1 Protein Engineering Network of Centres of Excellence, 713 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta, T6G 2S2 and 2 Biotechnology Research Institute, Montreal, Quebec, H4P 2R2, Canada
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: binding epitope/epidermal growth factor receptor/ligandreceptor interactions/NMR/transforming growth factor alpha
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
15N-labeled TGF and epidermal growth factor receptor extracellular domain (EGFR-ED) were prepared and purified according to previous methods (Hoyt et al., 1994
).
NMR sample preparation
The EGFR-ED (0.07 µmol) in a volume of 450 µl was dialyzed with a buffer containing 50 µM potassium phosphate, 10 mM potassium chloride, 1 mM ethylenediaminetetraacetic acid, 0.5 mM sodium azide, pH 6.5. A stock sample of 15N-labeled TGF was prepared by dissolving 3.8 mg in 100 µl of D2O containing 0.15 mM sodium 2,2-dimethyl-2-silapentane-5- sulfonate (DSS, internal standard). Successive aliquots of the stock TGF
were then added to the EGFR-ED to give TGF
:EGFR-ED ratios of 0.5:1, 1:1 (0.15 mM), 2:1, 3.5:1. 5:1 and 10:1, with the pH being adjusted to 6.0 after each addition of ligand. The volumes of the NMR sample ranged from 450 to 550 µl.
15N1H NMR spectroscopy
1H, 1-D and 15N1H 2-D HSQC spectra for TGF free in solution and at the various TGF
:EGFR-ED ratios were acquired at 600 MHz using a Varian Unity spectrometer equipped with a triple resonance probe and a z-axis pulse field gradient. These experiments were collected at 298 K and referenced relative to an internal DSS standard. The HSQC experiments incorporated a sensitivity-enhanced pulse sequence utilizing a selective pulse to prevent saturation of amide signals and pulse field gradients to effect suppression of the water resonance (Kay et al., 1992
; Muhandiram and Kay, 1994
) and were performed without presaturation during the recycle delay. The mixed States-TPPI method was employed to obtain sign discrimination in t1 (Marion and Wüthrich, 1983
)) and spectral widths of 7000 and 1600 Hz were used for the 1H and 15N dimensions, respectively. A total of 1024 t2 data points and 128 t1 increments were collected for the HSQC spectra and the signal was averaged over 256, 128, 80, 48, 32 and 16 transients for the 0.5:1 to 10:1 ligand:receptor ratios, respectively. The Fourier transformation of the spectra utilized shifted sine-bell and zero filling to 2Kx2K. HSQC crosspeaks were assigned based on the complete resonance assignment previously reported for TGF
at pH 6.0 (Hoyt et al., 1994
) and their volume integrated using the ll2d routine of VNMR (VNMR 5.1A, Varian Associates, Palo Alto, CA). Changes in the intensity of the HSQC crosspeaks over the course of the receptor titration were analyzed using the in-house program NHX written by Jianjun Wang and Campbell McInnes of the University of Alberta. This program calculates the ratio of the bound intensity of an HSQC crosspeak to its corresponding intensity in the spectrum of the free ligand.
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In order to obtain a greater understanding of the factors governing the interaction of TGF with the EGF receptor, a series of 2-D HSQC NMR experiments were acquired for the ligand free in solution and in the presence of receptor for a series of TGF
:EGF receptor ratios. For the initial data set where the ligand:receptor ratio was 0.5:1, no crosspeaks corresponding to the free ligand could be observed. Upon addition of further aliquots of ligand up to a maximum ratio of 10 mol of ligand to 1mol of receptor, a subset of crosspeaks matching the chemical shift of the free ligand appeared with each successive aliquot. In the final point of the titration, crosspeaks were observed for all amides expected to be present in the spectrum. Figure 1
depicts the HSQC spectrum of the TGF
EGF receptor complex at a 10:1 ratio and it is apparent from this spectrum that there is considerable variation between residues in the final intensity of the crosspeaks. This can be seen qualitatively from the labeled crosspeaks in Figure 1
. Table I
illustrates the changes that occur over the course of the titration as indicated by the residues for which crosspeaks appear at each ratio of ligand to receptor analyzed.
|
|
From initial examination of the data in Figure 1 and Table 1
, it is apparent that significant differences exist in the behavior of the amides from the various residues and different regions of this molecule. The crosspeaks that appear after initial additions of the ligand correspond to residues which exhibit behavior that is distinct from those crosspeaks which do not appear until the later points in the ligandreceptor titration. The conclusion that can be drawn from these data is that the wide variation in the ligand:receptor ratio required to effect reappearance of crosspeaks is correlated with the contribution of the various residues to the formation of the intermolecular complex. Our previous NMR studies on the TGF
EGFR complex indicated through analysis of changes in the intensity of intramolecular ligand NOEs which residues of TGF
formed the binding interface with the growth factor receptor (Hoyt et al., 1994
; McInnes et al., 1996
, 1998
). Despite the success of the NOE analysis method in defining the molecular requirements of complex formation, some limitations nonetheless exist. First, there are many factors which influence the intensity of an NOE crosspeak in a large complex. To name a few, these would include internuclear distance, mixing time and differential cross-relaxation and exchange rates in the free and bound ligand states. Second, TGF
is a dynamic molecule as the structural flexibility present in some regions is concurrent with rigidity in the ß-sheet and hairpin regions of the ligand (Moy et al., 1993
; Li and Montelione, 1995
). The consequence of the variation in structural plasticity is that there is a large difference in the number of NOEs per residue in different parts of the molecule (McInnes et al., 1996
). The result of this is that analysis of changes in NOE intensity on a per residue basis may not yield an accurate representation of the extent to which individual amino acids contribute to the binding interaction. To a significant degree, analysis of the changes in the intensity of HSQC crosspeaks obviates these limitations since it eliminates some of the factors inherent in the NOE analysis technique. In the first scenario, the influence of internuclear distance, NOE buildup due to intramolecular cross-relaxation need not be considered when using the HSQC experiment. In the second instance, the differential flexibility and consequent number of NOEs is no longer a factor since only a single crosspeak is analyzed corresponding to the amide nitrogen to amide proton correlation for each individual residue. The elimination of these factors makes the analysis of HSQC spectra for the free and bound ligand considerably more straightforward and in the case of TGF
yields even more conclusive and informative data on the residues comprising the binding determinants.
The loss in intensity of HSQC crosspeaks upon addition of EGFR is most likely due to the increased linewidths of the amide resonances in the less mobile states of the bound residues. The observed linewidth of a resonance is related a number of factors including the fraction of ligand bound, the ligandreceptor exchange rate, the chemical shift difference between the free and bound resonances and also the effective correlation time of the observed nuclei. In this case, amide groups which are sequestered in the binding site of the receptor will have a larger effective correlation time due to their reduced mobility and thus undergo more substantial broadening compared with those that are in more flexible non-bound regions. It is believed that there are very minimal changes in resonances of TGF upon complexation with its receptor, possibly indicating that the ligand adopts the same conformation when bound (Hoyt et al., 1994
). However the scenario of chemical shift perturbations upon binding cannot be excluded since the HSQC experiment discriminates for resonances with sharp lines and thus broad crosspeaks of the bound form of TGF
may not be observed in the spectrum. If this is the case, the interpretation of the data is unaffected since tightly bound residues will undergo larger chemical shift perturbations than those that are weakly bound and this also will lead to enhanced broadening of crosspeaks. The effect of line broadening in diminishing crosspeak intensity thus indicates contact sites of TGF
with the EGFR and in addition is apparently related to the contribution of the individual residues for the receptor binding site. Elucidation of the binding interface through interpretation of the loss in HSQC crosspeak intensity is made possible in this case by the fast exchange occurring between the free and receptor-bound growth factors, thus allowing information from the bound ligand to be retained and observed on the free molecule.
Quantitative determination of TGF binding determinants
As mentioned, an estimation of the relative contributions of ligand residues to receptor binding and activation can be obtained by observing the reappearance of the HSQC cross- peaks upon increase in the ligand concentration and hence ligand:receptor ratio. From Table I it is apparent that resonances reappear at different concentration of ligand and thus are involved to different degrees in contributing to receptor binding. This method gives an approximate indication of the variation between residues, but a less subjective determination would be to examine more accurately the changes in the intensity of the crosspeak and to compare them with the expected intensity in the free ligand. For the purpose of quantifying the changes in intensity of the HSQC crosspeaks and assessing the differential changes on a per residue basis, the crosspeaks from each successive data set over the course of the titration were volume integrated. The ratio of the HSQC crosspeak intensity in the free and bound species was then calculated for each residue and used on a comparative basis after scaling for differences in crosspeak intensity due to the increasing concentration of ligand after each addition of ligand aliquot.
The column graph in Figure 2 depicts the ratio of the bound intensity for the HSQC data set at a 10:1 ligand:receptor ratio to that of the free ligand for each residue observed for TGF
. In comparing the intensities of the free and bound ligand by expressing them as a ratio on a per residue basis, it is evident that the differential recovery of crosspeak intensity may be related to the relative contributions of residues of TGF
make to receptor recognition. From examination of these data, the contrast is apparent in terms of the wide variation in the amount of recovery of intensity for individual residues ranging from 0.94 for V25 to 0.17 for H12.
|
|
With the HSQC results, however, it can be observed that owing to the clustering of the various classes, four distinct regions are present on the receptor binding interface. These four regions incorporate residues H12 and F15 (red); L48 and A50 (red); G40, A41 and R42 (blue); and E44, H45 and L49 (green). The implication of the location of residues which embody the two red regions is that they provide the major contribution to the binding interaction of TGF with its receptor. This conclusion is validated by the mutagenesis data which supported the obligatory role of H12, F15 on the A loop helix and L48 on the flexible C-tail in binding and activity (Campion and Niyogi, 1994
; Groenen et al., 1994
).
The residues in the green region also play a role in the binding interaction although these residues make a secondary contribution. This is the case since the recovery of crosspeak intensity in this region is greater and thus these residues are not as tightly sequestered in the ligandreceptor complex. This is further supported by binding data available for H45 which suggested that this residue plays an important although not critical role in association with the EGF receptor (Campion and Niyogi, 1994; Groenen et al., 1994
). The fourth region on this face of the molecule is comprised of residues 4042 and owing to the intermediate recovery of intensity these residues make a moderate contribution in providing complementarity with the receptor binding pocket. Mutagenesis data for R42 purported an essential function for this residue; however, it is likely that it is vital in terms of its contribution to the structure of the binding epitope rather than making direct contact with the pocket. This conclusion can be drawn from the data demonstrating that even the conservative mutation of R42 to lysine completely abrogated receptor binding (Campion and Niyogi, 1994
; Groenen et al., 1994
).
From these results, in conjunction with the available mutagenesis data, the conclusion can be drawn that although the ligand face that contacts the receptor is comprised of a large number of residues, only a small subset which are distinct in locality on the growth factor provide the majority of the binding energy. This indicates the presence of two small functional epitopes which are separated from each other but are located on a larger structural epitope comprised of the face shown in the center of Figure 3. This conclusion is consistent with those taken from the study of ligandreceptor complexes such as the HGHreceptor (Clackson and Wells, 1995
; Clackson et al., 1998
) and EPOreceptor systems (Livnah et al., 1996
; Johnson et al., 1998
), which have resolved that while the ligand contact surface is formed by a large number of residues, only a small number of amino acids form `hydrophobic hot spots' and determine the affinity of the two molecules. In addition, the recent demonstration of a small non-peptidic compound that activates the receptor for granulocyte colony-stimulating factor (Tian et al., 1998
) further serves to reinforce this conclusion. These data also further corroborate the multidomain mode of receptor binding since they reveal that the two most important contact sites are located distinctly on the N- and C-terminal subdomains of TGF
.
Advantages of HSQC method for epitope mapping
These results definitively show that it is possible using heteronuclear NMR spectroscopy to delineate the receptor binding site on a ligand and to determine the relative contributions of the residues within this interface for the case of a small protein in complex with a large receptor molecule. The use of this technique presents an avenue to gain information on ligandreceptor interactions without crystallization of the complex and also obviates the need to generate multiple site-specific mutants and perform subsequent biological assays required to map the interface and determine the affinity that individual residues provide. The ability to generate this information in a relatively simple manner without the need to perform the lengthy methods mentioned above is an obvious advantage. Other NMR HSQC methods used to elucidate the nature of the ligandreceptor binding site have primarily focused on evaluating changes in the chemical shift of the amide group as a tool for establishing residues critical for binding and activity (Fejzo et al., 1996; Rajagopal et al., 1997
; Guo et al., 1998
). Interpreting these changes, however, as a means of determining the residues that are requisite for binding and their relative contributions can be complicated by conformational changes induced in the structure of the ligand upon complexation with the receptor (Osborne et al., 1997
). With this HSQC method, since the recovery of intensity to the values observed in the free ligand is being interpreted, binding-induced structural changes are not an issue and thus do not complicate the explanation of the data. Recently, Tomomori et al. (1999) used this approach to study the interaction between the EnvZ and OmpR proteins.
Implications for drug design
The separation of the two major regions contributing to the binding interface on the growth factor and the resulting multidomain interaction mode of the ligand present a considerable challenge for the design of small molecule mimics of TGF action. The increased resolution of the details of the binding interface should, however, permit the design of molecular architectures which present the ligand binding determinants in the correct conformation to effect antagonism or activation of the EGFR. This can possibly be assisted by simulation programs that design potential lead molecules through the generation of overlapping spheres to approximate the binding conformation and yield matches which are then scored and evaluated (Ewing and Kuntz, 1997
; Oshiro and Kuntz, 1998
). In addition, the distinct locality of the two binding regions may be especially suited to the SAR by NMR method where two low-affinity molecules are conjugated together and evaluated through NMR techniques to generate molecules of high binding affinity for possible evaluation as drug candidates (Shuker et al., 1996
; Hajduk et al., 1997
). In either case, the additional information generated on the ligand binding site presents a clearer picture of the molecular recognition events and contributes a greater understanding of ligandreceptor interactions as a whole.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clackson,T. and Wells,J.A. (1995) Science, 267, 383386.[ISI][Medline]
Clackson,T., Ultsch,M.H., Wells,J.A. and de Vos,A.M. (1998) J. Mol. Biol., 277, 11111128.[ISI][Medline]
Ewing,T.J.A. and Kuntz,I.D. (1997) J. Comput. Chem., 18, 11751189[ISI]
Fejzo,J., Lepre,C.A., Peng,J.W., Su,M. S.-S., Thompson,J.A. and Moore,J.M., (1996) Protein Sci., 5, 19171921.
Groenen,L.C., Nice,E.C. and Burgess,A.W. (1994) Growth Factors, 11, 235257.[ISI][Medline]
Guo,W., Sutcliffe,M.J., Cerione,R.A. and Oswald,R.E. (1998) Biochemistry, 37, 1403014037.[ISI][Medline]
Hajduk,P.J., Meadows R.P. and Fesik S.W. (1997) Science, 278, 497499
Hoyt,D.W., Harkins,R.N., Debanne,M.T., O'Connor-McCourt,M. and Sykes,B.D. (1994) Biochemistry, 33, 1528315292.[ISI][Medline]
Hynes,N.E. and Stern,D.F. (1994) Biochim. Biophys. Acta, 1198, 165184.[ISI][Medline]
Johnson,D.L. et al. (1998) Biochemistry, 37, 36993710.[ISI][Medline]
Kay,L.E., Keifer,P. and Saarinen,T. (1992) J. Am. Chem. Soc., 114, 1066310665[ISI]
Lax,I., Fischer,R., Ng,C., Segre,J., Ullrich,A., Givol,D. and Schlessinger,J. (1991) Cell Regul., 2, 337345.[ISI][Medline]
Li, Y-C. and Montelione,G.T. (1995) Biochemistry, 34, 24082423.[ISI][Medline]
Livnah,O., Stura,E.A., Johnson,D.L., Middleton,S.A., Mulcahy,L.S., Wrighton,N.C., Dower,W.J., Jolliffe,L.K. and Wilson,I.A. (1996) Science, 273, 464471[Abstract]
Marion,D. and Wüthrich,K., (1983) Biochem. Biophys. Res. Commun., 113, 967974[ISI][Medline]
McInnes,C. and Sykes,B.D. (1997) Biopolymers (Pept. Sci.), 43, 339366[ISI]
McInnes,C., Hoyt,D.W., Harkins,R.N., Pagila,R.N., Debanne,M.T., O'Connor-McCourt,M. and Sykes,B.D. (1996) J. Biol. Chem., 271, 3220432211.
McInnes,C., Wang,J., Al Moustafa, A.-E., Yansouni,C., O'Connor-McCourt,M. and Sykes,B.D. (1998) J. Biol. Chem., 273, 2735727363.
Moy,F.J., Li, Y-C., Ravenbuehler,P., Winkler,M.E., Scheraga,H.A. and Montelione,G.T. (1993) Biochemistry, 32, 73347353.[ISI][Medline]
Muhandiram,D. and Kay,L.E. (1994) J. Magn. Reson., Ser. B., 103, 203216[ISI]
Osborne,M.J., Wallis,R., Leung,K.-Y., Williams,G., Lian,L.-Y, James,R., Kleanthous,C. and Moore,G.R. (1997) Biochem. J., 323, 823831.[ISI][Medline]
Oshiro,C.M. and Kuntz,I.D. (1998) Proteins, 30, 321336.[Medline]
Prigent,S.A. and Lemoine,N.R., (1992) Prog. Growth Factor Res., 4, 124.[Medline]
Rajagopal,P., Waygood,E.B., Reizer,J., Saier,M.H. and Klevit,R.E. (1997) Protein Sci., 6, 26242627.
Richter,A., Conlan,J.W., Ward,M.E., Chamberlin,S.G., Alexander,P., Richards,N.G.J. and Davies,D.E. (1992) Biochemistry, 31, 95469554.[ISI][Medline]
Rusch,V., Mendelsohn,J. and Dmitrovsky,E. (1996) Cytokine Growth Factor Rev., 7, 133141.[Medline]
Salomon,D.S., Brandt,R., Ciardiello,F. and Normanno,N. (1995) Crit. Rev. Oncol. Hematol., 19, 183232.[ISI][Medline]
Shuker,S.B., Hajduk,P.J., Meadows R.P. and Fesik,S.W. (1996) Science, 274, 15311534.
Tian, S.-S. et al. ( 1998) Science, 281, 257259.
Tomomori,C. et al. (1999) Nature Struct. Biol., 6, 729734.[ISI][Medline]
Received July 21, 1999; revised November 12, 1999; accepted December 15, 1999.