J. Mol. Biol. (1986) 189, 377-382

Studies by ‘H Nuclear Magnetic Resonance and Distance Geometry of the Solution Conformation

of the a-Amylase Inhibitor Tendamistat

This is a preliminary report on the determination of the solution conformat,ion of the a-amylase inhibitor Tendamistat by nuclear magnetic resonance and distance geometry calculations. A characterization is given of the complete polypeptide backbone fold and the side-chains of the presumed active site in this protein. These results are based on complete sequence-specific resonance assignments, a list of 401 distance constraints from nuclear Overhauser effects, 168 distance constraints from hydrogen bonds and disulphide bridges, and 50 torsion angle constraints from measurements of spin-spin coupling constants.

Tendamistat (Hoe-467) is a polypeptide inhibitor that binds tightly to mammalian a-amylases (Aschauer et al., 1983; VBrtesy et al., 1984). The only information available on the conformation of this class of proteins (Murai et al., 1985; VBrtesy & Tripier, 1985) comes from earlier n.m.r.t studies (Kline & Wiithrich, 1985), which showed that nearly the entire polypeptide chain in Tendamistat is in two antiparallel P-pleated sheets. This letter presents t,he polypeptide backbone fold of Tenda- mistat in aqueous solution and the side-chain conformations of the proposed active-site residues.

The spatial structure determination relies on sequence-specific ‘H n.m.r. assignments (Wiithrich et al. ~ 1982), a list of NOESY cross peaks between pairs of protons that are close in space (Anil Kumar et al., 1981), and a computational method to calculate tertiary structures compatible with these distance constraints (Braun et al., 1981, 1983; Have1 & Wiithrich, 1984, 1985; Braun & Go, 1985). This letter gives a survey of the n.m.r. input constraints and the distance geometry calculations, and a characterization of the chain fold and the conforma- tion of the active site.

Tendamistat was studied in 5 mw-solution in H,O or ‘H,O, with the pH meter reading at room temperature adjusted to 3.2 with perchloric acid. All n.m.r. experiments were run on a Bruker WM-500 spectrometer, with the probe temperature regulated to 50°C. After complete exchange of the labile protons in ‘H,O, diagonal-suppressed NOESY spectra (Denk et al., 1985) were collected, while “regular” NOESY spectra were recorded in a mixed solvent of 90% H,O/lO”/o 2H20. Three different mixing times were used, i.e. 200, 100 and 50 milliseconds. The quality of the spectra obtained

t Abbreviations used: n.m.r., nuclear magnetic resonance; NOE, nuclear Overhauser enhancement; NOESY, 2-dimensional nuclear Overhauser enhancement spectroscopy; r.m.s.d., root-mean-square distance.

with these experimental conditions was shown by Kline & Wiithrich (1985).

The approach adopted for determining the upper distance limits corresponding to the NOESY cross peaks was similar to that used previously (Braun et al., 1983; Williamson et aE., 1985). The 200 millisecond spectra were used for the NOESY peak assignments, those at 50 and 100 milliseconds were helpful in eliminating spin diffusion peaks from the analysis (Anil Kumar et al., 1981). Using the 100 millisecond data, the NOESY cross peak intensities were grouped into three separate categories, and an upper distance limit was defined for each category. In the H,O spectra the cross peaks with amide protons had similar line shapes throughout, so that the peak height was used as a measure of the NOE intensity. Among the cross peaks with non-labile protons measured in the 2H20 spectra, the variation of the line shapes was far greater. Therefore, the area of the o2 cross sections was used as the intensity measurement. The choice of upper distance limits for the three categories was made for the cross peaks in the 2H20 spectra by comparing with the intensities of a number of peaks corresponding to distances fixed by the covalent structure, such as geminal methylene protons, or aH-/?CH3 in Ala. For the amide protons no covalently fixed distances exist, so the maximum and minimum intensities observed for d,, and d,,(i, i) cross peaks were used to calibrate the range of allowed distances, 2.2 to 3.6 A and 2.2 to 2.8 A, respectively. Further slight adjustments of these calibrations resulted from observations made during the distance geometry calculations. Also, at this stage two NOES with Trp18 were eliminated from the input (see below). Because no stereo- specific resonance assignments had been made, the pseudo-atom representation was used as required (Wiithrich et al., 1983). The NOE data used in the calculations have been summarized in Figure 1 as a diagonal plot. The lower left triangle shows all backbone-backbone NOE constraints, while the

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378 A. D. Kline et al

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Figure 1. Diagonal plot of the NOE distance constraints for Tendamistat. Both axes are calibrated with the sequence of the protein. The squares connect pairs of residues linked by one or more NOE constraints. The filled squares in the lower left part indicate constraints between backbone protons. The hatched squares and crossed squares in the upper right half indicate backbone-side-chain and side-chain-side-chain NOE constraints, respectively. Whenever a hatched and a crossed square occur at the same place, only the hatched square is shown.

upper right half contains the backbone-side-chain and the side-chain-side-chain constraints. Between sequentially neighbouring residues, 65 backbone- backbone, 48 backbone-side-chain and six side- chain-side-chain NOE constraints were identified. There are a further 51 backbone-backbone, 83 backbone-side-chain and 73 side-chain-side-chain medium-range and long-range NOE constraints, as well as 75 intra-residue constraints.

The NOE data were supplemented with addi- tional experimental observations using the same corresponding constraints as used previously (Williamson et al., 1985). This includes the two disulphide bonds 11-27 and 45-73 (Aschauer et al., 1983), 26 hydrogen bonds identified in the two antiparallel P-sheets, 43 backbone dihedral 4 angles corresponding to spin-spin coupling constants 3J nNa 2 8.0 Hz (Fig. 3 of Kline & Wiithrich, 1985), and the torsion angles x1 of Va112, Thr30, Thr32,

Va136, Va148, Ile53 and Thr55, which all correspond to spin-spin coupling constants 3J,, 2 8-O Hz. As was discussed by Williamson et al. (1985), some of these additional constraints are redundant with those imposed by ‘H-‘H NOES (for example the locations of the disulphide bonds and the hydrogen bonds could be identified independently on the basis of the NOE data), but the additional experimental observations impose tighter con- straints than the NOES alone.

In the calculations using the DISMAN program (Braun & Go, 1985), only the dihedral angles were varied, with the bond angles and bond lengths fixed at the standard ECEPP values (Momany et al.. 1975; Nemethy et al.: 1983). It is implicit in these computations that additional constraints arise because of the intrinsic rigidity of the polypeptide covalent structure and the van der Waals’ volumes of the individual atoms. “Core radii” as defined by

Letters to the Editor 379

Figure 2. Stereo view of the 4 solutions I to IV calculated by DISMAN, plotting only the backbone atoms N, C”, C’. The structures were superimposed so as to minimize the r.m.s.d. of the heavy atoms of the backbone with respect to structure I. Region 62 to 66 was eliminated from the best fit to better align the constrained areas. To simplify the presentation, the disulphide bridges were not drawn. In this particular orientation the 3 C” positions of Serl7, Lys34, and Asn25 form a vertical plane perpendicular to the proiection plane. Lys34 is in the back, and its projection is centred between the projections ofthe other 2 u-carbons. - -

Braun & Go (1985) were used to account for the repulsive forces between different atoms or groups of atoms. To avoid local minima the program uses a variable target function, which considers the shorter range constraints before proceeding to longer range constraints. The performance of this program was tested with calculations on the basic pancreatic trypsin inhibitor, using input data obtained from the crystal structure or from experimental n.m.r. distance constraints (Braun & G5, 1985; G. Wagner, W. Braun, T. F. Havel, T. Schaumann, N. Go & K. Wiithrich, unpublished results). These tests showed that, independent of the starting conformation, the algorithm converges to a solution if a complete set of short-range and long-range distance ‘information is used. If less complete constraint sets were used, the computations with some of the initial conforma- tions were occasionally trapped in local minima; these could then usually be excluded on the basis of the large residual distance and core violations.

For Tendamistat the experimental n.m.r. con- straints were used as input for ten separate structure computations, which started from different initial conformations and were pursued in parallel. In the initial phase, with the target function covering up to eight residues, local minima problems arising in the individual calculations were generally located in different regions of the polypeptide chain. On this level of local conforma- tions, these problems could be solved for all ten computations by re-randomization of the torsion angles weighted using the ranges in the untrapped structures. This approach was particularly helpful

in the regions of the tight turns. Subsequently, four calculations so far converged to a solution of the complete constraint list. These are the basis for the following discussions on the solution conformation of Tendamistat.

Figures 2 and 3 show, respectively, the backbone conformations of the four solutions and a schematic drawing of structure “I”. This orientation illus- trates the two-sheet architecture of the protein, with one sheet consisting of the strands 12 to 17, 20 to 25 and 52 to 58 in the foreground, and the other with the strands 30 to 37, 41 to 49 and 67 to 73 behind. In the previous study of the secondary structure, Tendamistat was given the domain classification of a Greek-key P-barrel (Kline & Wiithrich, 1985). The + 1. +3. - 1, - 1, +3 topology pattern recognized in Figures 2 and 3 supports the classification as a Greek-key barrel (Richardson, 1981). Though similar barrel topolo- gies have been identified, it appears that none of the other proteins has an identical chain fold to that of Tendamistat. The b-barrel domain structure is also readily apparent from the diagonal presenta- tion of the backbone-backbone NOES in Figure 1. It is also reflected in the NOES involving the side- chain protons (upper right in Fig. 1).

The quality of the structures obtained was judged from the residual violations of the n.m.r. and core radii constraints, and from the r.m.s.d. between the different solutions in Figure 2. Table 1 lists the average violation for n.m.r. distance constraints in both the initial and final structures, and thus shows that on average the

380 A. D. Kline et al.

Figure 3. Schematic drawing of the backbone topology obtained using the co-ordinates of structure I. The ribbon drawing was generated by the CONFOR program (Billeter et aZ., 1985), using the same orientation as in Fig. 2. Modifications to this computer drawing were added by hand to give the picture a 3-dimensional effect. The arrowed ribbons indicate the position and direction of the p-sheet strands. The rope structure was used for the loop areas, with the broken lines indicating those places where the data did not give a well-confined solution. The disulphide bridges are indicated by lightning bolts.

calculations had reduced the violations by a factor of about 500. The four solutions all had similar average violations, indicating that each structure was an equally credible solution. Table 1 also shows that in the initial structures a large number of

constraints were violated by 10.0 A or more, indicating that the starting conformations were not at all solutions to the constraints. In the final structures, all but one of the residual violations were smaller than 0.5 A. This indicates that after the elimination of two NOES with the indole ring of Trpl8 (see below) the experimental distance constraints contained no serious inconsistencies. Relative to the constraints by the core radii. all four solutions were adequate as there were no violations of the contact limits over 0.5 A, and only two violations exceeded O-3 A in all four of the solutions. This is a good result considering that t,here are at least 2500 potential core contacts in t,he folded molecule. Further refinement of the van der Waals’ interactions is envisaged with the use of a proper energy function (Momany et al., 1975; Nemethy et al., 1983).

In Figure 2 the four conformers are superimposed for minimum r.m.s.d. between the backbone heavy atoms of structure I and the other structures. Clearly, a larger spread is observed for the terminal residues 1 to 5 and 74, which are not confined by any long-range NOES (Fig. 1). Similarly, the increased variability of the loop region 62 to 66 is consistent with this region’s low density of interconnecting NOE data (Fig. 1). For the residues 6 to 73 in the four structures, the average r.m.s.d. for the backbone heavy atoms of the six pair-wise comparisons is I.6 A. This compares with 15.2 A for the same comparison of the corresponding starting structures. The average r.m.s.d. values thus show that the starting structures were not related to each other, whereas the final structures have a similar global conformation. Apart from the terminal regions and the loop 62 to 66, the distance geometry calculations indicate that the conformation of the polypeptide backbone is well-defined by the n.m.r. constraints.

The following are some additional features of the Tendamistat backbone fold. The previously identi- fied antiparallel b-sheets were found to be consis- tent with the additional, newly acquired n.m.r. constraints and the covalent structure of the molecule. A right-handed twist is seen for both sheets, which is consistent with the handedness in

Table 1 Table 1 Computations of the Tendamistat conformation using DISMAN: comparison of the Computations of the Tendamistat conformation using DISMAN: comparison of the violations of distance constraints ,imposed by the n.m.r. data for the initial and jkal violations of distance constraints ,imposed by the n.m.r. data for the initial and jkal

structures structures

(:omputation

Initial structure Final structure

Average Number of violations Average Number of violationt violation? violations

(4 > 10.0 A >0.5 A (4 >0.5 A

I 12.5 236 314 0.026 0 II 12.5 228 301 0.024 0 III 10.4 226 304 0.026 0 IV 15.0 234 303 0.029 1l

t Sum of all distance violations divided by the total number of n.m.r. distance constraints. i.e. 569. 1 The single violation was 0.53 A.

Letters to the Editor 381

Figure 4. Stereo view taken from the 4 structures in Fig. 2, showing the complete residues Serl7. Trpl8, Argl9, and Tyr20. The structures were superimposed so as to minimize the r.m.s.d. of the backbone atoms with respect to structure I. For the backbone and the indole ring all atoms are shown, for the rest of the structure the hydrogen atoms have been omitted. (This orientation of the indole ring is not unique, see the text.)

other proteins (Schulz & Schirmer, 1979; Richardson, 1981). In the structures in Figures 2 and 3 the predominantly hydrophobic sides of the two /?-sheets face each other, forming a hydro- phobic interior. A tentative identification of the sequence locations and types of tight turns was obtained from the dihedral angles of the correspon- ding residues. Two non-hairpin /?-turns occur at positions 10-I 1 and 50-51 (lower left and lower right, respectively, in Fig. 3). The lo-11 turn was classified as a type I turn, the 50-51 turn as type II (Richardson, 1981). Two hairpin turns at 18-19 and 37-41 are at the top of the molecule in Figure 3. The two-residue turn appears to be of type I rather than the more commonly observed type I’ for hairpin turns (Sibanda & Thornton, 1985). A classification of the turn at 37-41 would be premature, even though the NOE pattern is consistent with a class of recently identified five- residue turns (Sibanda & Thornton, 1985). Another interesting observation is the formation of the 45-73 disulphide link to the outside of the b-sheet rather than on the inside.

The understanding of cc-amylase inhibition by Tendamistat and related proteins centres on a highly conserved sequence of Trp-Arg-Tyr (Aschauer et al., 1983; Murai et al., 1985; Vertesy & Tripier, 1985). These amino acid types have also been implicated by inactivation studies on one member of the inhibitor family (Arai et aE., 1985). Tn Tendamistat these residues are in positions 18 to 20 and form part of the protein surface. Figure 4 shows this peptide segment for the four structures of Figure 2. This specific conformation is consistent with the majority of the NOES. The location of the Arg between the two rings is also manifested in the high field shifts of the Arg resonances (NH, 6.73; C”H, 3.74; @H,, 0.41, -0.17; CYH2, 0.37, 0.37). Despite the surface location, the Trpl8 side-chain is confined by nine NOES, so that its conformation

appears particularly well-defined in Figure 4. How- ever, there were two additional NOES that were found not to be consistent with the ensemble of all other NOES by exploratory initial distance geo- metry calculations. Therefore, it cannot be excluded that there are multiple conformers that differ locally by the orientation of the indole ring of Trp18.

This study on the solution conformation of Tendamistat has been carried out without any information from crystallographic studies on this or homologous proteins. To facilitate t,he comparison of the solution and crystal structures of this protein, the orientation of the molecule described in Figure 2 is also used in the Figures of the accompanying paper on the X-ray data on Tendamistat (Pflugrath et al., 1986).

This letter is a progress report on the n.m.r. determination of the solution conformation of Tendamistat. Additional work will include a re-examination of the NOESY spectra using the present molecular model in conjunction with the sequence-specific resonance assignments to com- plete the assignments of the NOESY cross peaks, more quantitative evaluation of the NOE intensi- ties, stereospecific assignments of the interior leucine and valine methyl groups and collection of more complete data on side-chain orientations by further measurements of J-couplings. Combined with energy minimization these additional data can be expected to yield refinements of the backbone conformation, characterization of the interior amino acid side-chains, and provide a basis for further studies of the protein surface.

An ample supply of Tendamistat (Hoe-467) was obtained from Hijchst Aktiengesellschaft, Frankfurt am Main. All calculations were done at the Zentrum fiir Tnteraktives Rechnen, ETH-Hiinggerberg. Financial support by the Schweizerischer Kationalfonds is grate-

382 A. D. Kline et al.

fully acknowledged (project 3.284.82 and international Denk, W., Wagner, G., Rance. M. & Wiithrich, K. (1985). postdoctoral fellowship to A.K.). We thank Mrs E. Huber J. Magn. Res. 62, 336-340. and Mrs E.-H. Hunziker for the careful preparation of the Havel, T. & Wiithrich, K. (1984). Bull. Math,. Biol. 46. manuscript and the illustrations. 673-698.

Allen D. Kline Werner Braun Kurt Wiithrich

Institut fiir Molekularbiologie und Biophysik Eidgeniissische Technische Hochschule-HGnggerberg CH-8093 Ziirich, Switzerland

Received 19 December 1985

References

Anil Kumar, Wagner, G., Ernst, R. R. & Wiithrich, K. (1981). J. Amer. Chem. Sot. 103, 3654-3658.

Arai, M., Oouchi, N., Goto, A., Ogura, S. & Murao, S. (1985). Agric. Biol. Chem. 49, 1523-1524.

Aschauer, H., VBrtesy, L., Nesemann, G. & Braunitzer, G. (1983). Hoppe-Seyler’s 2. Physiol. Chem. 364, 1347-1356.

Billeter, M., Engeli, M. & Wiithrich, K. (1985). J. Mol. Graph. 3, 79-83.

Braun, W. & Go, N. (1985). J. Mol. Biol. 186, 611-626. Braun, W., BBsch, C., Brown, L. R., GG, N. & Wiithrich,

K. (1981). Biochim. Biophyys. Actu, 667, 377-396. Braun, W., Wider, G., Lee, K. H. $ Wiithrich, K. (1983).

J. Mol. Biol. 169, 921-948.

Havel, T. & Wiithrich, K. (1985). J. Mol. Biol. 182, 281- 294.

Kline, A. D. & Wiithrich, K. (1985). J. Nol. Biol. 183. 503-507.

Momany. F. A., McGuire. F. F.. Burgess. A. W. $ Scheraga. H. A. (1975). J. Phys. Chem. 79, 2361- 2381.

Murai, H., Hara, S., Ikenaka, T., Goto, A.. Arai, M. di Murao, S. (1985). J. Biochem. 97, 1129-1133.

Nbmethy, G., Pottle, M. S. & Scheraga. H. A. (1983). J. Phys. Chem. 87, 1883-1887.

Pflugrath, J., Wiegand, E., Huber, R. & VBrtes?-. I,. (1986). J. Mol. Biol. 189, 383-386.

Richardson, J. S. (1981). Advnn. Protein Ohem. 34. 167- 339.

Schulz, G. E. & Schirmer. R. H. (1979). Prinkpleu of Protein Structure, Springer-Verlag, New York.

Sibanda, B. L. 6 Thornton, ,J. M. (1985). Nutuw (London), 316, 170-174.

VBrtesy, L. & Tripier, D. (1985). FEBS Letters, 185, 187- 190.

VBrtesy. L., Oeding, V., Bender, R.. Zepf, K. & Nesemann, G. (1984). Eur. J. Biochem. 141, 505-512.

Williamson, M. P.. Havel, T. F. & Wiithrich, K. (1985). J. Mol. Riol. 182, 295-315.

Wiithrich, K.. Wider, G., Wagner, G. & Braun. W. (1982). J. Mol. Biol. 155, 311-319.

Wiithrich, K., Billeter, M. & Braun, W. (1983). J. Xol. Biol. 169, 949-961.

Edited by M. F. Moody