T h e EMBO Journal vol.5 no.2 pp.409-413, 1986 Location o f 'continuous' antigenic determinants i n t h e protruding regions o f proteins J.M.Thornton, M.S.Edwards, W.R.Taylor a n d D.J.Barlow Laboratory of Molecular Biology, Department o f Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, UK Communicated b y D.C.Phillips A simple method i s described to locate 'antigenic' peptides from t he a-carbon co-ordinates o f a protein, based on pro- trusion from t h e protein's globular surface. A good corre- lation is found between those parts o f a protein which protrude a n d t he experimentally determined antigenic pep- tides i n myoglobin, lysozyme and myohemerythrin. A com- parison i s made between t h e u se o f protrusion index, mobility, solvent accessibility a n d hydrophilicity fo r predicting t h e most likely antigenic peptides. K e y words: antigenicity/peptides/prediction/protrusion index/con- tinuous determinants Introduction Although t h e immune response t o a foreign protein i s complex a n d incompletely understood, i t ha been shown that short pep- tides taken from a protein will cross-react with antibodies raised against t h e complete protein (Atassi, 1984; Milton a n d V an Regenmortel, 1979). Furthermore i t h a s been proposed that some peptides c an b e used t o raise anti-peptide antibodies, which recog- nize t he complete protein an d could therefore b e used t o generate synthetic vaccines (Lerner, 1982). T o recent publications have emphasized t h e importance of peptide flexibility (Westof et al., 1984; Tainer et al., 1984) a n d have shown that i n the protein t h e regions corresponding t o t he antigenic peptides are usually highly mobile. An alternative viewpoint i s that these regions (which we will term 'peptide determinants') are antigenic because they ar e t h e most accessible t o th e large antibody molecule. I n this paper we show that f o r lysozyme, myoglobin a n d myohem- erythrin, t h e experimentally determined peptide determinants cor- respond predominantly t o those parts of t he structure which protrude, a s defined by generating an equi-momental ellipsoid t o f i t t h e protein a n d calculating those segments which 'stick out'. T h e correlation with t h e experimental data is superior t o that obtained using hydrophilicity (Hopp a n d Woods, 1981) a n d com- parable with t h e results presented n mobility (Westof et al., 1984; Tainer e t al., 1984, 1985). To test t he hypothesis that peptide determinants protrude from t h e surface o f t h e protein, it wa s necessary t o construct an ap - proximation t o that surface. Since most proteins ar e distinctly ellipsoidal i n shape (Taylor e t al., 1983; Prabhakaran a n d Pon- nuswamy, 1982), t h e ellipsoid was calculated which h a s t h e same moments of inertia a s t he protein structure (the equi-momental ellipsoid), using a standard analytical method involving Cauchy's Momental ellipsoid (Taylor e t al., 1983). This method determines t he ratios between t h e lengths o f t h e principal axes a , b , c a n d their directions, b u t t he absolute size o f t h e ellipsoid i s arbitrari- l y chosen t o include a specified percentage of atoms. Fo r exam- O I R L Press Limited, Oxford, England ple, t he 9 0 %llipsoid will include 9 0 %o f t h e atoms, with 10 % lying outside or protruding from t he globular shape. Therefore we c an simply assign t o each residue a 'protrusion index' or P I specifying t h e % ellipsoid a t which that residue first becomes external. F o r example, a l l residues which a r e outside t he 9 0 % ellipsoid ar e assigned a PI=9; those which are outside t h e 8 0 % ellipsoid (but n o t th e 9 0% ellipsoid) have P I= 8 , etc. Thus f o r each protein the ratios b / a a n d c/a, a n d the directions o f t h e principal axis, are calculated from the co-ordinates (all atoms or just a-carbon co-ordinates c an be included). Then t he 90%, 80%, 70%, etc. ellipsoids ar e generated a n d t he PI assigned t o each residue. Ellipsoids c an b e generated fo r each domain o f a multi-domain protein or f o r t he whole structure. Th e method was applied t o three proteins f o r which t h e a- carbon co-ordinates were available as well as some evidence t o Table I . Averaged values of P I , accessibility, mobility over t h e length of each peptide Protein a d Protrusion Accessibility Mobility Hydrophilicity peptide index P I % A CC % B % H % Myoglobin 1- 6 6. 8 1 8 7 7 12 1 7 5 0 . 5 3 5 15-22 5.5 32 56 42 1 2 2 3 0 . 6 3 0 56-62 6 . 9 1 6 6 5 2 8 8 73 a 1 . 4 3 94-99 4. 2 3 6 75 1 4 1 2 19 0.5 3 8 113-119 3. 6 6 4 a 5 5 4 6 1 4 12 -0.2 7 6 a 121-127 7 . 4 8 5 0 5 2 a 1 5 8 0 . 4 4 2 145-151 8.3 2 8 5 5 13 14 0 68 a Lysozyme 38-54 4 . 6 4 3 5 2 4 6 1 5 8 2 a 0.19 25 64-80 5 .6 15 6 4 1 0 2 5 9 3 0 Myohemerythrin Highb 3-16 5 . 6 1 2 3 1 1 2 0.16 5 2 a 7-16 4 . 8 4 0 29.5 3 4 -0.19 7 8 a 37-46 5 . 8 1 8 29.5 3 3 0.35 3 5 57-66 6 . 5 9 2 9 4 3 0.74 11 Mediumb 63-72 7 . 2 1 3 2 7 0 . 3 3 8 69-82 5 . 3 2 1 30 26 0 . 6 11 73-82 4.1 58 28.6 4 8 0.84 8 Lowb 22-35 2 . 1 98 22 9 5 0.78 4 26-35 1.7 9 8 21.7 9 2 0.24 4 4 42-51 3 . 3 73 29 3 9 -0.5 9 2 96-109 2 . 6 9 0 22 93 -0.11 8 6 100-109 2 . 7 8 0 2 1 9 8 0 6 5 S e e legend t o Figure 1 for data sources. T h e % columns represent t h e percentage of peptides, o f equal length t o t h e epitope, with higher average values than t h e epitope. F o r example, 18 % o f hexapeptides i n myoglobin have P I 2 6 . 8 aActive peptides where percentile is >50%. bFor definition o f high, medium a n d l o w activity se e legend t o Figure i c . 4 09
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8/6/2019 Location of 'continuous' antigenic determinants in the protruding regions of proteins
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T h e EMBO J o u r n a l v o l . 5 n o . 2 p p . 4 0 9 - 4 1 3 , 1 9 8 6
L o c a t i o n o f ' c o n t i n u o u s ' a n t i g e n i c d e t e r m i n a n t s i n t h e p r o t r u d i n g
r e g i o n s o f p r o t e i n s
J . M . T h o r n t o n , M . S . E d w a r d s , W . R . T a y l o r a n dD . J . B a r l o w
L a b or a to r y o f M o l e c u l a r B i o l o g y , D e p a r t m e n t o f C r y s t a l l o g r a p h y , B i r k b e c k
C o l l e g e , M a l e t S t r e e t , L o n d o n WC1E 7 H X , UK
C o m m u n i c a t e d b y D . C . P h i l l i p s
A s i m p l e m e t h o d i s d e s c r i b e d t o l o c a t e ' a n t i g e n i c ' p e p t i d e s
f r o m t h e a - c a r b o n c o - o r d i n a t e s o f a p r o t e i n , b a s e d on pro-
t r u s i o n f r o m t h e p r o t e i n ' s g l o b u l a r s u r f a c e . A g o o d corre-
l a t i o n i s f o u n d b e t w e e n t h o s e p a r t s o f a p r o t e i n w h i c h
p r o t r u d e a n d t he e x p e r i m e n t a l l y d e t e r m i n e d a n t i g e n i c p e p -
t i d e s i n m y o g l o b i n , lysozyme a n d m y o h e m e r y t h r i n . A c o m -
p a r i s o n i s made b e t w e e n t h e use o f p r o t r u s i o n i n d e x , m o b i l i t y ,s o l v e n t a c c e s s i b i l i t y a n d h y d r o p h i l i c i t y f o r p r e d i c t i n g t h e m o s t
l i k e l y a n t i g e n i c p e p t i d e s .K e y w o r d s : a n t i g e n i c i t y / p e p t i d e s / p r e d i c t i o n / p r o t r u s i o n i n d e x / c o n -t i n u o u s d e t e r m i n a n t s
I n t r o d u c t i o n
A l t h o u g h t h e i m m u n e response t o a f o r e i g n p r o t e i n i s c o m p l e xa n d i n c o m p l e t e l y u n d e r s t o o d , i t h a s b e e n s h o w n t h a t s h o r t pep-
t i d e s t a k e n f r o m a p r o t e i n w i l l c r o s s - r e a c t w i t h a n t i b o d i e s r a i s e da g a i n s t t h e c o m p l e t e p r o t e i n ( A t a s s i , 1 9 8 4 ; M i l t o n a n d V a n
R e g e n m o r t e l , 1 9 7 9 ) . F u r t h e r m o r e i t h a s b e e n p r o p o s e d t h a t som e
p e p t i d e s can b e u s e d t o r a i s e a n t i - p e p t i d e a n t i b o d i e s , w h i c h recog-
n i z e t h e c o m p l e t e p r o t e i n a n d c o u l d t h e r e f o r e b e u s e d t o g e n e r a t e
s y n t h e t i c v a c c i n e s ( L e r n e r , 1 9 8 2 ) . Two r e c e n t p u b l i c a t i o n s h a v e
e m p h a s i z e d t h e i m p o r t a n c e o f p e p t i d e f l e x i b i l i t y ( W e s t o f e t a l . ,1 9 8 4 ; T a i n e r e t a l . , 1 9 8 4 ) a n d h a v e s h o w n t h a t i n t h e p r o t e i nt h e r e g i o n s c o r r e s p o n d i n g t o t h e a n t i g e n i c p e p t i d e s are u s u a l l yh i g h l y m o b i l e . An a l t e r n a t i v e v i e w p o i n t i s t h a t t h e s e regions
( w h i c h we w i l l t e r m ' p e p t i d e d e t e r m i n a n t s ' ) are a n t i g e n i c b e c a u s e
t h e y are t h e most a c c e s s i b l e t o t h e l a r g e a n t i b o d y m o l e c u l e . I nt h i s paper we s h o w t h a t f o r l y s o z y m e , m y o g l o b i n a n d m y o h e m -e r y t h r i n , t h e e x p e r i m e n t a l l y d e t e r m i n e d p e p t i d e d e t e r m i n a n t s c o r -
r e s p o n d p r e d o m i n a n t l y t o t h o s e p a r t s o f t h e s t r u c t u r e w h i c h
p r o t r u d e , a s d e f i n e d b y g e n e r a t i n g an e q u i - m o m e n t a l e l l i p s o i dt o f i t t h e p r o t e i n a n d c a l c u l a t i n g t h o s e s e g m e n t s w h i c h ' s t i c k o u t ' .T h e c o r r e l a t i o n w i t h t h e e x p e r i m e n t a l d a t a i s s u p e r i o r t o t h a to b t a i n e d u s i n g h y d r o p h i l i c i t y ( H o p p a n d W o o d s , 1 9 8 1 ) a n d c om-
p a r a b l ew i t h t h e
r e s u l t s p r e s e n t e don
m o b i l i t y ( W e s t o fe t
a l . ,1 9 8 4 ; T a i n e r e t a l . , 1 9 8 4 , 1 9 8 5 ) .To t e s t t h e h y p o t h e s i s t h a t p e p t i d e d e t e r m i n a n t s p r o t r u d e f r o m
t h e s u r f a c e o f t h e p r o t e i n , i t was necessary t o c o n s t r u c t an ap-
p r o x i m a t i o n t o t h a t s u r f a c e . S i n c e most p r o t e i n s are d i s t i n c t l ye l l i p s o i d a l i n s h a p e ( T a y l o r e t a l . , 1 9 8 3 ; P r a b h a k a r a n a n d P o n -
nuswamy, 1 9 8 2 ) , t h e e l l i p s o i d was c a l c u l a t e d w h i c h h a s t h e same
moments o f i n e r t i a a s t h e p r o t e i n s t r u c t u r e ( t h e e q u i - m o m e n t a l
e l l i p s o i d ) , u s i n g a s t a n d a r d a n a l y t i c a l m e t h o d i n v o l v i n g C a u c h y ' sM o m e n t a l e l l i p s o i d ( T a y l o r e t a l . , 1 9 8 3 ) . T h i s m e t h o d d e t e r m i n e s
t h e r a t i o s b e t w e e n t h e l e n g t h s o f t h e p r i n c i p a l axes a , b , c a n d
t h e i r d i r e c t i o n s , b u t t h e a b s o l u t e s i z e o f t h e e l l i p s o i d i s a r b i t r a r i -
l y c h o s e n t o i n c l u d e a s p e c i f i e d p e r c e n t a g e o f a t o m s . F o r exam-
O IRL P r e s s L i m i t e d , O x f o r d , E n g l a n d
p l e , t h e 9 0% l l i p s o i d w i l l i n c l u d e 9 0%o f t h e a t o m s , w i t h 10%
l y i n g o u t s i d e or p r o t r u d i n g f r o m t h e g l o b u l a r s h a p e . T h e r e f o r e
we can s i m p l y a s s i g n t o e a c h r e s i d u e a ' p r o t r u s i o n i n d e x ' or P I
s p e c i f y i n g t h e % e l l i p s o i d a t w h i c h t h a t r e s i d u e f i r s t b e c o m e s
e x t e r n a l . F o r e x a m p l e , a l l r e s i d u e s w h i c h a r e o u t s i d e t h e 90%e l l i p s o i d are a s s i g n e d a P I = 9 ; t h o s e w h i c h are o u t s i d e t h e 80%e l l i p s o i d ( b u t n o t t h e 90% e l l i p s o i d ) h a v e PI= 8 , e t c . T h u s f o re a c h p r o t e i n t h e r a t i o s b / a a n d c / a , a n d t h e d i r e c t i o n s o f t h ep r i n c i p a l a x i s , are c a l c u l a t e d f r o m t h e c o - o r d i n a t e s ( a l l a t o m s
or j u s t a - c a r b o n c o - o r d i n a t e s can b e i n c l u d e d ) . T h e n t h e 9 0 % ,
8 0 % , 7 0 % , e t c . e l l i p s o i d s are g e n e r a t e d a n d t h e P I a s s i g n e d t o
e a c h r e s i d u e . E l l i p s o i d s can b e g e n e r a t e d f o r e a c h d o m a i n o f a
m u l t i - d o m a i n p r o t e i n or f o r t h e w h o l e s t r u c t u r e .
T h e m e t h o d was a p p l i e d t o t h r e e p r o t e i n s f o r w h i c h t h e a-
c a r b o n c o - o r d i n a t e s we re a v a i l a b l e as w e l l as some e v i d e n c e t o
T a b l e I . A v e r a g e d v a l u e s o f P I , a c c e s s i b i l i t y , m o b i l i t y a n d h y d r o p h i l i c i t y c a l c u l a t e do v e r t h e l e n g t h o f e a c h p e p t i d e
P r o t e i n a n d P r o t r u s i o n A c c e s s i b i l i t y M o b i l i t y H y d r o p h i l i c i t yp e p t i d e i n d e x
P I % ACC % B % H %
M y o g l o b i n
1-6 6 . 8 1 8 7 7 1 2 1 7 5 0 . 5 3 5
1 5 - 2 2 5 . 5 3 2 5 6 4 2 1 2 2 3 0 . 6 3 0
56-62 6 . 9 1 6 6 5 2 8 8 7 3 a 1 . 4 3
94-99 4 . 2 3 6 7 5 1 4 1 2 1 9 0 . 5 3 8
1 1 3 - 1 1 9 3 . 6 6 4 a 5 5 4 6 1 4 1 2 - 0 . 2 7 6 a
1 2 1 - 1 2 7 7 . 4 8 5 0 5 2 a 1 5 8 0 . 4 4 21 4 5 - 1 5 1 8 . 3 2 8 5 5 1 3 1 4 0 6 8 a
L y s o z y m e
38-54 4 . 6 4 3 5 2 4 6 1 5 8 2 a 0 . 1 9 2 5
6 4 - 8 0 5 . 6 1 5 6 4 1 0 2 5 9 0 . 1 7 3 0
M y o h e m e r y t h r i n
H i g h b
3-16 5 . 6 1 2 3 1 1 2 0 . 1 6 5 2 a
7-16 4 . 8 4 0 2 9 . 5 3 4 - 0 . 1 9 7 8 a37-46 5 . 8 1 8 2 9 . 5 3 3 0 . 3 5 3 5
5 7 - 6 6 6 . 5 9 2 9 4 3 0 . 7 4 1 1
M e d i u m b
63-72 7 . 2 1 3 2 7 0 . 3 3 8
69-82 5 . 3 2 1 3 0 2 6 0 . 6 1 1
7 3 - 8 2 4 . 1 5 8 2 8 . 6 4 8 0 . 8 4 8
L o w b
2 2 - 3 5 2 . 1 9 8 2 2 9 5 0 . 7 8 4
26-35 1 . 7 9 8 2 1 . 7 9 2 0 . 2 4 4 4
4 2 - 5 1 3 . 3 7 3 2 9 3 9 - 0 . 5 9 2
9 6 - 1 0 9 2 . 6 9 0 2 2 9 3 - 0 . 1 1 8 6
1 0 0 - 1 0 9 2 . 7 8 0 2 1 9 8 0 6 5
S e e l e g e n d t o F i g u r e 1 f o r d a t a s o u r c e s .T h e % c o l u m n s r e p r e s e n t t h e p e r c e n t a g e o f p e p t i d e s , o f e q u a l l e n g t h t o t h ee p i t o p e , w i t h h i g h e r a v e r a g e v a l u e s t h a n t h e e p i t o p e . F o r e x a m p l e , 18% o f
h e x a p e p t i d e s i n m y o g l o b i n h a v e P I 2 6 . 8a A c t i v e p e p t i d e s w h e r e p e r c e n t i l e i s > 5 0 % .b F o r d e f i n i t i o n o f h i g h , m e d i u m a n d l o w a c t i v i t y s e e l e g e n d t o F i g u r e i c .
4 0 9
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J . M . T h o r n t o n e t a t .
d e f i n e t h e l o c a t i o n o f t h e p e p t i d e d e t e r m i n a n t s ( A t a s s i , 1 9 8 4 ;M i l t o n a n d V a n R e g e n m o r t e l , 1 9 7 9 ; W e s t o f e t a l . , 1 9 8 4 ; T a i n e re t a l . , 1 9 8 4 , 1 9 8 5 ) ( s e e F i g u r e 1 ) . A g r a p h i c s a l g o r i t h m( E L L I P S E ) w a s a l s o u s e d t o d i s p l a y a n d r o t a t e t h e p r o t e i n a n d
i t s e q u i - m o m e n t a l e l l i p s o i d ( F i g u r e 2 a ) . T h e p e p t i d e s a r e i d e n -t i f i e d e i t h e r b y s u c c e s s f u l c o m p e t i t i o n w i t h p r o t e i n - a n t i b o d y c o m -
p l e x f o r m a t i o n , o r b y r a i s i n g a n t i p e p t i d e a n t i b o d i e s , w h o s e
i n t e r a c t i o n w i t h t h e c o m p l e t e p r o t e i n w a s d e t e r m i n e d ( s e e l e g e n dt o F i g u r e 1 ) .
R e s u l t s
C o r r e l a t i o n b e t w e e n p r o t r u s i o n i n d e x a n d t h e l o c a t i o n o f t h e p e p -
t i d e d e t e r m i n a n t s
F o r e a c h p r o t e i n a p l o t i s m a d e o f t h e ' p r o t r u s i o n i n d e x ' ( P I )a g a i n s t t h e r e s i d u e n u m b e r , a s s h o w n i n F i g u r e l a - c . A l s os h o w n a r e t h e v a r i a t i o n s a l o n g t h e c h a i n o f s o l v e n t a c c e s s i b i l i t y( L e e a n d R i c h a r d s , 1 9 7 1 ) , h y d r o p h i l i c i t y ( H o p p s a n d W o o d s ,
1 9 8 1 ) a n d m o b i l i t y ( B - v a l u e s ) ( W e s t o f e t a l . , 1 9 8 4 ; T a i n e r e ta l . , 1 9 8 4 ) . T a b l e I g i v e s t h e a v e r a g e s o f t h e s e f o u r v a r i a b l e s a n d
a MYOGLOBIN
3 -
2 - i ° W W
.4
70 -
60 -
% 5 0 -
°4 0;
o 3 0
. 2 0
E, 1 0
1 1 3 - 1 2 1 - 1 4 6 -
E p i t o p * s i 6 1 ; - 2 2 - 5 5 5 6 - 6 2 7 2 89 9 4 - 1 t 9 t 2 7
5 -
22gS1
1 1 0 20 30 40 50 60 7 0 8 0 90 1 O O 1 1 0 1 2 0 1 3 0 140 1S 3
R e s i d u e n o .
a r a n k e d p e r c e n t i l e ( s e e b e l o w ) c a l c u l a t e d f o r e a c h p e p t i d e d e -t e r m i n a n t .
F o r a l l t h e p r o t e i n s t h e r e i s a m a r k e d c o r r e l a t i o n b e t w e e n h i g hP I v a l u e s a n d t h e l o c a t i o n o f t h e ' a n t i g e n i c p e p t i d e s ' . T h e p l o t ss u g g e s t t h a t m o s t o f t h e s e p e p t i d e s h a v e P I > 5 , a n d o f t e n c o r r e -s p o n d t o a p e a k i n t h e P I p l o t ( s e e T a b l e I ) . F o r m y o g l o b i n , a l l
t h e a n t i g e n i c p e p t i d e s , e x c e p t p e r h a p s r e s i d u e s 1 1 3 - 1 1 9 , c o r r e -s p o n d t o a p e a k i n t h e P I p l o t s ( F i g u r e l a ) . A l l i n c l u d e a t l e a s to n e r e s i d u e w i t h P I > 7 , a n d f o u r o f t h e s e v e n p e p t i d e s i n c l u d e
t h e m o s t p r o t r u s i v e r e s i d u e s ( P I = 9 ) . F o r l y s o z y m e ( F i g u r e l b )t w o o f t h e p r o t r u d i n g s e g m e n t s c o r r es p o n d a l m o s t e x a c t l y t o t h et w o i d e n t i f i e d c o n t i n u o u s a n t i g e n i c r e g i o n s ( r e s i d u e s 3 8 - 5 4 a n d
6 4 - 8 0 ) . E v e n m o r e i n t e r e s t i n g l y t h e l o w r e s o l u t i o n c r y s t a l s t r u c -t u r e o f t h e l y s o z y m e - a n t i b o d y c o m p l e x s h o w s t h a t r e s i d u e s 1 8 -
2 7 a n d 1 1 6 - 1 2 8 , t w o o f t h e o t h e r e x t e r n a l s e g m e n t s , f o r m t h en o n - c o n t i n u o u s d e t e r m i n a n t w h i c h i n t e r a c t s w i t h t h e a n t i b o d y( A m i t e t a l . , 1 9 8 5 ) .
F o r m y o h e m e r y t h r i n t h e d a t a d e f i n e w h i c h p e p t i d e s a r e e f f e c -t i v e i n r a i s i n g a n t i b o d i e s w h i c h i n t e r a c t w i t h t h e w h o l e p r o t e i n .T h e r e i s a c l e a r c o r r e l a t i o n b e t w e e n t h e a c t i v e p e p t i d e s a n d h i g h
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F i g . 1 . V a r i a t i o n o f t h e h y d r o p h i l i c i t y , s o l v e n t a c c e s s i b i l i t y , m o b i l i t y a n d p r o t r u s i o n i n d e x a l o n g t h e sequence f o r m y o g l o b i n , lysozyme a n d m y o he m er y t hr i n .
F o r e a c h p r o t e i n t h e e q u i m o m e n t a l e l l i p s o i d i s g e n e r a t e d as d e s c r i b e d i n T a y l o r et a l . ( 1 9 8 3 ) a n d t h e P I i s c a l c u l a t e d as d e s c r i b e d i n t h e t e x t . T h e r e s i d u ea c c e s s i b i l i t y i s c a l c u l a t e d u s i n g t h e m e t h o d o f K a b s c h a n d S a n d e r ( 1 9 8 3 ) w i t h t h e i r P a s c a l program w h i c h i s a v a i l a b l e on t h e P r o t e i n D a t a B a n k ( B e r n s t e i n e t
a l . , 1 9 7 7 ) . T h e h y d r o p h i l i c i t y v a l u e s are c a l c u l a t e d u s i n g t h e h e x a p e p t i d e a v e r a g i n g m e t h o d o f Hopp a n d Woods ( 1 9 8 1 ) . T h i s a v e r a g i n g s m o o t h s o u t t h e p l o t ,
c o m p a r e d w i t h t h e o t h e r v a r i a b l e s , w h i c h are n o t a v e r a g e d . T h e d a t a f o r t h e l o c a t i o n o f t h e ' a n t i g e n i c ' p e p t i d e s are t a k e n f r o m Young e t a l . ( 1 9 8 3 ) ; L e a c h
( 1 9 8 3 ) , I b r a h i m i et a l . ( 1 9 8 0 ) ; A t a s s i ( 1 9 8 4 ) a n d M i l t o n a n d V a n R e g e n m o r t e l ( 1 9 7 9 ) a n d t h e y are i n d i c a t e d b y s o l i d b a r s a b o v e t h e P I p l o t i n a a n d b .( a ) M y o g l o b i n . C o - o r d i n a t e d a t a a n d B - v a l u e s f r o m t h e P r o t e i n D a t a B a n k f i l e 1MBD. T h e P I v a r i a t i o n i s c a l c u l a t e d f r o m a - c a r b o n atom c o - o r d i n a t e s . T h e
d a s h e d l i n e i n d i c a t e s an e p i t o p e o f u n c e r t a i n l o c a t i o n . P e p t i d e s 1-6 a n d 1 2 1 - 1 2 7 d o n o t compete w i t h w h o l e p r o t e i n - a n t i b o d y c o m p l e x f o r m a t i o n , b u t are
e f f e c t i v e i n r a i s i n g a n t i b o d i e s w h i c h r e c o g n i z e t h e w h o l e p r o t e i n ( Y o u n g e t a l . , 1 9 8 3 ) . ( b ) Hen e g g w h i t e lysozyme. C o - o r d i n a t e d a t a f r o m P r o t e i n D a t a B a n k
f i l e 2LYZ. T h e P I p l o t i s c a l c u l a t e d f r o m a - c a r b o n c o - o r d i n a t e s . T h e d o t t e d l i n e i n d i c a t e s p o r t i o n s o f a d i s c o n t i n u o u s e p i t o p e i d e n t i f i e d b y c r y s t a l l o g r a p h y( A m i t et a l . , 1 9 8 5 ) . B - v a l u e s are f r o m B e r t h o u et a l . ( 1 9 8 3 ) . ( c ) M y o h e m e r y t h r i n . C o - o r d i n a t e d a t a f r o m P r o t e i n D a t a B a n k f i l e 1MHR. T h e B - v a l u e s a r e
t a k e n f r o m S h e r i f f et a l . ( 1 9 8 5 ) a n d a r e c o r r e c t e d f o r c r y s t a l c o n t a c t s . T h e P I p l o t i s c a l c u l a t e d f r o m a - c a r b o n c o - o r d i n a t e s . T h e d a t a f o r a l l t h e a t o m s were
n o t a v a i l a b l e a n d t h e r e f o r e t h e s o l v e n t a c c e s s i b i l i t y p l o t c o u l d n o t b e c a l c u l a t e d . A s d e s c r i b e d i n T a i n e r et a l . ( 1 9 8 4 ) 1 2 p e p t i d e s were s y n t h e s i z e d a n d t h e i r
r e a c t i v i t i e s , d e f i n e d f r o m i m m u n o p r e c i p i t a t i o n r e s u l t s , are i n d i c a t e d as f o l l o w s : t h e m o s t a c t i v e p e p t i d e s ( > 4 0 0 0 c . p . m . ) ; a r m m e d i u m - a c t i v i t y p e p t i d e s(200-4000 c.p.m.); low-activity peptides (<200 c.p.m.).
P I v a l u e s . T h e f o u r most a c t i v e p e p t i d e s ( 3 - 1 6 , 7 - 1 6 , 37-46a n d 5 7 - 6 6 ) , a s d e t e r m i n e d f r o m t h e i m m u n o p r e c i p i t a t i o n r e s u l t s ,are a l s o t h e r e g i o n s w h i c h p r o t r u d e f a r t h e s t f r o m t h e m o l e c u l a r
s u r f a c e , i n c l u d i n g t h e r e s i d u e s w i t h P I = 9 . T h e ' c o l d ' n o n - a c t i v ep e p t i d e s ( 2 2 - 3 5 , 2 6 - 3 5 , 96-109 a n d 1 0 0 - 1 0 9 ) are t h e l e a s t
p r o t r u s i v e ( P I A V <3 ) d e s p i t e b e i n g p a r t i a l l y a c c e s s i b l e t o s o l v e n t
as h e l i c a l r e g i o n s . T h i s p l o t s h o w s t h a t t h e N - t e r m i n a l p e p t i d e( 1 - 6 ) i s very p r o t r u s i v e , as h a s b e e n f o u n d f o r ma n y p r o t e i n s( T h o r n t o n a n d S i b a n d a , 1 9 8 3 ) , a n d t h e r e f o r e p r e d i c t s t h a t i
s h o u l d b e e f f e c t i v e i n r a i s i n g a n t i b o d i e s t o t h e w h o l e p r o t e i n .T h e P I p l o t s d e r i v e d u s i n g a l l t h e a t o m s , i n s t e a d o f j u s t t h e
a - c a r b o n s , are b r o a d l y c o m p a r a b l e ( c o r r e l a t i o n c o e f f i c i e n t =
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J . M . T h o r n t o n e t a l .
T a b l e H . C o r r e l a t i o n s a b e t w e e n p r o t r u s i o n i n d e x , a c c e s s i b i l i t y , m o b i l i t y a n d
h y d r o p h i l i c i t y p l o t s f o r m y o g l o b i n
p l b A c c e s s i b i l i t y c M o b i l i t y d H y d r o p h i l i c i t y e A n t i g e n i c i t y f( x 2 )
P I 1 . 0 0 . 6 3 0 . 4 6 0 . 3 7 2 8 . 5
A c c e s s i b i l i t y 1 . 0 0 . 4 0 0 . 6 4 8 . 6
M o b i l i t y 1 . 0 0 . 0 7 3 3 . 2
H y d r o p h i l i c i t y 1 . 0 0 . 7 6
a T h e c o r r e l a t i o n c o e f f i c i e n t i s c a l c u l a t e d u s i n g t h e s t a n d a r d f o r m u l a t i o n . I t s
v a l u e v a r i e s f r o m -1 ( a n t i - c o r r e l a t i o n ) t o + 1 ( c o m p l e t e c o r r e l a t i o n ) .b T h e P I i s c a l c u l a t e d f r o m a - c a r b o n c o - o r d i n a t e s a s d e s c r i b e d i n t h e t e x t .
c T h e a c c e s s i b i l i t y i s t h e a c c e s s i b l e s u r f a c e a r e a f o r e a c h r e s i d u e , c a l c u l a t e df r o m a l l - a t o m c o - o r d i n a t e s u s i n g t h e m e t h o d o f K a b s c h a n d S a n d e r ( 1 9 8 3 ) .d M o b i l i t y i s d e f i n e d b y t h e c r y s t a l l o g r a p h i c B - v a l u e s , o b t a i n e d f r o m h i g h l yr e f i n e d p r o t e i n s t r u c t u r e s ( s e e t e x t ) .e H y d r o p h i l i c i t y i s c a l c u l a t e d f r o m t h e s e q u e n c e u s i n g t h e m e t h o d o f H o p pa n d W o o d s ( 1 9 8 1 ) .f T h e x 2 v a l u e s a r e f r o m 2 x 2 c o n t i n g e n c y t a b l e s o f e a c h p a r a m e t e r a g a i n s tt h e o b s e r v e d l o c a t i o n o f a n t i g e n i c r e s i d u e s . E a c h r e s i d u e i s s c o r e d 1 o r 0
f o r i t s a n t i g e n i c i t y ( a n t i g e n i c = 1 ; n o n - a n t i g e n i c = 0 ) . S i m i l a r l y , e a c h
r e s i d u e i s s c o r e d 1 o r 0 a c c o r d i n g t o w h e t h e r i t s p a r a m e t e r v a l u e i s g r e a t e rt h a n o r l e s s t h a n a t h r e s h o l d , s e t a t t h e o v e r a l l a v e r a g e f o r t h a t p a r a m e t e r
( s o t h a t e q u a l t o o r g r e a t e r t h a n = 1 ; l e s s t h a n = 0 ) . T h e l a r g e r t h e v a l u e
o f X 2 t h em o r e t h e p a r a m e t e r d i f f e r e n t i a t e s b e t w e e n
a n t i g e n i ca n d n o n -
a n t i g e n i c r e s i d u e s .
a
0 . 8 4 ) b u t l e s s s e n s i t i v e . T h e a d v a n t a g e o f t h e a - c a r b o n p l o t s i s
t h a t t h e y h i g h l i g h t s e gm e nt s o f c h a i n w h i c h p r o t r u d e , r e g a r d l e s so f s i d e - c h a i n c o n f o r m a t i o n , w h i c h w i l l a l m o s t c e r t a i n l y b e f l e x i b l ei n s o l u t i o n . N e v e r t h e l e s s t h e e l l i p s o i d i s c l e a r l y a g r o s s a p p r o x i -m a t i o n t o t h e c o m p l e x s u r f a c e o f t h e p r o t e i n a n d t h e r a t h e r g o o d
a g r e e m e n t s u g g e s t s t h a t t h e p e p t i d e d e t e r m i n a n t s m u s t o f t e n b e
l o c a t e d o n g r o s s p r o t r u s i o n s .
C o m p a r i s o n o f p r o t r u s i o n i n d e x w i t h o t h e r p a r a m e t e r s
We h a v e a t t e m p t e d t o m a k e a r i g o r o u s c o m p a r i s o n b e t w e e n t h eu s e o f P I , m o b i l i t y , s o l v e n t a c c e s s i b i l i t y a n d h y d r o p h i l i c i t y f o rp r e d i c t i o n b y c a l c u l a t i n g f o r e a c h e p i t o p e i t s r a n k p o s i t i o n o n
t h e d i f f e r e n t s c a l e s ( s e e T a b l e I ) . F o r a g i v e n e p i t o p e , t h e a v e r a g e
P I i s c a l c u l a t e d f o r e a c h r e s i d u e s e g m e n t o f t h e s a m e l e n g t h i nt h e s e q u e n c e a n d t h e n t h e % o f s e g m e n t s w i t h P I h i g h e r t h a nt h a t c a l c u l a t e d f o r t h e e p i t o p e i s g i v e n . T h i s p r o c e d u r e i s r e p e a t e df o r a c c e s s i b i l i t y , m o b i l i t y a n d h y d r o p h i l i c i t y . F o r e x a m p l e , f o rr e s i d u e s 1-6 i n m y o g l o b i n , 1 2 % o f 6 - r e s i d u e s e g m e n t s h a v e
a h i g h e r a c c e s s i b i l i t y , 5% h a v e h i g h e r m o b i l i t y , 3 5 % h i g h e rh y d r o p h i l i c i t y a n d 1 8 % h i g h e r P I . A l t h o u g h t h i s i s s e n s i t i v e t ot h e l e n g t h o f t h e p e p t i d e s , w h i c h a r e n o t w e l l d e f i n e d , i t p r o v i d e sa q u a n t i t a t i v e c o m p a r i s o n o f t h e p a r a m e t e r s .
I n s p e c t i o n o f T a b l e s I a n d I I i n d i c a t e s t h a t P I , m o b i l i t y a n d
a v e r a g e d a c c e s s i b i l i t y h a v e b r o a d l y c o m p a r a b l e s u c c e s s r a t e s ,
b
- 4 6 - 6 '
1 - 6 X '..
d
F i g . 2 . P l o t s o f t h e p r o t e i n b a c k b o n e s h o w i n g l o c a t i o n o f t h e c o n t i n u o u s a n t i g e n p e p t i d e s ( d a s h e d ) a n d t h e e l l i p s o i d w i t h -50% a t o m s i n s i d e , d e r i v e d f r o m
c - c a r b o n c o - o r d i n a t e s . T h e s e f i g u r e s a r e p l o t t e d u s i n g o u t p u t f r o m t h e p r o g r a m E L L I P S E w h i c h a l l o w s t h e s t r u c t u r e a n d i t s e l l i p s o i d t o b e r o t a t e d i n t h r e ed i m e n s i o n s u s i n g t h e t r a c k e r b a l l . F o r e a c h p r o t e i n t h e r a t i o s o f t h e e l l i p s o i d a x i s l e n g t h s a , b , c a r e g i v e n b e l o w . ( a ) M y o g l o b i n a n d i t s t h r e e - d i m e n s i o n a le l l i p s o i d a s s e e n o n t h e E v a n s a n d S u t h e r l a n d P S 2 g r a p h i c s s y s t e m , w h e r e c o l o u r s ( n o t s h o w n h e r e ) a r e u s e d t o d i f f e r e n t i a t e e p i t o p e s . ( b ) M y o g l o b i n .
b / a = 0 . 9 , c / a = 0 . 5 . ( c ) L y s o z y m e . b / a = 0 . 6 , c / a = 0 . 6 . T h e d i s c o n t i n u o u s e p i t o p e ( 1 8 - 2 7 ; 1 1 6 - 1 2 9 ) i s a l s o s h o w n . ( d ) M y o h e m e r y t h r i n . b / a = 0 . 6 , c / a = 0 . 5 .T h e c o l d ' n o n - a c t i v e ' p e p t i d e s ( r e s i d u e s 2 2 - 3 5 a n d 96- 1 0 9 ) a r e s h o w n d o t t e d .
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L o c a t i o n o f ' c o n t i n u o u s ' a n t i g e n i c d e t e r n i n a n t s
a n d w h i l s t a l l a r e b e t t e r t h a n t h e s e q u e n c e - d e r i v e d h y d r o p h i l i c i t yi n d e x , n o n e i s d e f i n i t i v e . F o r e a c h m e a s u r e t h e r e i s a t l e a s t o n e
e p i t o p e w h i c h i s n o t r e c o g n i z e d ( i n d i c a t e d b y s u p e r s c r i p t a i nT a b l e I ) . H o w e v e r , t h e b r o a d a g r e e m e n t u n d e r l i n e s t h e c o r r e l a -t i o n b e t w e e n t h e s e v a r i a b l e s ( s e e T a b l e I I ) . F o r m y o g l o b i n t h ec o r r e l a t i o n c o e f f i c i e n t s b e t w e e n t h e P I , a c c e s s i b i l i t y a n d m o b i l i t yr a n g e f r o m 0 . 4 t o 0 . 6 3 . S u r p r i s i n g l y , t h e r e i s l i t t l e c o r r e l a t i o nb e t w e e n h y d r o p h i l i c i t y a n d m o b i l i t y . I n g e n e r a l , t h o u g h n o ta l w a y s , t h e p e p t i d e d e t e r m i n a n t s w h i c h p r o t r u d e a r e h y d r o p h i l i c
w i t h h i g h e r t h a n a v e r a g e a c c e s s i b i l i t i e s , a n d a r e m o b i l e . F o r m a n yo f t h e p e p t i d e s t h e p e r c e n t i l e v a l u e s a r e q u i t e h i g h u s i n g a n y o f
t h e m e a s u r e s . T h i s s u g g e s t s t h a t t h e s p e c i f i c s e q u e n c e m u s t b e
i m p o r t a n t a s w e l l a s t h e p r o t r u s i o n , h y d r o p h i l i c i t y , e t c . T h er e l a t i v e i m p or t a n c e o f f l e x i b i l i t y v e r s u s a n t i b o d y a c c e s s i b i l i t y i s
n o t r e s o l v e d a n d n e e d s m o r e w e l l - d e f i n e d i m m u n o l o g i c a l a n d
c r y s t a l l o g r a p h i c d a t a .
D i s c u s s i o n
A l t h o u g h t h e w h o l e s u r f a c e o f a p r o t e i n i s p r o b a b l y i m m u n o g e n i c
( B e n j a m i n e t a l . , 1 9 8 4 ) , m o s t o f t h e p e p t i d e d e t e r m i n a n t s p r o t r u d ef r o m t h e g l o b u l a r s u r f a c e . T h i s o b s e r v a t i o n c a n b e m o s t e a s i l ye x p l a i n e d i n t e r m s o f c o n t i n u o u s a n d d i s c o n t i n u o u s d e t e r m i n a n t s ,
r a t h e r t h a n a g e n e r a l i z e d s h a p e o f t h e a n t i b o d y c o m b i n i n g s i t e( D a v i e s a n d M e t z g e r , 1 9 8 3 ) . A p a r t f r o m t h e p r o t r u d i n g l o o p
r e g i o n s , t h e m a j o r i t y o f t h e s u r f a c e p a t c h e s o n a p r o t e i n w i l l c o m -p r i s e a m i n o a c i d s f r o m r e s i d u e s d i s t a n t i n t h e l i n e a r s e q u e n c e .G i v e n t h e l a r g e a r e a o f c o n t a c t f o u n d i n m o s t p r o t e i n - p r o t e i n
i n t e r a c t i o n s a n d e x p e c t e d f o r a n t i b o d y - a n t i g e n c o n t a c t s ( A m i te t a l . , 1 9 8 5 ) , a l l t h e e p i t o p e s w i l l p r o b a b l y b e d i s c o n t i n u o u s t os o m e e x t e n t . H o w e v e r , m o s t o f t h e e x p e r i m e n t a l m e t h o d s u s e d
t o d e f i n e p e p t i d e d e t e r m i n a n t s i n e v i t a b l y l o c a t e o n l y t h o s e e p i -t o p e s w h e r e t h e p r i m a r y s i t e o f i n t e r a c t i o n , e s s e n t i a l f o r r e c o g -n i t i o n , d e r i v e s f r o m s e q u e n t i a l a m i n o a c i d s . T h e s e s e g m e n t s w i l la l m o s t a l w a y s b e p r o t r u d i n g l o o p s , s i n c e e l s e w h e r e a m i n o a c i d sd i s t a n t i n t h e s e q u e n c e w i l l b e i n c l o s e p r o x i m i t y .
I n c o n c l u s i o n , t h e c a l c u l a t i o n o f P I p r o v i d e s a s i m p l e m e t h o d
f o r l o c a t i n g t h e p r o t r u d i n g p a r t s o f a p r o t e i n f r o m t h e a - c a r b o nc o - o r d i n a t e s . M o s t o f t h e p e p t i d e d e t e r m i n a n t s c o r r e s p o n d t o p r o -t r u s i o n s , a n d c a n b e e a s i l y l o c a t e d f r o m t h e t h r e e - d i m e n s i o n a ls t r u c t u r e o f t h e a n t i g e n . We a r e c u r r e n t l y d e v e l o p i n g a s i m p l em e t h o d , s i m i l a r t o t h e h y d r o p h i l i c i t y p l o t s ( H o p p a n d W o o d s ,1 9 8 1 ) , t o p r e d i c t p r o t r u s i o n f r o m s e q u e n c e . W i t h r e g a r d t o d i s -c o n t i n u o u s d e t e r m i n a n t s , t h e s e p r o t r u d i n g l o o p s a r e h i g h l y a v a i l -a b l e f o r i n t e r a c t i o n w i t h a n a n t i b o d y a n d a r e t h e s i t e s o f m a x i m u ms e q u e n c e v a r i a t i o n . T h e r e f o r e o n e m i g h t e x p e c t t h e m t o m a k ea m a j o r c o n t r i b u t i o n t o t h e a n t i b o d y r e c o g n i t i o n r e s p o n s e . [ S e ef o r e x a m p l e t h e r e c e n t s t u d y o f t h e r h i n o v i r u s ( R o s s m a n n e t a l . ,1 9 8 5 ) . ] H o w e v e r , i t m a y b e t h a t t h e y a r e t o o f l e x i b l e o r t o o p o l a rt o m a k e t h e b e s t r e c o g n i t i o n s i t e s f o r a n a n t i b o d y . D a t a t o a n s w e r
t h e s e q u e s t i o n s s h o u l d b e a v a i l a b l e i n t h e n e a r f u t u r e .
A c k n o w l e d g e m e n t s
We t h a n k Mr F r e d d i e E l l i o t t f o r e x c e l l e n t p h o t o g r a p h y , a n d t h e SERC f o r f i n a n -c i a l s u p p o r t . M . S . E . h o l d s a CASE S t u d e n t s h i p i n c o l l a b o r a t i o n w i t h D r P e t e rM u r r a y - R u s t a t G l a x o .
R e f e r e n c e s
A m i t , A . G . , M a r i u z z a , R . A . , P h i l i p s , S . E . U . a n d P o l j a k , R . J . ( 1 9 8 5 ) N a t u r e , 3 1 3 ,1 5 6 - 1 5 8 .
A t a s s i , M . Z . ( 1 9 8 4 ) E u r . J . B i o c h e m . , 1 4 5 , 1 - 2 0 .
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( 1 9 8 4 ) A n n u . R e v . I m m u n o l . , 2 , 6 7 - 1 0 1 .
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1 7 9 - 1 8 4 .
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H e c h t , H - J . , J o h n s o n , J . E . , K a me r , G . , L u o , M. , M o s s e r , A . G . , R u e c k e r t , R . R . ,S h e r r y , B . a n d V r i e n d , G . ( 1 9 8 5 ) N a t u r e , 3 1 7 , 1 4 5 - 1 5 3 .
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Y o u n g , C . R . , S c h m i t z , H . E . a n d A t a s s i , M . Z . ( 1 9 8 3 ) M o l . I m m u n o l . , 2 0 ,5 6 7 - 5 7 0 .
R e c e i v e d o n 2 8 O c t o b e r 1 98 5 ; r e v i s e d o n 1 7 D e c em b e r 1 9 8 5
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