Bioscience Reports I, 461-468 (1981)
Printed in Great Britain
~61
I n t r a m o l e c u l a r g e n e r a l a c i d c a t a l y s i s in t h e binding
r e a c t i o n s of ~ - m a c r o g l o b u l i n and c o m p l e m e n t
components
C 3 and C/~
Stephen G. DAVIES ~ and Robert B. SIM%w
*The Dyson Perrins Laboratory, South Parks Road, Oxford OXl 3QY;
and %MRC Immunochemistry Unit, Department of Biochemistry,
University of Oxford, South Parks Road, Oxford OXI 3QU, U.K.
(Received 20 May 1981)
The c o m p l e m e n t s y s t e m p r o t e i n s C3 and C4 and the
plasma protease inhibitor c~2-macroglobulin~ when
a c t i v a t e d by limited proteolysis~ can bind covalently to
other macromolecules. The t h r e e proteins also e x h i b i t
an unusual internal peptide-bond cleavage reaction when
denatured.
The covalent binding r e a c t i o n is l i k e l y to
o c c u r by a t r a n s a c y l a t i o n m e c h a n i s m i n v o l v i n g an
internal thioiester in the t hr e e proteins.
However, the
a c t i v a t e d s p e c i e s of t h e s e p r o t e i n s a r e m uch m o r e
r eac t i ve than simple thiolesters.
Studies of m o l e c u l a r
m o d e l s of t h e t h i o l e s t e r r e g i o n in C3 show that an
i n t r a m o l e c u l a r acid c a t a l y s i s m e c h a n i s m
can both
a c c o u n t for the exceptional r e a c t i v i t y of the act i vat ed
form of these proteins and provide an e x p l a n a t i o n f o r
the denaturation-induced peptide bond cleavage.
The complement system in blood is a group of about 20 proteins
involved in the clearance of foreign materials.
During activation of
t h e c o m p l e m e n t s y s t e m by~ f or e x a m p l e , i m m u n e c o m p l e x e s or
invading micro-organism% the complement proteins C3 and C4 become
d e p o s i t e d on t h e s u r f a c e of t h e a c t i v a t i n g materials (1-3).. This
deposition is an essential signal for phagocytosis, which is stimulated
by interaction of the deposited C3 with specific receptors on phagocytic cells (4).
Although the phenomenon of C3 and C4 d e p o s i t i o n
has been known for many years9 it has only recent l y been shown that
these proteins form covalent ester or amide bonds between themselves
and t h e s u r f a c e s to which t h e y bind (5-8).
The plasma protease
inhibitor o~macroglobulin (eeM)9 a l t h o u g h s u p e r f i c i a l l y not c l o s e l y
related to the complement proteins~ is similar in monomer size to C3
and C4, and is also known to bind covalently to other proteins (9~10).
Chemical and amino acid sequence studies on C3 and oeM have shown
th at both are likely to contain an internal thiolester, formed between
the Cys residue and the second Glu residue in the sequence Gly-CysGly-GIu-Glu ( l l - 1 3 ) .
The c o v a l e n t binding r e a c t i o n is t h o u g h t t o
w
whom reprint requests should be addressed.
9
The Biochemical Society
462
DAVIES & SIM
involve acyl transfer from the thiolester to a nucleophilic group (-OH
or -NHg) on the binding 'substrate' (Fig. 1). The act i vat ed forms of
t h e s e p r o t e i n s a r e , h o w e v e r , very much more react i ve than simple
thiolesters. Structural considerations described here indicate a simple
g e n e r a l aci d catalysis mechanism which accounts for the exceptional
r e a c t i v i t y of the proteins, and also p r o v i d e s an e x p l a n a t i o n for an
unusual a u t o l y t i c p e p t i d e - b o n d cleavage reaction which is unique to
this family of proteins, i.e. C3, C~, and c~M.
Background
C3, C#, and c~M all exist in plasma in unactivated 'native' forms.
Consistent with the evidence that these proteins contain an i n t e r n a l
thiolester, the native forms can be inactivated slowly (i.e. lose their
ability to bind to macromolecules) when they are incubated with high
concentrations of low-molecular-weight nucleophiles (e.g. methylamine,
ammonia, hydrazine, or hydroxylamine) (l l , l t ~-16).
Inactivation with
m e t h y l a m i n e leads to covalent incorporation of t h e methylamine into
the proteins (Fig. 1) (11,15,16).
Studies on loss of act i vi t y of C3,
Cg, and a2M when s t o r e d in a q u e o u s s o l u t i o n s u g g e s t t h a t slow
cleavage of the internal thiolester by water occurs over a period of
weeks at 4~
(2,8,17).
The rates of reaction of the native proteins
with water and small amine nucleophiles are generally similar to the
rates of cleavage of low-molecular-weight thiolesters by these reagents
(lg-20).
B e f o r e t he p h y s i o l o g i c a l r e a c t i o n of covalent binding to macromolecules occurs, each of the three p r o t e i n s must be a c t i v a t e d by
limited proteolysis. Activation of C3 and C4 is mediated by cleavage
of a single peptide bond by specific complement system proteases (1).
A c t i v a t i o n of ea2M occurs by cleavage of peptide bonds in the 'bait'
region of 0~!M by any of a wide range of proteases (9,13).
In each
case, proteolysis occurs at a site distant in the primary st ruct ure from
the internal thiolester ( i 0 ) .
The a c t i v a t e d s p e c i e s of C3 is v e r y
reactive, with a lifetime of about 10-4 s in aqueous solution (g). In
contrast to the native protein, a c t i v a t e d C3 will r e a c t with a v e r y
wide r a n g e of o x y g e n , n i t r o g e n , and sulphur n u c l e o p h i l e s (g,21).
Activated u~M has a longer lifetime in aqueous solution [about 2 min
( 2 2 ) ] , but this is still 104- to 105-fold less stable than the unactivated
species. The r eact i vi t y of the a c t i v at ed proteins is much great er than
would be expect ed for a simple thiolester.
A further f e a t u r e of the chemistry of this group of p r o t e i n s is
t h a t , in d e n a t u r i n g c o n d i t i o n s , t h e proteins undergo cleavage of a
peptide bond to produce a new blocked N-terminal amino acid ( i 0 , 1 2 ) .
Peptide bond cleavage is preceded by the appearance of a t i t r a t a b l e
SH group (23).
This reaction does not occur when the nucleophileinactivated
f o r m s of t h e p r o t e i n s are d e n a t u r e d ( 1 0 , 1 2 ) .
The
mechanism suggested by several workers (10,12,13,22) to explain this
p r o c e s s r e q u i r e s n u c l e o p h i l i c a t t a c k on the internal thiolester by a
nitrogen atom which is in a peptide bond. Such a reaction is unusual
in that the peptide bond nitrogen atom is not normally very nucleophilic.
REACTIVITY OF
C3,
C4, AND &2M
463
CH3NH2
C3
9~ . .
/C3Q§
SH Xph
Fig. i.
The i n t e r a c t i o n of C3 and activated C3 with
nucleophiles.
(a) Inactivation of 'native' C3 by methylamine.
(b) Activation of C3 by proteolytic cleavage to the
protein fragments C3a + C3b~ and subsequent reaction of C3b
with (i) an OH group on a complement-activating surface, (ii)
the competing nucleophile phenylhydrazine~ or (iii) water.
The native and activated forms of ~2 M and C4 undergo the same
series of reactions.
Hypothesis
We report here a simple consideration which accounts for the very
high r e a c t i v i t y of the thiolester in the a c t i v a t e d forms of C3, C4, and
e~zM, and for the nucleophilic nitrogen necessary f o r the denaturationinduced peptide bond cleavage. We suggest that the r e a c t i v i t y of the
a c t i v a t e d p r o t e i n s is a result of hydrogen bonding between the free
acid group of the first glutamic acid residue in the Gly-Cys-Gly-GluGlu s e q u e n c e , and t h e c a r b o n y l oxygen of the thiolester (Fig. 2).
Formation of a hydrogen bond in this way would increase the e l e c t r o p h i l i c i t y of t he t h i o l e s t e r .
Intramolecular general acid catalysis of
this type is well-documented in model systems (2#,25), and can result
in up to 10IS-fold e n h a n c e m e n t of n u c l e o p h i l i c a t t a c k on e s t e r
carbonyls.
Lt64
DAVIES & SIM
.....H-~C)\~0
n
,0
"~
cys H /7
CI
0
H
gin
~ N
glu
gly
REACTIVITY OF
C3,
C#, AND O~2M
#65
Fig. 2.
A space-filling CPK molecular model
(Corey-Pauling Models, California Institute of
Technology, Pasadena, California) of the sequence
Gly-Cys-Gly-Glu-Glu-Asn-Met~ which is present in
both C3 and ~2 M is shown.
A thiolester bond
between the Cys and second Glu residue can be
formed without imposing any unusual constraints on
the structure.
Formation of a hydrogen bond from
the free hydroxyl of the first Glu residue to the
thiolester causes only the minor constraint of
distortion of the nitrogen atom of the peptide
bond between the two Glu residues towards the
non-planar sp 3 hybridized form.
(a) Conformational drawing of part of the model, showing the
position of the thiolester and hydrogen bond; (b)
photograph of the model at the same orientation as
(a); (c) photograph of the model as in (b) but
turned through 150 ~ clockwise to illustrate the
hydrogen bond.
M o l e c u l a r models ( F i g . 2) of the t h i o i e s t e r derived from the
pentapeptide Gly-Cys-Giy-Glu-Glu indicate that the f o r m a t i o n of the
hydrogen bond as described above constrains the Glu-Glu peptide bond
such that it is no longer planar. Loss of planarity results in a change
in the character of the nitrogen of this peptide bond from its normal
a m i d e - l i k e planar n o n - n u c l e o p h i l i c f o r m t o w a r d s an a m i n e - l i k e
tetrahedral nucleophilic form (Fig. 2).
The energy of hydrogen bond
formation would be sufficient to allow a considerable change in the
character of the nitrogen under discussion. The denaturation-induced
peptide cleavage r e a c t i o n would then i n v o l v e the a d d i t i o n of the
n u c l e o p h i l i c n i t r o g e n to the hydrogen bond-activated thiolester, with
formation of a favourable five-membered imide ring and release of
thiol.
Subsequent hydrolysis of the imide would cause peptide bond
cleavage and f o r m a t i o n of a new c y c l i c / ~ - t e r m i n a l p y r o g ~ u t a m y l
residue (Fig. 3).
As shown in Fig. 2b,c, one f a c e of the planar carbonyl of the
thiolester is completely shielded by the G l y - G l u - G l u s e c t i o n of t h e
peptide chain.
This f a c e is t h e r e f o r e not available for a t t a c k by
external nucleophiles. We propose that in unactivated native forms of
C3, C#, and ~2M, the other f a c e is similarly sterically p r o t e c t e d by
a n o t h e r r e g i o n of t h e p o l y p e p t i d e chai n.
A p p a r e n t i n c r e a s e in
r e a c t i v i t y on a c t i v a t i o n t h e r e f o r e simply requires a conformational
change that exposes this face of the thiolester to external nucleophiles
a l l o w i n g t he binding r e a c t i o n to o c c u r .
Exposure is likely to be
co mp lete in C3 and p a r t i a l in cz2M. C o n f o r m a t i o n a l c h a n g e s and
a l t e r a t i o n in a n t i g e n i c i t y of t h e s e proteins on activation has been
documented (1#,17).
The simple mechanism we have proposed has the essential f e a t u r e
of an i n t r a m o l e c u l a r h y d r o g e n bond.
F o r m a t i o n of such a bond
e x p l a i n s for t h e f i r s t t i m e bot h t h e exceptional r e a c t i v i t y of the
activ ated proteins and the occur r ence of the unusual autolytic cleavage
reaction.
466
DAVIES
f.--~...H--O\ /,,0
~ .o"
0
H
"K/
0
H
H S ~
H20
i
H S ~
(~
0
HOOC0
& SIM
REACTIVITY
OF C3, C4, AND a2M
467
Fig. 3. Mechanism of autolytic cleavage reaction of
C3, C4, and ~2 M.
The denaturation-induced peptidebond cleavage reaction in these proteins is likely to
occur as follows:
Conformational changes during
denaturation allow nucleophilic attack (A) by the
nitrogen of the distorted p e p t i d e b o n d on the
thiolester, with formation of the cyclic imide (B).
Further denaturation allows access of water, and
hydrolysis of the original peptide bond occurs, with
formation of a new N-terminal pyroglutamyl residue
(C).
Although relatively little is known of the amino acid sequence of
C3, it is already evident that sequence identity between c~M and C3
is limited, and may be confined to a few areas of the proteins (refs.
26,27; cf. 2g).
We s u g g e s t t h a t t h e c h e m i c a l r e a c t i v i t y in t h e
c o v a l e n t binding r e a c t i o n can be explained by consideration of the
conserved sequence Gly-Cys-Gly-Glu-Glu alone, a l t h o u g h it is l i k e l y
that other highly conserved regions, involved in shielding and exposure
of the thio[ester, and possibly also in secondary stabilization, will be
found.
Acknowledgements
We are very grateful to Dr. B. 3. Sutton for assistance with model
building and to Professor R. R. Porter, Professor 3. R. Knowles, Dr.
T. M. Twose, and Dr. E. Sim for valuable advice and discussion.
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