c o n s t a n t was used i n the c a l c u l a t i o n s ( 6 ) . I n a d d i t i o n t o t h e u s u a l terms f o r t h e c o v a l e n t and noncoval e n t i n t e r a c t i o n s , a c o n s t r a i n t energy p e n a l t y term was added t o the p o t e n t i a l f u n c t i o n f o r some o f t h e MD/EM runs i n t h e p r o t o c o l as d e s c r i b e d above. A semiharmonic form was used f o r t h i s term: E(NOE) = K/2 = 0

( r - r ) 0

2

for r > r for r £ r

0

(1)

0

1

2

where Κ i s t h e f o r c e c o n s t a n t (3 k c a l / m o l - A - i n the p r e s e n t c a l c u l a t i o n s ) , r i s t h e i n t e r p r o t o n d i s t a n c e , and r i s t h e NOE c u t o f f d i s t a n c e . A v a l u e o f 3·5 X has been used f o r r i n o u r calculations. 0

0

NMR

Spectroscopy:

The t e s t o l i g o s a c c h a r i d e , GXS, was c h a r a c t e r i z e d e x t e n s i v e l y on a B r u k e r WH-400 NMR s p e c t r o m e t e r ( o p e r a t i n g f r e q u e n c y 400 MHz) by 1Dand 2D-NMR s p e c t r t o s c o p y . The s y n t h e s i s (13) and the NMR s p e c t r o s c o p i c c h a r a c t e r i z a t i o n (13» 1^) o f GXS has been r e p o r t e d elsewhere. To g e n e r a t e t h e d i s t a n c e c o n s t r a i n t s t o be used i n t h e con s t r a i n t energy f u n c t i o n , E(N0E), t h e s p a t i a l c o n t a c t s were e s t a b l i s h e d on t h e b a s i s o f a 2D-N0ESY experiment w i t h a 400 ms m i x i n g time performed on a Bruker WH-400 NMR s p e c t r o m e t e r . The f o l l o w i n g


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Table I . L i s t o f P a r t i a l Atomic Charges f o r GXS


Residue Name Gal Gal Gal Gal Gal Gal Gal Gal Gal Gal Gal Gal Gal Gal Gal Gal Gal Gal Gal Gal Gal Gal xyi xyi xyi Xyl Xyi Xyl Xyl Xyl Xyl Xyl Xyl xyi xyi xyi xyi xyi xyi Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser

Atom Name cr H1* C2» 0 H H2' C3 0 H H3 C4' 0 H H4» C5 Η5· C6' 0 Η Η6· Η7' 05· 0 C1» Η1' C2 0 H H2 C3 0 H H3 C4' H4' C5 H5' H5" 05 0 CB HB HB" Ca Ha N H H C 0 0 H 1

f

f

f

f

f

f

f

1

f

Atom Form C1 H1 C2 02 H9 H2 C3 03 H10 H3 CM 04 H11 H4 C5 H5 C6 06 H12 H6 H7 05 040 C10 H8 C20 020 H22 H21 C30 030 H32 H31 C40 H41 C50 H51 H52 010 060 C60 H61 H62 C70 H71 N1 H72

H73 C80 080 090 H90

Atom Type CS1 HC CS1 OA HO HC CS1 OA HO HC CS1 OA HO HC CS1 HC CS2 OA HO HC HC OS OS CS1 HC CS1 OA HO HC CS1 OA HO HC CS1 HC CS1 HC HC OS OS CH2 HC HC CH1 HC NT H H C 0 OA HO

Sequence Number 1 2 3 4 5 6

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

Atom Partial Charge 0.199 0.050 0.026 -0.386 0.268 0.093 0.065 -0.356 0.232 0.062 0.056 -0.360 0.241 0.069 0.061 0.059 0.020 -0.529 0.385 0.081 0.051 -0.270 -0.268

0.212 0.045 0.027 -0.333 0.223 0.100 0.168 -0.301 0.215 -0.053 0.101 0.008 0.002 0.088 0.043 -0.267 -0.257 -0.095 0.110 0.115 -0.005 0.107 -0.465

0.131 0.230

0.381 -0.294 -0.400

0.317


231

232


i n t e r r e s i d u e NOE c o n t a c t s were observed: Gal-H1 t o Xyl-H4; Gal-H1 t o Xyl-H5 ; and Xyl-H1 t o Ser-H ·. I n a d d i t i o n , i n t r a r e s i d u e con t a c t s Gal-?I1 t o Gal-H3, Gal-H1 ^ o Gal-H5, Xyl-H1 t o Xyl-H3, and Xyl-H1 t o Xyl-H5 were a l s o observed. A l l these i n t e r and i n t r a r e s i d u e s p a t i a l c o n t a c t s were used i n the c a l c u l a t i o n s . Results: G e n e r a t i o n of S t a r t i n g Conformations

(Stage 1 ) :

X

On the b a s i s o f the H v i c i n a l c o u p l i n g c o n s t a n t a n a l y s i s (13,14), both the sugars were assumed t o adopt the C c h a i r conformations as s t a r t i n g s t r u c t u r e s i n Stage 1. No sugar r e p u c k e r i n g was observed i n any of the s i m u l a t i o n s . The e x o c y c l i c t o r s i o n a n g l e θ ( 0 5 - C 5 - C 6 - 0 6 ) f o r the g a l a c t o s e r e s i d u e has been a s s i g n e d a s t a r t i n g v a l u e o f 178.2° as observed i n the c r y s t a l s t r u c t u r e f o r 3-D-galactose ( 1 5 ) . An a n a l y s i s of the v i c i n a l c o u p l i n g c o n s t a n t d a t a ( J , J ") f o r the s i d e c h a i n of the s e r i n e r e s i d u e i n d i c a t e d t n a t the s i d e c h a i n e x i s t e d p r e d o m i n a n t l y i n the "C" rotamer. Hence a v a l u e o f χ = 60° was used f o r a l l the Stage 1 conforma t i o n s . I n d e f i n i n g the Stage 1 c o n f o r m a t i o n s , the t o r s i o n a n g l e s f o r each l i n k a g e ( i . e . , φ , ψ f o r X-S and φ , ψ f o r G-X) were g i v e n v a l u e s t h a t c o r r e s p o n d t o the gauche+, gauche- and t r a n s c o n f i g u r a tions. T h i s procedure generated 9 c o n f o r m a t i o n s f o r each l i n k a g e (see T a b l e s I I and I I I ) . H


X

?

f

f

f

f

1

Χ

Χ

2

2

MP/EM C a l c u l a t i o n s : The i n t e r r e s i d u e l i n k a g e s X-S and G-X were s u b j e c t e d s e p a r a t e l y t o the p r o t o c o l shown i n F i g u r e 1 and the v a r i a t i o n s i n the t o r s i o n a n g l e s due t o r e f i n e m e n t a t d i f f e r e n t s t a g e s a r e shown i n T a b l e s I I and and I I I , r e s p e c t i v e l y . The s i d e c h a i n o r i e n t a t i o n of s e r i n e d e f i n e d by the t o r s i o n a n g l e χ remains r e l a t i v e l y i n v a r i a n t a t the v a r i o u s s t a g e s of the r e f i n e m e n t (see T a b l e I I ) . The e x o c y c l i c t o r s i o n a n g l e , θ (OS'-CS'-Co'-Oo*) foç the g a l a c t o s e r e s i d u e groups i n t o two v a l u e s c e n t e r e d around 177.2 and 60.7°in the f i n a l s t a g e . On the o t h e r hand, the l i n k a g e t o r s i o n a n g l e s φ and and φ and ψ e x p e r i e n c e c o n s i d e r a b l e v a r i a t i o n s a t v a r i o u s s t a g e s of the MD/EM r e f i n e m e n t . These v a r i a t i o n s a t d i f f e r e n t s t a g e s are a l s o p l o t t e d i n F i g u r e s 2 and 3 t o emphasize the convergence of n i n e s t a r t i n g c o n f o r m a t i o n s i n t o d i s t i n c t f a m i l i e s . The c o n f o r m a t i o n s f o r X-S converge i n t o t h r e e d i s t i n c t f a m i l i e s , A B and C . For the o t h e r l i n k a g e , G-X, the c o n f o r m a t i o n s converge i n t o two d i s t i n c t f a m i l i e s , A and B . I n each c a s e , t h e r e was o n l y one s e t o f c o n f o r m a t i o n s i n t h e f i n a l s t a g e t h a t c o r r e c t l y reproduced the observed NOESY c o n t a c t s (see T a b l e I V ) . These a r e the A c o n f o r m a t i o n s f o r X-S and t h e A c o n f o r m a t i o n s f o r G-X. A p p a r e n t l y the o t h e r c o n f o r m a t i o n s r e p r e s e n t models t h a t are t r a p p e d i n l o c a l energy minima. S i n c e they do not s a t i s f y the observed NOE c o n t a c t s , t h e s e o t h e r c o n f o r m a t i o n s (B Cj and B ) a r e i g n o r e d i n the remainder of the a n a l y s i s . From t h e s e r e s u l t s , the c o n f o r m a t i o n f o r the GXS m o l e c u l e was c o n s t r u c t e d as A A Here the average l i n k a g e t o r s i o n a n g l e s f o r the A (G-X) and Aj (X-S) f a m i l i e s have been used. A v a l u e of 1

2

2

l f

2

l f

2

x

l f

2

2

l

i e

2


2

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233


Table II ; Results of MD/EM Calculations on the XS Linkage

Torsion Angles in X-S*

Stage 1

Stage 2

Stage 3

-63.4 +175.5 +63.5

Stage 4

Stage 5

Final Family

Φι Φι Χ

+60.0 +60.0 +60.0

-62.0 +179.8 +55.1

-61.6 +178.6 +61.1

-59.9 +178.7 +61.3

Bi

Φι Ψι Χ

+60.0 -60.0 +60.0

+ 165.Κ-194.9) +165.6C-194.4) -177.7 +92.4 +68.7 +94.7 +69.8 +57.9 +58.9

-178.3 +67.9 +58.0

Αι

Φι Φι Χ

+60.0 ±180.0 +60.0

+5.8 +160.3 +73.5

-60.5 +179.2 +62.2

-58.7 +179.6 +63.1

Β»

Φι Φι Χ

-60.0 +60.0 +60.0

+144.5C-215.5) +107.7C-192.3) -175.2 +95.2 +80.8 +73.1 +61.2 +61.6 +68.7

-175.8 +73.2 +60.6

Αχ

Φι Φι Χ

-60.0 -60.0 +60.0

-68. 4 -140.8 +70.0

-63.3 -92.2 +64.1

-70.8 -76.8 +67.3

-67.9 -75.7 +66.7

Ci

Φι Φι Χ

-60.0 ±180.0 +60.

-49.3 -175.8 +62.7

-64.5 +176.9 +58.1

-61.4 ±180.0 +62.1

-61 .4 ±180.0 +62.1

Bi

Φι Φι Χ

±180.0 +60.0 +60.0

-120.5 +63.7 +50.9

+167.4(-192.6) -177.5 +70.9 +85.5 +61.7 +58.7

-177.9 +69.3 +57.8

Ai

Φι Φι Χ

±180.0 -60.0 +60.0

-110.6 +50.4 +64.7

-118.8 +59.4 +52.0

-172.2 +81.8 +59.8

-171.7 +81.4 +59.8

Ai

Φι Φι Χ

±180.0 ±180.0 +60.0

+175.9C-184.1) +163.9C-196.1 ) +179.8(-180.2) -177.5 +82.4 +95.9 +69.7 +71.7 +58.0 +60.2 +58.5 +58.9

Ai

-54.5 +174.8 +55.8

f

* φ (05'-C1'-Ο-Οβ) and ψ (C1 -0-CB-Ca) define the linkage torsion angles between Xyl and Ser. x(0-C&-Ca-N) defines the side chain orientation of Ser. The angles are expressed i n degrees. χ

1



2

2

2

2

2

f

-60.9 +123.2

-1.2

2

-78.9 +138.4

-65.7 + 158.2

±180.0 -60.0

±180.0 ±180.0

-56.9 +134.5

-55.8 +140.6

-77.6 -67.6

-49.8 +118.2

±180.0 +60.0

-60.0 ±180.0

-60.0 -60.0

-60.0 +60.0

f

-57.3 +127.6

-59.5 +123.8

-60.3 + 119.5

-58.9 +120.9

-71.8 -61.4

-60.9 +126.9

-80.9 -63.3

-51.2 -40.2

+60.0 -60.0

+132.5

-56.9 +115.0

-46.2 +99.5

+60.0 +60.0

+60.0 ±180.0

Stage 3

Stage 2

Stage 1

-56.9 +124.7

+ 131

-63.2 .9

-64.2 +129.3

-57.1 +124.4

-77.8 -55.9

-57.4 +124.9

-57.8 +123.7

-69.0 -51.9

-64.9 +127.2

Stage 4

-59.6 +127.0

-65,9 +132.2

-64.7 +128.9

-60.7 +126.4

-76.0 -55.1

-59.4 +126.8

-62.1 +127.6

-75.2 -54.8

-67.1 +129.0

Stage 5

2

φ ( 0 5 ' - Π -0-C4') and ψ (C1'-0-C4'-C3 ) define the linkage t o r s i o n angles between Gal and X y l . The angles are expressed i n degrees.

Φ Ψ

2

2

2

2

2

2

2

2

2

2

2

Φ Ψ

Φ Ψ

Φ Ψ

Φ Ψ

Φ Ψ

Φ Ψ

2

Φ* Ψ

Φ Ψ

Linkage Torsion Angles in G-X*

Table I I I ; Results of MD/EM C a l c u l a t i o n s on the GX linkage


A

A

A

A

2

2

2

2

2

2

2

2

2

B

A

A

B

A

Final Family


F i g u r e 2: V a r i a t i o n i n the l i n k a g e t o r s i o n a l a n g l e s ( φ ^ ψ χ ) f o r the XS l i n k a g e o f GXS a t d i f f e r e n t s t a g e s o f the MD/EM p r o t o c o l i n f i g u r e 1. French and Brady; Computer Modeling of Carbohydrate Molecules ACS Symposium Series; American Chemical Society: Washington, DC, 1990.



236

ψ

2

Figure 3: Variation i n the linkage torsional angles (φ ,ψ ) for the GX linkage of GXS at d i f f e r e n t stages of the MD/EM protocol i n figure 1. 2


2

14.


KRISHNA ET A L

237

177.2° was used f o r t h e e x o c y c l i o c t o r s i o n a n g l e o f g a l a c t o s e i n the A c o n f o r m a t i o n s . To account f o r any l o n g range e f f e c t s on charge d i s t r i b u t i o n and t o r e l i e v e any s t e r i c c o n f l i c t s t h a t might a r i s e due t o t h e modular c o n s t r u c t i o n o f t h e c o n f o r m a t i o n o f t h e whole m o l e c u l e , t h e f a m i l y o f c o n f o r m a t i o n s A A i was s u b j e c t e d t o additional energy m i n i m i z a t i o n w i t h o u t NOE c o n s t r a i n t s . The r e s u l t i n g f a m i l y , A 'h \ i s r e p r e s e n t a t i v e o f the conformations o f GXS c o m p a t i b l e w i t h t h e e x p e r i m e n t a l d a t a . I n t h e s e c o n f o r m a t i o n s ( A A ) , one weak i n t e r r e s i d u e hydrogen bond between X y l 3*0H and G a l 0 5 has been d e t e c t e d (0...H 2.6 A , 0-H...0 114°). T h i s was an a r t i f a c t stemming from t h e i n vacuo c a l c u l a t i o n s , and c o u l d be overcome by c h o o s i n g an a p p r o p r i a t e d i e l e c t r i c c o n s t a n t . F i g u r e 4 shows a t y p i c a l example from t h e f i n a l A A set of structures f o r GXS. Even though t h e A 'k * f a m i l y e x p l a i n s the observed NOE cont a c t s i n GXS, we have a l s o examined whether a c o n f o r m a t i o n a l exchange i n v o l v i n g d i f f e r e n t f a m i l i e s c o u l d a l s o e x p l a i n t h e observed i n t e r r e s i d u e NOE c o n t a c t s . T a b l e IV shows a comparison o f the c a l c u l a t e d i n t e r r e s i d u e p r o t o n d i s t a n c e s f o r t h e f a m i l i e s A Bj, C A and B , t o g e t h e r w i t h t h e average t o r s i o n a n g l e s . The B f a m i l y p r e d i c t s t h a t both 3» and 3" p r o t o n s w i l l e x p e r i e n c e a s i g n i f i c a n t NOE c o n t a c t w i t h Xyl-H1' p r o t o n , whereas t h e C f a m i l y p r e d i c t s a NOE c o n t a c t between t h e X y l - H 1 and S e r H&". These cont a c t s were n o t observed i n t h e experiment. The B f a m i l y f o r t h e GX l i n k a g e p r e d i c t s t h a t both t h e H5 and H5" p r o t o n s o f x y l o s e w i l l e x p e r i e n c e s t r o n g NOE c o n t a c t w i t h G a l - H 1 p r o t o n . This too i s a t v a r i a n c e w i t h t h e experiment. Thus a c o n f o r m a t i o n a l exchange w i t h f a m i l i e s such as A B , B ^A , B C e t c would have r e s u l t e d i n NOE c o n t a c t s t h a t were n o t observed i n t h e experiment on GXS. T h i s suggests t h a t these f a m i l i e s do not make a major cont r i b u t i o n t o t h e c o n f o r m a t i o n a l e q u i l i b r i u m o f GXS. However, f o r o t h e r types o f o l i g o s a c c h a r i d e s , such a c o n f o r m a t i o n a l exchange s h o u l d a l s o be c o n s i d e r e d i n t h e modeling c a l c u l a t i o n s . I n c o n c l u s i o n , we have developed a g e n e r a l methodology p a r t i c u l a r l y s u i t a b l e f o r modeling o f unbranched complex c a r b o h y d r a t e s , s i n c e i t l e n d s i t s e l f t o a modular a n a l y s i s o f a l o n g c a r b o h y d r a t e chain. T h i s methodology i s based on MD/EM c a l c u l a t i o n s w i t h NMR d e r i v e d c o n s t r a i n t s introduced i n t o the c a l c u l a t i o n s t o generate conformations compatible with the experimental data. Our approach d i f f e r s from t h a t o f S c a r s d a l e e t a l (7) i n some e s s e n t i a l d e t a i l s . The form o f t h e pseudoenergy f u n c t i o n chosen i n our work t o r e p r e s e n t NOE c o n s t r a i n t s i s s i m i l a r t o t h a t used by K a p t e i n e t a l (5) i n t h e i r p r o t e i n modeling s t u d i e s , b u t d i f f e r s from t h a t o f S c a r s d a l e e t a l . The l a t t e r a u t h o r s have used a p o t e n t i a l f u n c t i o n t h a t h a s a n e g a t i v e minimum a t r - r , approaches z e r o f o r r >> r and i s p o s i t i v e f o r r

Computer Modeling of Carbohydrate Molecules - American Chemical

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