(Received for publication, December 21, 1994)
From the
To examine the effect of a conformational constraint introduced
into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we
constructed a mutant protein by inserting an RGD-containing sequence
flanked by two Cys residues between Val and Asn
of human lysozyme. The CRGDSC-inserted lysozyme was expressed in
yeast, purified, and designated as Cys-RGD4. Using baby hamster kidney
cells, Cys-RGD4 was shown to possess even higher cell adhesion activity
than that of the RGDS-inserted lysozyme, RGD4.
The Cys-RGD4 protein
was co-crystallized with a lysozyme inhibitor,
tri-N-acetylchitotriose, and the three-dimensional structure
was determined at 1.6-Å resolution by x-ray crystallography. In
contrast to RGD4, the inserted RGD-containing region of Cys-RGD4 was
well defined. The structural analysis revealed that the two inserted
Cys residues form a new disulfide bond in Cys-RGD4, as expected, and
that the RGD region assumes a type II` -turn conformation of
Gly-Asp with a hydrogen bond between the C=O of Arg and the
H-N of Ser. In addition, it was confirmed that two more hydrogen
bonds are present in the RGD region of the Cys-RGD4 lysozyme. These
results suggest that the conformation of the RGD-containing region is
rigid and stable in the Cys-RGD4 molecule and that the type II`
-turn structure of RGD is essential for binding to integrins with
high affinity.
The Arg-Gly-Asp (RGD) ()sequence is a well known site
in cell adhesive proteins, such as fibronectin (Pierschbacher and
Ruoslahti, 1984), vitronectin (Suzuki et al., 1985), and
fibrinogen (Watt et al., 1979), for binding to their
receptors, the integrins (Hynes, 1987; Hemler, 1991). In addition to
these cell adhesive proteins, a number of other proteins have been
found to contain the RGD sequence, but only a limited number of them
possess cell adhesion activity. This fact could be explained if only an
RGD sequence with an appropriate conformation can interact with the
receptor molecule (Ruoslahti and Pierschbacher, 1987). To determine the
functional conformation of RGD, we previously constructed a mutant
protein (RGD4) by inserting the RGDS sequence of human fibronectin
between Val
and Asn
of human lysozyme, using
a yeast expression system (Yamada et al., 1993). We have
already examined the three-dimensional structure of the RGD4 lysozyme
by x-ray crystallographic and two-dimensional NMR techniques and shown
that the RGD-containing region is conformationally flexible (Yamada et al., 1993).
Next, to examine the effect of a conformational constraint introduced into the RGD sequence on cell adhesion activity, we inserted the RGDS sequence flanked by two Cys residues at the aforementioned site in human lysozyme. This design is based on the fact that the cyclic form of an RGD-containing peptide has much higher affinity to integrins than the linear counterpart (Pierschbacher and Ruoslahti, 1987; Kumagai et al., 1991). Here we report the functional evaluation and the x-ray structural analysis of the conformationally constrained RGD sequence in the mutant lysozyme, Cys-RGD4.
Cell adhesion activity was determined using baby hamster kidney (BHK) cells as described (Maeda et al., 1989; Yamada et al., 1994). The amount of lysozyme adsorbed onto a plate was estimated by subtracting the unadsorbed amount from the added amount of lysozyme in the assay. The unadsorbed amount was determined based on the lytic activity (Taniyama et al., 1990) remaining in the sample solution after binding to the plate. The results indicated that the adsorption efficiency was in the range of 60-80% at the concentrations shown in Fig. 1, for both native and mutant lysozymes.
Figure 1:
Cell spreading assay on the substrates
coated with each mutant lysozyme. The plastic substrates were coated
with different concentrations of native lysozyme (), RGD4
(
), and Cys-RGD4 (
). BHK cells were incubated on the
substrates for 60 min at 37 °C in a CO
incubator. The
extent of cell spreading was expressed as the number of cells adhered
per unit surface area (cm
).
Prior to the co-crystallization, we
tried to crystallize Cys-RGD4 using 2.5 M NaCl as a
precipitant, which is the standard protocol for the crystallization of
native human lysozyme (Inaka et al., 1991), but could not.
This was because the solubility of the protein is very low under such
conditions. The addition of the (GlcNAc) molecule to
Cys-RGD4 was quite effective in increasing its solubility, although the
reason remains to be explained.
The x-ray intensity data were collected by an automated oscillation camera system (DIP-320, MAC Science) equipped with a cylindrical imaging plate (Miyahara et al., 1986). The structure refinements were carried out using the program package, X-PLOR, version 3.0 (Brünger et al., 1987), and the modified program, PROLSQ (Hendrickson, 1985).
We obtained a mutant lysozyme, Cys-RGD4, by inserting the
CRGDSC sequence between Val and Asn
of human
lysozyme. In the Cys-RGD4 protein, no free thiol group was detected by
the 5,5`-dithiobis(nitrobenzoic acid method (Ellman, 1959). The peptide
mapping analysis (data not shown) indicated that the two inserted Cys
residues in Cys-RGD4 are linked to each other without any effects on
the four disulfide bonds present in native human lysozyme.
Cell
adhesion activity of Cys-RGD4 was assayed using BHK cells. As shown in Fig. 1, the Cys-RGD4 protein possessed a high level of activity,
which corresponds to one-tenth that of human vitronectin. This high
activity is surprising, considering that Cys-RGD4 contains only four
residues, RGDS, from human fibronectin. Fig. 1also shows that
Cys-RGD4 is 3-fold more effective than the RGDS-inserted mutant
lysozyme, RGD4, at 1000 nM. The cell adhesion activities of
both mutant lysozymes were completely inhibited by the addition of
either GRGDSP peptide or polyclonal antibody against vitronectin
receptor, as was the case for the vitronectin activity (data not
shown). The results suggest that the cell adhesion signals in these
mutant proteins are transduced to BHK cells through the interaction
with the vitronectin receptor, the integrin
.
To determine the conformation of
the inserted RGD region, the Cys-RGD4 protein was co-crystallized with
a lysozyme inhibitor, tri-N-acetylchitotriose
((GlcNAc)), and the three-dimensional structure was
elucidated crystallographically. The co-crystal was isomorphous to that
of the native protein with (GlcNAc)
, and the structure was
determined by the molecular replacement method at 1.6-Å
resolution. The crystal data and the refinement parameters are
summarized in Table 1.
The inserted
Cys-Arg
-Gly
-Asp
-Ser
-Cys
region in the Cys-RGD4 molecule demonstrated continuous electron
densities (Fig. 2C), and a clear model could be built (Fig. 2B). The positions of the preceding residues,
Thr
-Pro
-Gly
-Ala
-Val
,
were somewhat uncertain because of the lack of continuous electron
densities (Fig. 2C), as was the case for those of
Ala
-Val
in RGD4 (Yamada et al.,
1993). The conformational model (Fig. 2B) and the
(
,
) angles (Table 2) of the inserted residues
indicate that the RGD region contains a type II`
-turn of
Gly
-Asp
and a type I
-turn of
Ser
-Cys
, with two hydrogen bonds,
Ser
N-Arg
O (2.87 Å) and
Asn
N-Asp
O (2.88 Å). In addition,
another hydrogen bond, Arg
N-Ser
O
(2.58 Å), and a disulfide bond,
Cys
-Cys
, were also shown to exist in
this region (Fig. 2B). The presence of these chemical
bonds suggests that the conformation of the RGD-containing region is
rigid and stable in the Cys-RGD4 lysozyme. The side chain of
Arg
is flexible and could be modeled in two ways (Fig. 2B), while the Asp
side chain is
conformationally restricted because of a possible interaction with the
side chain of His
(His
N
2-Asp
O
1, 2.70 Å) (Fig. 2C). The elimination of this interaction in the
Cys-RGD4 protein, if possible, might result in an increase in cell
adhesion activity, because the side chains of Asp and Arg are essential
for binding to the integrins.
Figure 2:
Crystal structures of native lysozyme and
Cys-RGD4. A, the backbone models of native lysozyme (blue
line) and Cys-RGD4 (yellow line). Val in
each lysozyme is labeled. The models were well superimposed, except for
the regions around the inserted residues. B, the stereo
drawing of the conformational model in the inserted region of Cys-RGD4.
The skeleton structure of the inserted region is shown in red.
The structural model of the Arg
side chain can be built
in two ways. The disulfide bond and the hydrogen bonds in the RGD
region are shown by a green line and broken lines,
respectively. Val
in Cys-RGD4 is labeled. C, the
(2F
- F
) electron density map in the RGD region
of Cys-RGD4. The skeleton structure of the inserted region is shown in red. Val
in Cys-RGD4 is labeled. The electron
densities at the upper left and right sides belong to
those of the neighboring protein molecules in the
crystal.
We have constructed a mutant lysozyme, Cys-RGD4, in which the
CRGDSC sequence is inserted between Val and Asn
of human lysozyme. Cell adhesion assays using BHK cells revealed
that the mutant exhibits even higher activity than that of the
RGDS-inserted mutant, RGD4. The x-ray structural analysis, as well as
the peptide mapping analysis (data not shown), indicated that the
Cys-RGD4 mutant possesses a new disulfide bond between the two inserted
Cys residues, in addition to the four native disulfide bonds of human
lysozyme. These results demonstrate that the introduction of a
conformational constraint into the RGD sequence of the mutant lysozyme
significantly increases the affinity to the integrins, as is the case
for an RGD-containing peptide.
The Cys-RGD4 mutant was successfully
co-crystallized with a lysozyme inhibitor, (GlcNAc). The
x-ray analysis suggested that the inserted RGD region is distant from
the (GlcNAc)
binding cleft of human lysozyme (Fig. 2A). In addition, the (GlcNAc)
molecule had no effects on the cell adhesion activity of Cys-RGD4
at the concentration (6.0 mg/ml) used for the crystallization (data not
shown). These results suggest that the RGD region assumes a
biologically active conformation in the complex of Cys-RGD4 with
(GlcNAc)
.
Recently, several researchers have reported structural analyses of RGD-containing proteins and discussed the functional conformation of the RGD sequence. NMR (Main et al., 1992) and x-ray (Dickinson et al., 1994) studies on the RGD-containing tenth type III module of human fibronectin have shown that the RGD region lies on a conformationally mobile loop. Similar results have been reported for the NMR solution structures of the disintegrins, a family of RGD-containing integrin antagonists from snake venoms (Adler et al., 1991; Saudek et al., 1991). We have also described that the RGD region of the RGDS-inserted mutant lysozyme, RGD4, is conformationally flexible and that such flexibility could allow the RGD conformation to be induced to fit into the binding pocket of the integrin receptor (Yamada et al., 1993). In these cases, however, it is conceivable that the RGD region, which is highly flexible by nature, assumes a fixed conformation when it binds to integrins to form a ligand-receptor complex. In addition, we cannot completely rule out the possibility that the RGD sequence in these proteins has a rigid and specific conformation by itself and that it was ill defined because of its location at the apex of a flexible, long loop.
The present structural analysis of Cys-RGD4 has shown
that the RGD sequence resides within a stable type II` -turn of
Gly-Asp, with a hydrogen bond between the C=O of Arg and the
H-N of Ser. Thus, it seems likely that the new disulfide bond
between the two inserted Cys residues influences the RGD region to
assume the turn structure with higher biological activity. Leahy et
al.(1992) have solved the x-ray crystal structure of the
fibronectin type III domain from tenascin and reported that the RGD
region is located in an extended type II`
-hairpin loop. Quite
recently, Krezel et al. (1994) have examined the NMR solution
structure of the leech protein decorsin, a potent integrin antagonist
related to the disintegrins, and suggested the presence of a distorted
type II` turn of Gly-Asp in the RGD-containing region. In the three
cases including ours, the main chain structures of RGD bear a
resemblance to one another (Table 2), although the side chain
structures of RGD are unclear because of their flexibility. These
results strongly suggest that the type II`
-turn conformation of
Gly-Asp in RGD is essential for binding to integrins with high
affinity.
In the present investigation, we have succeeded in determining the functional conformation of RGD in a cell adhesive lysozyme, Cys-RGD4. It is well known that RGD-dependent cell adhesion plays important roles during various physiological phenomena, such as tissue remodeling, platelet aggregation, bone resorption, and tumor metastasis. The information described here could be quite helpful in designing a drug to modulate these functions.