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J. Biol. Chem., Vol. 277, Issue 17, 14942-14953, April 26, 2002
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From the
Received for publication, January 2, 2002, and in revised form, February 13, 2002
We have previously identified compstatin, a
13-residue cyclic peptide, that inhibits complement activation by
binding to C3 and preventing C3 cleavage to C3a and C3b. The structure
of compstatin consists of a disulfide bridge and a type I Compstatin, a 13-residue cyclic peptide, is a novel complement
inhibitor with the potential to be developed into a therapeutic agent
(1). Compstatin binds to C3, which is a central complement component,
through which all three pathways of complement activation, the
classical, alternative, and lectin, converge (2). Although complement
is part of the innate immune system while acting as a bridge between
the innate and adaptive immune systems, and its activation is an
important line of defense against invading foreign pathogens (3-5),
its inappropriate activation can result in host cell damage (6). This
is the case in more than 25 pathological conditions, including a number
of autoimmune diseases, burn injuries, ischemia reperfusion injuries,
stroke, dialysis, and cardiopulmonary bypass (see Ref. 7 for a complete
list and references). The following have been shown. (a)
Compstatin inhibits complement activation in vitro in human
serum (8). (b) Compstatin totally inhibits in
vivo heparin/protamine-induced complement activation in primates
without side effects, in a situation that is typical in cardiac surgery
(9). (c) Compstatin inhibits complement activation without
toxicity in whole blood, in models of extracorporeal circuits, which
resemble those used in cardiopulmonary bypass, dialysis, and
plasmapheresis (10). (d) Compstatin prolongs the lifetime of
porcine-to-human ex vivo perfused kidney xenograft model
with human blood (11). (e) Finally, compstatin does not appear to have significant cytotoxicity as it showed little or no
inhibition of clotting (12).
Compstatin was first identified as a 27-residue peptide using
combinatorial phage-displayed random peptide library and was subsequently truncated, without loss of activity, to a 13-residue peptide (8). An attempt to further truncate the 13-residue peptide
yielded inactive fragments (13). The sequence of compstatin is
Ile1-Cys2-Val3-Val4-Gln5-Asp6-Trp7-Gly8-His9-His10-Arg11-Cys12-Thr13-NH2,
where Cys2 and Cys12 are disulfide-bonded.
Reduction and alkylation of the two cysteines result in loss of
inhibitory activity (8). We have determined previously the
three-dimensional structure of compstatin in solution using NMR and a
restrained hybrid distance geometry/simulated annealing methodology
(14) and an alternative refinement methodology using global
optimization (15). Compstatin forms a type I Because of its activity, low toxicity, and structural simplicity
compstatin is a promising candidate for drug development (1, 12, 13).
It is not unusual for a short cyclic peptide to become an oral drug; a
successful example is the widely used immunosuppressant cyclosporin
(16). Also, it is known that several immunogenic peptides adopt
functional With the goal to improve on the activity of compstatin, we have
initiated the rational design of compstatin analogs based on the
available solution structure and function data. We have constructed 7 analogs by introducing perturbations in the key components of the
structure of compstatin, which we have studied using homonuclear
two-dimensional NMR spectra. Our goal is to classify which residues are
essential for structural stability, activity, or both. Also, we aim to
elucidate the roles of the disulfide bridge, of the hydrophobic patch,
and of the charges of compstatin in both structural stability and
binding/inhibitory activity. Certain substitutions have resulted in
retaining or slightly increasing the inhibitory activity while
maintaining the structural stability. Other substitutions have resulted
in loss of inhibitory activity while maintaining the characteristic Despite the presence of multiple conformations (due to flexibility) of
small peptides in solution, NMR has been widely used to determine the
presence of conformers with distinguishable populations in linear and
cyclic peptides, peptide fragments derived from protein sequences,
immunogenic peptides, and peptides of de novo design
(e.g. see Refs. 17-23, and references therein). These
studies typically require the use of two-dimensional correlation
spectroscopy (TOCSY,1
DQF-COSY) for resonance assignments and residue identification and
two-dimensional NOE/ROE spectroscopy (NOESY and ROESY) to establish NOE
connectivity patterns that are consistent with specific structure (20).
In certain cases, coupling constants, chemical shifts, and temperature
coefficients of chemical shifts are also helpful.
Based on the structural data we present here and our
earlier experimental work on structure, binding kinetics, and activity, we propose a model for recognition and binding of compstatin to C3.
Peptide synthesis and purification was performed as described
previously (8, 14). Inhibitory activity of compstatin and its analogs
on the complement system was studied by measuring their effect on the
alternative pathway. Complement activation inhibition was assessed by
measuring the lysis of rabbit erythrocytes in normal human serum, as
described previously (8, 14). All analogs were examined by mass
spectroscopy and found to be monomeric. The analogs of compstatin used
for NMR studies were acetylated with the exception of the C2A/C12A
analog. The NMR samples were prepared in 90% H2O, 10%
D2O buffer containing 50 mM potassium phosphate, 100 mM potassium chloride, 0.1% sodium azide,
and 10 µM EDTA. Sample pH was ~6 for the C2A/C12A,
Ac-V3A, Ac-W7F, Ac-Q5G/D6A/W7A, and Ac-Q5G/D6P/W7F analogs and 6.5 for
the Ac-H9A and Ac-V4A/H9A/T13I analogs. Peptide concentrations were in
the range of 1.5-5 mM, depending on the analog. NMR
spectra were recorded at 5 °C.
NMR spectra for the Ac-V3A, Ac-W7F, Ac-H9A, C2A/C12A, Ac-V4A/H9A/T13I,
and Ac-Q5G/D6A/W7A analogs were collected using Bruker DMX 500 MHz
spectrometer and for the Ac-Q5G/D6P/W7F were collected using a Bruker
AMX 400 MHz spectrometer. DQF-COSY, TOCSY, and NOESY spectra were
collected for all analogs using standard pulse sequences (see Ref. 24
and references therein). All spectra were collected using the 3-9-19 pulse sequence with gradients for water suppression (25). The NOE
mixing time was 500 ms and the TOCSY mixing time was 60 ms for the
Ac-V3A, Ac-W7F, Ac-H9A, C2A/C12A, Ac-V4A/H9A/T13I, and Ac-Q5G/D6A/W7A
analogs. The NOE mixing time was 400 ms, and the TOCSY mixing time was
75 ms for the Ac-Q5G/D6P/W7F analog.
Spectral processing was performed using matNMR, which is a
toolbox for processing NMR/EPR data (www.nmr.ethz.ch/matnmr; written by
Jacco van Beek) under MATLAB (The Mathworks, Inc., Natick, MA) and for
the Ac-Q5G/D6P/W7F analog using Felix (Molecular Simulations Inc., San
Diego, CA). In t2 dimension apodization was
performed using Gaussian window functions for the DQF-COSY and NOESY
spectra and cosine squared window functions for the TOCSY spectra. In t1 dimension cosine squared window functions
were used for all spectra. The solvent was deconvoluted from the
spectra using the time domain convolution method of Marion et
al. (26) with a sine bell function.
Table I shows the analogs of
compstatin we have studied by NMR and their inhibitory activities.
These are the single replacement analogs Val3
The Structural Basis of Compstatin Activity Examined by
Structure-Function-based Design of Peptide Analogs and NMR*,
§,
**,,
Department of Chemical and Environmental
Engineering, University of California, Riverside, California 92521, the ¶ Department of Chemistry and Biochemistry, University of
California at San Diego, La Jolla, California 92093, the
National Centre for Cell Science, Pune University Campus,
Ganeshkhind, Pune 411007, India, the
Department of Biological Applications and
Technologies, University of Ioannina, GR 45110 Ioannina, Greece,
§§ Walter Reed Army Institute of Research,
Silver Spring, Maryland 20910, and the ¶¶ Department of
Pathology and Laboratory Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-turn
located at opposite sides to each other. The disulfide bridge is part
of a hydrophobic cluster, and the -turn is part of a polar surface. We present the design of compstatin analogs in which we have introduced a series of perturbations in key structural elements of their parent
peptide, compstatin. We have examined the consistency of the structures
of the designed analogs compared with compstatin using NMR, and we have
used the resulting structural information to make structure-complement
inhibitory activity correlations. We propose the following. 1) Even in
the absence of the disulfide bridge, a linear analog has a propensity
for structure formation consistent with a turn of a
310-helix or a -turn. 2) The type I -turn is a
necessary but not a sufficient condition for activity. 3) Our
substitutions outside the type I -turn of compstatin have altered
the turn population but not the turn structure. 4) Flexibility of the
-turn is essential for activity. 5) The type I -turn introduces
reversibility and sufficiently separates the two sides of the peptide,
whereas the disulfide bridge prevents the termini from drifting apart,
thus aiding in the formation of the hydrophobic cluster. 6) The
hydrophobic cluster at the linked termini is involved in binding to C3
and activity but alone is not sufficient for activity. 7) -Turn
residues Gln5
(Asn5)-Asp6-Trp7(Phe7)-Gly8
are specific for the turn formation, but only
Gln5(Asn5)-Asp6-Trp7-Gly8
residues are specific for activity. 8) Trp7 is likely to be
involved in direct interaction with C3, possibly through the formation
of a hydrogen bond. Finally we propose a binding model for the
C3-compstatin complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-turn spanning residues
Gln5-Asp6-Trp7-Gly8.
Measurements of inhibitory activities of compstatin analogs designed
using an alanine scan in the sequence between residues Cys2
and Cys12 have shown that Val3 and the four
residues of the -turn are essential for retaining activity (14).
Alanine replacement for each one of the remaining residues resulted in
reduced but not lost inhibitory activity (14). Fig.
1, A and C,
shows the hydrophobic character of the structure of compstatin. We
observe a hydrophobic clustering forming a patch at the linked termini
involving residues Ile1, Cys2,
Val3, Val4, Cys12,
Thr13, whereas the ring of Trp7 caps the
-turn with orientation toward the hydrophobic clustering. We have
hypothesized that this hydrophobic clustering may be essential for
structural stability and inhibitory activity of compstatin (1, 14).
Fig. 1B shows the electrostatic character of compstatin. Positive charges from His9, His10 (each about
50% protonated at the experimental pH), and Arg11 and a
negative charge from Asp6 are observed at the opposite side
of the hydrophobic cluster. However, a positive charge from the amino
terminus is disrupting the hydrophobic cluster. This charge was
neutralized by acetylation of the amino terminus in our effort to avoid
cleavage of the peptide bond between Ile1 and
Cys2 during the biotransformation of compstatin (13).
Interestingly, upon acetylation activity of compstatin was increased by
a factor of 3 (13). We then designed our subsequent studies using the more effective sequence of
Ac-Ile1-Cys2-Val3-Val4-Gln5-Asp6-Trp7-Gly8-His9-His10-Arg11-Cys12-Thr13-NH2.
View larger version (23K):
[in a new window]
Fig. 1.
A, the hydrophobic surface
of compstatin. Hydrophobic residues are drawn in green, and
the rest are drawn in gray. B, the
electrostatic surface of compstatin, where blue and
red correspond to residues with positive and negative
charges, respectively. The orientation is the same as in A. C, a van der Waals sphere representation of compstatin
that shows the continuity of the hydrophobic patch in a different view
than in A and B. Residues Trp7 and
Thr13 are colored in light green to denote less
hydrophobicity, compared with Ile1, Val3,
Val4, Cys2, Cys12 (darker
green). Note that this structure corresponds to non-acetylated
compstatin at the amino terminus. The lowest energy structure of
compstatin has been used from the ensemble of NMR structures deposited
in the Protein Data Bank under code 1A1P (14). The program MOLMOL (29)
has been used for molecular representation. Nter, amino
terminus; Cter, carboxyl terminus.
-turn structures (17, 18). In an earlier study we
designed and tested a number deletion analogs of compstatin in an
attempt to truncate further the 13-residue peptide, but none of them
was active (13). Neither a retro-inverso analog nor -turn analogs,
with substitutions that we expected to enhance a type I or a type II
-turn, were active (13).
-turn found in compstatin. Finally, some substitutions have resulted in loss of both structural stability and activity. We present here the
design of the 7 analogs, one of which is slightly more active than
compstatin and one that is equally as active as compstatin.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Ala
(Ac-V3A), Trp7 Phe (Ac-W7F), and His9 Ala (Ac-H9A), the double replacement analog Cys2 Ala/Cys12 Ala (C2A/C12A), and the triple replacement
analogs Val4 Ala/His9 Ala/Thr13 Ile (Ac-V4A/H9A/T13I), Gln5 Gly/Asp6 Ala/Trp7 Ala (Ac-Q5G/D6A/W7A),
and Gln5 Gly/Asp6 Pro/Trp7
Phe (Ac-Q5G/D6P/W7F). Given that we know the solution structure of
compstatin (14) we can now examine by using NMR the following: 1) if
the designed analogs form structures consistent with the type I
-turn of compstatin or with some other type of turn or secondary
structure; 2) the role of the disulfide bridge in the formation of
structure in compstatin; 3) the role of the hydrophobic and polar
surfaces in the structural stability of compstatin; and 4) which of the
above structural characteristics are essential for function.
Inhibitory activities of compstatin analogs studied by NMR and their
parent peptidesa
We begin our analysis by focusing on the two major types of -turns,
type I and II. We test the consistency of the observed NOEs with the
expected NOEs for type I and type II -turns. This can be
accomplished by examining the following NOEs:
HN(2)-HN(3),
HN(3)-HN(4),
H
(2)-HN(3),
H(3)-HN(4), and
H(2)-HN(4), where the numbers in parentheses
refer to residues 2, 3, and 4 of the -turn (20). NOEs
H(2)-HN(3) and
H(3)-HN(4) should always be present in the
case of a -turn, but they are also generic NOEs in the case of
extended structure. The weak NOE H(2)-HN(4)
is characteristic of the presence of a -turn of both types, type I
and type II. A strong HN(3)-HN(4) NOE is also
characteristic of the presence of a -turn of both types, type I and
type II. The NOE that distinguishes a type I from a type II -turn is
the HN(2)-HN(3), which has strong intensity in
the case of a type I -turn and weak intensity in the type II
-turn. The turn assignment can be further refined by looking at the
intensities of the H(2)-HN(3) NOE, which
should be medium for a type I -turn and strong for type II -turn
(20).
The Linear Analog with Cys2 Ala/Cys12 Ala
(C2A/C12A) Double Replacement--
In the C2A/C12A
analog the two cysteines are replaced by alanines, making it a linear
peptide. This is an inactive peptide (Table I). The rationale for the
design of the linear C2A/C12A analog was to test the significance of
the disulfide bridge in restricting the conformational space of
compstatin and in the formation of the type I -turn.
Fig. 2A shows the
H/side
chain(
1)-HN(2) region of the
TOCSY spectrum of the C2A/C12A analog, where all residues with the exception of Ile1 have been identified. (Note that this analog is not
acetylated which makes the amide of the of the first residue unobservable because of fast exchange with the solvent.) Chemical shift
overlaps in both the H and HN of
Val3/Val4, and to a lesser extent
Arg11/Ala12 are present. Also, chemical shift
overlaps in the H of Trp7/His9,
and to a lesser extent
Asp6/Trp7/His9 are present.
Finally, chemical shift overlaps in the HN of
Ala2/His10 are present. These overlaps are
characteristic of the flexibility of this analog.
|
Fig. 2B shows the
H/H
(1)-HN(2)
region of the NOESY spectrum of the C2A/C12A analog. Sequential
connectivities corresponding to intra-residue
H(i)-HN(i) and short
range H(i)-HN(i + 1)
NOE cross-peaks are marked in Fig. 2B, but only the
intra-residue cross-peaks are labeled for simplicity. Unfortunately,
because of the overlap of the Hs of
Asp6/Trp7, we cannot distinguish the presence
of a weak H(i)-HN(i + 2) cross-peak for the Asp6 H-Gly8
HN NOE from the strong Trp7
H-Gly8 HN cross-peak (Fig.
2B, arrow), which would be consistent with
-turn of the parent peptide, compstatin. This led us to look closer at the
H(1)/HN(2)
region of the spectrum where indeed a very weak
H(i)-HN(i + 2)
cross-peak is present corresponding to the Asp6
H-Gly8 HN NOE (Fig. 2,
B and C). This cross-peak is consistent with the presence of a turn population in the segment
Gln5-Asp6-Trp7-Gly8 as
in parent peptide compstatin. Further examination of
HN(1)-HN(2)
region of the NOESY spectrum (Fig. 2D) reveals a medium
Trp7 HN-Gly8 HN
cross-peak. An Asp6 HN-Trp7
HN cross-peak is also present but somehow obscured by the
diagonal (Fig. 2D). These two cross-peaks are consistent
with the presence of a type I -turn in the segment
Gln5-Asp6-Trp7-Gly8, as
in parent peptide compstatin. Closer examination of the spectra in Fig.
2, B and D, reveals the presence of
Gln5 H-Trp7 HN and
Gln5 HN-Asp6 HN
cross-peaks, which in combination with the Asp6
HN-Trp7 HN cross-peak are
characteristic of the presence of a type I -turn in the segment
Val4-Gln5-Asp6-Trp7.
Alternatively, we can say that two fused type I -turns are present
in the segment
Val4-Gln5-Asp6-Trp7-Gly8.
A weak H(i)-HN(i + 3)
cross-peak, Gln5 H-Gly8
HN (Fig. 2B), in combination with the
H(i)-HN(i + 2) and
HN(i)-HN(i + 1)
cross-peaks mentioned above for the fused -turns, is suggestive of
the presence of a population forming a turn of a 310-helix
(20) in the segment
Gln5-Asp6-Trp7-Gly8.
Based on the observed NOEs for the C2A/C12A analog and the -turn
forming analogs (see below), we conclude that the linear sequence of
compstatin has a propensity to form a 310-helix, which upon
formation of the disulfide bond is reduced to a -turn.
Interestingly, a type III -turn is classified as a turn of a
310-helix (19, 27). A type III -turn has characteristic
dihedral angles for residues 2 and 3 of the turn
(
2, 
2) = (
60o,
30o) and (3, 3) = (60o, 30o) compared with a type I -turn
which has dihedral angles (2, 2) = (60o, 30o) and (3,
3) = (90o, 0o). However,
because there is a statistical variation associated with dihedral
angles of ±30o (which is also the error of calculated
dihedral angles from NMR data), the type III -turn is often
classified under the type I -turn category. In this report we do not
distinguish a type III from a type I -turn. The structure of the
C2A/C12A analog is not sufficient for C3 binding and activity (Table
I), which makes the disulfide bridge necessary for activity. Based on
the structure of compstatin (Fig. 1), we propose that the disulfide bridge brings together the termini of compstatin thus contributing to
the formation of the hydrophobic patch, which in turn is necessary for
recognition and binding to C3.
The Single Replacement His9 Ala (Ac-H9A)
Analog--
In the Ac-H9A analog the His9, right outside
the C-terminal residue of the -turn, is replaced by Ala. This is an
analog slightly more active than its parent peptide, Ac-compstatin
(Table I). The rationale for the design of the Ac-H9A analog was to
introduce additional conformational freedom at one end of the type I
-turn by removing the bulky histidine and replacing it with the
smaller alanine. This accomplishes a test for the turn stability
without losing activity, given that a non-acetylated ring-only form of this analog (with missing residues Ile1 and
Thr13 outside the peptide ring) was active, although less
active than its parent peptide ring-only compstatin (Table I of Ref.
14).
Fig. 3A shows the
H(1)-HN(2)
region of the DQF-COSY spectrum of the Ac-H9A analog, where all
residues have been identified. Chemical shift overlaps in the
H are observed for Cys2/Cys12
and Gln5/Ala9. Chemical shift overlaps in the
HN are observed for of Ala9/Arg11
and Gln5/Thr13.
|
Fig. 3B shows the
H(1)-HN(2)
region of the NOESY spectrum of the Ac-H9A analog. Sequential
connectivities corresponding to intra-residue
H(i)-HN(i) and short
range H(i)-HN(i + 1)
NOE cross-peaks are marked in the figure. In addition, a weak
H(i)-HN(i + 2)
cross-peak corresponding to Asp6
H-Gly8 HN is observed
(circled in Fig. 3B), which is consistent with
the presence of a -turn. Fig. 3C shows the
HN(1)-HN(2)
region of the NOESY spectrum of the Ac-H9A analog where observed HN(i)-HN(i + 1)
cross-peaks are labeled. Medium intensity cross-peaks corresponding to Asp6 HN-Trp7
HN and Trp7 HN-Gly8
HN NOEs are observed which are consistent with the presence
of a type I -turn in the segment
Gln5-Asp6-Trp7-Gly8, as
in compstatin. In compstatin, a very weak
H(i)-HN(i + 2) NOE of
Asp6 H-Gly8 HN was
also observed (14), which is absent in the Ac-H9A analog (data not
shown). This suggests a slightly increased
flexibility or alternatively slightly reduced population of the type I
-turn in Ac-H9A analog compared with compstatin.
The above NOE data are consistent with the presence of a weak (with
small but observable population) type I -turn in the segment
Gln5-Asp6-Trp7-Gly8 of
Ac-H9A analog, as was the case of parent peptide compstatin. In
compstatin, the presence of Gly8 at position 4 of the type
I -turn was deemed necessary to reduce side chain steric hindrance
for turn reversal. By replacing the next residue His9 by
Ala, we introduced additional flexibility outside one end of the
-turn by increasing the available conformational space. This
additional flexibility did not alter the type I -turn structure, although it slightly reduced its population. It is worth noting that
the population of the type -turn in compstatin was estimated at
42-63% (14), which suggests flexibility in the parent peptide as
well. This analog possesses slightly higher inhibitory activity than
compstatin (Table I), which suggests that flexibility outside the
-turn contributes to activity.
The Triple Replacement Val4 Ala/His9
Ala/Thr13 Ile (Ac-V4A/H9A/T13I) Analog--
In the
Ac-V4A/H9A/T13I analog not only His9 but also
Val4, right outside both ends of the -turn, are replaced
by alanines, with the rationale of introducing even more conformational
freedom outside the turn, than in the Ac-H9A analog. In addition,
Thr13 is replaced with the more hydrophobic isoleucine to
enhance the hydrophobic clustering at the termini. This analog is
equally active as compstatin (Table I).
Fig. 4A shows the
H(1)-HN(2)
region of the DQF-COSY spectrum of the Ac-V4A/H9A/T13I analog, where
all residues except Ile1 have been identified
(Ile1 has been identified in the TOCSY spectrum, see the
Supplemental Material). Chemical shift overlap in the H
is observed for Asp6/His10. Chemical shift
overlaps in the HN are observed for
Ala4/Gly8, Gln5/Ile13,
and Ala9/Arg11. Despite the overlaps in
individual dimensions, the resulting cross-peaks are distinct (Fig.
4A).
|
Fig. 4B shows the
H(1)-HN(2)
region of the NOESY spectrum of the Ac-V4A/H9A/T13I analog. All
sequential connectivities corresponding to intra-residue
H(i)-HN(i) and short
range H(i)-HN(i + 1)
NOE cross-peaks are observed and marked in the figure. However, a weak
H(i)-HN(i + 2)
cross-peak for the Asp6 H-Gly8
HN NOE is not observed (position indicated by an
arrow in Fig. 4B). Fig. 4C shows the
HN(1)-HN(2)
region of the NOESY spectrum of the Ac-V4A/H9A/T13I analog, where
observed HN(i)-HN(i + 1)
NOEs are labeled. Medium/weak intensity cross-peaks corresponding to
Asp6 HN-Trp7 HN and
Trp7 HN-Gly8 HN NOEs
are observed, which are consistent with the presence of a type I
-turn in the segment
Gln5-Asp6-Trp7-Gly8, as
in compstatin. It should be noted that the absence of the Asp6 H-Gly8 HN NOE
suggests a reduced turn population in the Ac-V4A/H9A/T13I analog
compared with the Ac-H9A analog.
The above NOE data are consistent with the presence of a small
population of type I -turn in the
Gln5-Asp6-Trp7-Gly8
segment of the Ac-V4A/H9A/T13I analog, which is smaller than the turn
population in the same segment of the Ac-H9A analog. Despite the
increase of conformational freedom and weakening of the type I
-turn, this peptide is an analog equally active to compstatin (Table
I).
The fact that both Ac-H9A and Ac-V4A/H9A/T13I but not the linear
C2A/C12A analog are active suggests that flexibility between the
-turn and disulfide bridge structures is necessary for binding activity.
The Single Replacement Val3 Ala (Ac-V3A)
Analog--
In the Ac-V3A analog Val3 is replaced by Ala.
The rationale for this replacement was to weaken the hydrophobic
clustering at the termini. This replacement resulted in an inactive
analog (Table I).
Fig. 5A shows the
H(1)-HN(2)
region of the DQF-COSY spectrum of the Ac-V3A analog, where all
residues have been identified. Chemical shift overlaps in both
H and HN are observed for
Asp6/His9. Chemical shift overlaps in the
H are observed for Cys2/Cys12
and to a lesser extent for Ala3/Arg11. Chemical
shift overlaps in the HN are observed for
Asp6/Trp7/His9 and
Gln5/Arg11. Interestingly, the Ac-V3A
replacement had an impact in the chemical shift of Asp6,
which is shifted upfield (Fig. 5A) compared with the other
analogs (Figs. 2A, 3A, and
4A).
|
Fig. 5B shows the
H(1)-HN(2)
region of the NOESY spectrum of the Ac-V3A analog. All sequential
connectivities corresponding to intra-residue
H(i)-HN(i) and short
range H(i)-HN(i + 1)
NOE cross-peaks are observed and marked in the figure. In addition, a
medium H(i)-HN(i + 2)
cross-peak is observed corresponding to NOE Asp6
H-Gly8 HN, which is characteristic of the
presence of a -turn in the segment Gln5-Asp6-Trp7-Gly8.
Fig. 5C shows the
HN(1)-HN(2)
region of the NOESY spectrum of the Ac-V3A analog, where observed
HN(i)-HN(i + 1) NOEs are
labeled. Two strong cross-peaks corresponding to NOEs between
Gln5 HN-Asp6 HN and
Trp7 HN-Gly8 HN are
observed, but because of the overlap of the amide protons of
Asp6 and Trp7 we cannot determine or exclude
the presence of the Asp6
HN-Trp7 HN cross-peak. Because of
the intensities of the observed NOEs, we deduce that the observed
structure is consistent with the presence of a strong type I -turn
in the segment
Gln5-Asp6-Trp7-Gly8, as
in parent peptide compstatin.
Based on the NOE data of the Ac-V3A analog, the Val3 Ala replacement has resulted in a stronger (of higher population) type I -turn. It appears that the remote, in relation with the -turn of compstatin, residue Val3 somehow influences the -turn
stability in the parent peptide. Because the activity of the Ac-V3A
analog is lost (Table I), we can say that Val3 influences
the binding of the parent peptide compstatin to C3, possibly either
through the hydrophobic clustering of the termini or through some type
of stabilization of the -turn. The simultaneous presence of both
effects cannot be excluded.
The Single Replacement Trp7 Phe (Ac-W7F)
Analog--
In the Ac-W7F analog Trp7 is replaced by Phe.
The rationale for this replacement was to enhance the hydrophobicity of
the -turn and participation of residue 7 in the hydrophobic
clustering, if any (Fig. 1A). This replacement resulted in
an inactive analog (Table I).
Fig. 6A shows the
H(1)-HN(2)
region of the DQF-COSY spectrum of the Ac-W7F analog, where all
residues have been identified. Chemical shift overlaps in the
H are observed for Cys2/Cys12
and Gln5/Thr13. Chemical shift overlaps in the
HN are observed for Phe7/His9,
Gln5/Arg11, and
His10/Cys12. Despite the overlaps in individual
dimensions, the resulting cross-peaks are distinct (Fig.
6A).
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Fig. 6, B and C, shows the
H(1)-HN(2) and
HN(1)-HN(2)
regions, respectively, of the NOESY spectrum of the Ac-W7F analog. All
sequential connectivities corresponding to intra-residue
H(i)-HN(i) and short
range H(i)-HN(i + 1)
NOE cross-peaks are observed and marked in Fig.
6B. In addition, a medium/weak intensity
H(i)-HN(i + 2)
cross-peak is observed corresponding to NOEs of Asp6
H-Gly8 HN, which is
characteristic of the presence of -turn in the segment Gln5-Asp6-Trp7-Gly8.
The segment
Gln5-Asp6-Trp7-Gly8 can
be further assigned as a type I -turn because of the presence of the
medium intensity
HN(i)-HN(i + 1)
cross-peaks of Asp6 HN-Phe7
HN and Phe7 HN-Gly8
HN NOEs (Fig. 6C).
Based on the NOE data it appears that Trp7
specifically is not unique for the formation of the type I -turn, as
the turn is present in the Ac-W7F analog as well. The need for
participation of an aromatic residue at position 7 in the formation of
the type I -turn cannot be excluded. It is also possible that an
aromatic residue at position 7 simultaneously influences the structure of the amino-terminal half of compstatin, which is part of the hydrophobic cluster. Because of loss of inhibitory activity in the
Ac-W7F analog, it is possible that Trp7 participates in
binding with C3. This could be possible through a hydrogen bond
involving the indole amide of Trp7 as a donor.
The Triple Replacement Gln5 Gly/Asp6 Ala/Trp7 Ala (Ac-Q5G/D6A/W7A)
Analog--
In the Ac-Q5G/D6A/W7A analog Gln5 is replaced
by Gly and Asp6 and Trp7 are replaced by Ala.
The rationale for this replacement was to introduce steric
simplification and flexibility within the -turn of the parent
peptide, compstatin. This replacement resulted in an inactive analog
(Table I).
Fig. 7A shows the
H(1)-HN(2)
region of the DQF-COSY spectrum of the Ac-Q5G/D6A/W7A analog, where all
residues have been identified. Chemical shift overlaps in the
HN are observed for Cys2/Gly5,
Val3/Arg11, and
His10/Cys12 and in the H for
Ala6/Ala7 and
His9/His10. Despite the H
overlaps the resulting cross-peaks are distinct (Fig.
7A).
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Fig. 7, B and C, shows the
H(1)-HN(2) and
HN(1)-HN(2)
regions, respectively, of the NOESY spectrum of the Ac-Q5G/D6A/W7A analog. All sequential connectivities corresponding to intra-residue H(i)-HN(i) and short
range H(i)-HN(i + 1)
NOE cross-peaks are observed and marked in Fig. 7B. A H(i)-HN(i + 2)
cross-peak corresponding to Ala6
H-Gly8 HN NOE that if present
would be consistent with the -turn of the parent peptide,
compstatin, cannot be assigned with certainty, as it would overlap with
the strong intensity Ala7 H-Gly8
HN cross-peak (marked with arrow in Fig.
7B). To have a type I -turn in the same segment as the
parent peptide compstatin,
HN(i)-HN(i + 1)
cross-peaks corresponding to Ala6
HN-Ala7 HN and Ala7
HN-Gly8 HN NOEs should be present
in Fig. 7C. From these cross-peaks only the Ala6
HN-Ala7 HN NOE is present, thus
excluding structural similarity with the parent peptide compstatin. The
presence of sequential Gln5 HN-Ala6
HN and Ala6 HN-Ala7
HN NOEs could be consistent with a type I -turn in the
segment Val4-Gly5-Ala6-Ala7,
but because of missing of the accompanying Gly5
H(i)-Ala7
HN(i + 2) NOE in Fig. 7B, we conclude
that the Ac-Q5G/D6A/W7A analog is unstructured.
Based on the NOE data, the introduction of flexibility within
the type I -turn of compstatin resulted in both loss of the structural stability and loss of inhibitory activity. This suggests that either the type I -turn or specific residues in the turn segment or both are necessary for C3 binding and inhibitory activity.
The Triple Replacement Gln5 Gly/Asp6
Pro/Trp7 Phe (Ac-Q5G/D6P/W7F) Analog--
In the
Ac-Q5G/D6P/W7F analog Gln5 is replaced by Gly;
Asp6 is replaced by Pro; and Trp7 is replaced
by Phe. The rationale for this replacement was to enforce the -turn
and test relation to activity. We hypothesized that this could be
achieved by the following: (a) removal of steric hindrance
imposed on the backbone from the side chain of the first residue of the
type I -turn by replacing Gln5 with Gly; (b)
introduction of a proline that provides a backbone bend frequently
observed in type II -turns in proteins (28); and (c)
replacement of Trp7 by Phe to retain, to a certain extent,
bulkiness at position 3 of the turn of parent peptide, while
maintaining hydrophobicity.
Fig. 8, A and
C, shows the
H(1)-HN(2) and
HN(1)-HN(2)
regions, respectively, of the NOESY spectrum of the Ac-Q5G/D6P/W7F analog. Fig. 8B is a portion of Fig. 8A including
NOEs involving the HN of Gly8, plotted at lower
contour level. All sequential connectivities corresponding to
intra-residue
H(i)-HN(i) and short
range H(i)-HN(i + 1)
NOE cross-peaks are observed and marked in Fig. 8A, except for the lack of amide in Pro6 which introduces a break. An
extremely weak
H(i)-HN(i + 2)
cross-peak between Pro6 H-Gly8
HN is present in Fig. 8B, which is consistent
with the presence of a very weak turn segment in
Gly5-Pro6-Phe7-Gly8, as
was the case in the corresponding segment of compstatin. In addition,
we observe a medium intensity
H(i)-HN(i + 2)
cross-peak and weak intensity
H(i)-HN(i + 3) and
H(i)-HN(i + 4)
cross-peaks corresponding to NOEs of Gly5
H-Phe7 HN, Gly5
H-Gly8 HN, and Gly5
H-His9 HN, respectively. These NOEs could be
characteristic of a turn of an 
-helix in the segment
Gly5-Pro6-Phe7-Gly8-His9,
but the assignment cannot be completed using
HN-HN NOEs because of lack of amide proton in
proline. Thus, we conclude that these NOEs are consistent with the
presence of undetermined structure in the segment
Gly5-Pro6-Phe7-
Gly8-His9.
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Based on the NOE data the radical triple replacement in the
Ac-Q5G/D6P/W7F analog produced a structured segment
Gly5-Pro6-Phe7-Gly8-His9,
in which a small turn populations in the segment
Gly5-Pro6-Phe7-Gly8
cannot be excluded. The loss of activity and possible introduction of
additional structure in the Ac-Q5G/D6P/W7F analog points out the
specificity of the four residues of the type I -turn of the parent
peptide compstatin for structural stability and inhibitory activity.
Activity Studies of Compstatin Analogs with Conservative Replacements-- We have constructed a number of conservative replacement compstatin analogs and have tested their activity (Table II), without performing the NMR analysis, as it was deemed unnecessary. Some of these analogs are active, but only one of them has shown slightly higher activity than Ac-compstatin (Table II). When comparing the Ac-V3L, Ac-Q5N, and R11K compstatin analogs to compstatin, it appears that Val is slightly preferred than Leu at position 3; Asn is equally preferred as Gln at position 5; and Arg is more preferred than Lys at position 11 (Table II). Also, the double replacement in the Ac-V3L/Q5N analog resulted in a 2-fold lower activity than Ac-compstatin (Table II). To address the issue of proteolytic cleavage at Arg11 during biotransformation of compstatin (13), we have prepared the Ac-R11S, Ac-Q5N/R11A, Ac-H9A/R11A compstatin analogs, none of which was more active than compstatin (Table II). It appears that Arg is a preferred amino acid at position 11, although the double replacement analog Ac-H9A/R11A showed only about 2-fold reduced activity than compstatin because of compensation from the H9A substitution. Also, replacement of Arg11 with dArg resulted to total loss of inhibitory activity.
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To test further our hypothesis of hydrophobic clustering at the
termini, we prepared the Ac-T13I analog, where Thr was replaced with
the more hydrophobic Ile to enhance hydrophobicity. This analog showed
slightly higher activity than compstatin (Table II), consistent with
our hypothesis.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We have designed seven analogs of compstatin in which we have
introduced a series of perturbations in key structural elements of
their parent peptide, compstatin. The design was based on the previously determined three-dimensional structure of compstatin (14).
The structure of compstatin consists of a disulfide bridge on one side
and a type I -turn on the opposite side. The disulfide bridge is
part of a hydrophobic cluster, and the type I -turn is part of a
polar surface. The structural perturbations were made to test the
effect of specific residue replacements in the structural stability of
compstatin and their contributions in binding to C3 and complement
inhibitory activity. We know that the disulfide bridge is essential for
activity, and we have hypothesized that both the type I -turn and
the hydrophobic clustering at the termini are necessary for activity
(1, 13, 14). Our first goal was to correlate structure with activity
for each analog and for compstatin. Our second goal was to design
analogs that are equally active to or more active than compstatin.
Specifically, our structural perturbations were made for the following
reasons: 1) to test the significance of the disulfide
bridge in the formation of structure in compstatin; 2) to locally alter
the structure of compstatin, outside and inside the -turn, to test
the structural stability of the -turn; and 3) to locally alter the
hydrophobic clustering to test its contribution in the formation of the
-turn. The structural analysis of the compstatin analogs was made
using NMR data. We used correlation spectroscopy for proton assignments and NOE spectroscopy to elucidate NOE connectivity patterns that are
consistent with structure. The known three-dimensional structure of
compstatin (14) was used as a base-line structural template for our
analysis. Fig. 9 summarizes the backbone
NOE connectivity patterns observed for the seven compstatin analogs
studied by NMR. The higher intensities of the
H(i)-HN(i + 1)
compared with HN(i)-HN(i + 1) NOEs suggest NOE averaging because of the presence of extended
conformations, in addition to the observable populations of the
proposed structures. This is expected because of flexibility and
conformational interconversion that is present in small peptides in
solution.
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The C2A/C12A linear analog showed a structure consistent with the presence of a turn of a 310-helix. A 3<
-Phe7 HN are observed.
B, portion of A containing Gly8
plotted at lower contour level, which reveals the weak Gly5
H