©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure of a Conformationally Constrained Arg-Gly-Asp Sequence Inserted into Human Lysozyme (*)

(Received for publication, December 21, 1994)

Takao Yamada (§) Haiwei Song Koji Inaka (¶) Yoshimi Shimada Masakazu Kikuchi Masaaki Matsushima

From the Protein Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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` beta-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` beta-turn structure of RGD is essential for binding to integrins with high affinity.


INTRODUCTION

The Arg-Gly-Asp (RGD) (^1)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.


MATERIALS AND METHODS

Vector Construction

Oligonucleotides were chemically synthesized using an automated DNA synthesizer (model 380B, Applied Biosystems). The double-stranded DNA encoding CRGDSC was obtained by annealing 5`-TGCCGCGGTGATTCTTGT-3` and 5`-ACAAGAATCACCGCGGCA-3`. M13mpXhLZM was ligated with the CRGDSC-coding gene after digesting with HincII (Yamada et al., 1993). The sequence of the mutant gene thus obtained was confirmed by dideoxy sequencing. The genes encoding the signal sequence and the mutated human lysozyme were combined with an XhoI-SmaI large fragment from pERI8602 (Taniyama et al., 1990) to construct the expression plasmid.

Expression and Purification of Cys-RGD4

The Cys-RGD4 mutant was expressed in yeast as described previously (Yoshimura et al., 1987), with a productivity of 1.7 mg/liter. The secreted Cys-RGD4 protein was purified essentially as described (Taniyama et al., 1990). HPLC was performed using a cation-exchange column (Asahipak ES-502C, Asahikasei Co. Ltd., Japan) and a hydroxyapatite column (TAPS-020810, Tonen K. K., Japan).

Measurement of Cell Adhesion Activity

Protein was determined by measuring the weight in the freeze-dried form of each mutant lysozyme.

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 (circle), RGD4 (bullet), and Cys-RGD4 (up triangle). BHK cells were incubated on the substrates for 60 min at 37 °C in a CO(2) incubator. The extent of cell spreading was expressed as the number of cells adhered per unit surface area (cm^2).



X-ray Crystallography

Crystallization was carried out using a vapor diffusion technique. To obtain co-crystals of Cys-RGD4 with tri-N-acetylchitotriose ((GlcNAc)(3)), the protein solution (25 µl) contained 20 mg/ml Cys-RGD4, 2.0 M NaCl, 50 mM sodium phosphate (pH 6.0), and 6.0 mg/ml (GlcNAc)(3) (Seikagaku-kogyo Co. Ltd., Japan). The molar ratio of (GlcNAc)(3) to the enzyme was 7:1. This solution was diffused against a solution (1 ml) of 2.7 M NaCl and 50 mM sodium phosphate (pH 6.0) in a chamber maintained at 13.0 °C. The co-crystals achieved a size large enough for x-ray diffraction experiments within 2 weeks.

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)(3) 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).


RESULTS

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 alpha(v)beta(3).

To determine the conformation of the inserted RGD region, the Cys-RGD4 protein was co-crystallized with a lysozyme inhibitor, tri-N-acetylchitotriose ((GlcNAc)(3)), and the three-dimensional structure was elucidated crystallographically. The co-crystal was isomorphous to that of the native protein with (GlcNAc)(3), 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` beta-turn of Gly-Asp and a type I beta-turn of Ser-Cys, with two hydrogen bonds, SerN-ArgO (2.87 Å) and AsnN-AspO (2.88 Å). In addition, another hydrogen bond, ArgN-SerO (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 (HisN 2-AspO 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.






DISCUSSION

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)(3). The x-ray analysis suggested that the inserted RGD region is distant from the (GlcNAc)(3) binding cleft of human lysozyme (Fig. 2A). In addition, the (GlcNAc)(3) 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)(3).

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` beta-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` beta-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` beta-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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-06-872-8200; Fax: 81-06-872-8210.

Present address: Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Kanagawa 227, Japan.

(^1)
The abbreviations used are: RGD, Arg-Gly-Asp; (GlcNAc)(3), tri-N-acetylchitotriose; BHK, baby hamster kidney.


ACKNOWLEDGEMENTS

We thank Drs. M. Ikehara, J. Sugai, and K. Sekiguchi for their interest and encouragement throughout this work. We also thank Dr. A. Kidera for helpful discussions and A. Uyeda for technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.