MiAMP1, a novel protein from Macadamia integrifolia adopts a greek key β-barrel fold unique amongst plant antimicrobial proteins 1

Article No. jmbi.1999.3163 available online at http://www.idealibrary.com on

J. Mol. Biol. (1999) 293, 629±638

MiAMP1, a Novel Protein from Macadamia integrifolia Adopts a Greek Key b -Barrel Fold Unique Amongst Plant Antimicrobial Proteins Ailsa M. McManus1, Katherine J. Nielsen1, John P. Marcus2, Stuart J. Harrison2, Jodie L. Green2, John M. Manners2 and David J. Craik1* 1

Centre for Drug Design and Development, The University of Queensland, Brisbane Queensland 4072, Australia 2

Cooperative Centre for Tropical Plant Pathology, The University of Queensland Brisbane, Queensland 4072, Australia

MiAMP1 is a recently discovered 76 amino acid residue, highly basic protein from the nut kernel of Macadamia integrifolia which possesses no sequence homology to any known protein and inhibits the growth of several microbial plant pathogens in vitro while having no effect on mammalian or plant cells. It is considered to be a potentially useful tool for the genetic engineering of disease resistance in transgenic crop plants and for the design of new fungicides. The three-dimensional structure of MiAMP1 was determined through homonuclear and heteronuclear (15N) 2D NMR spectroscopy and subsequent simulated annealing calculations with the ultimate aim of understanding the structure-activity relationships of the protein. MiAMP1 is made up of eight b-strands which are arranged in two Greek key motifs. These Greek key motifs associate to form a Greek key b-barrel. This structure is unique amongst plant antimicrobial proteins and forms a new class which we term the b-barrelins. Interestingly, the structure of MiAMP1 bears remarkable similarity to a yeast killer toxin from Williopsis mrakii. This toxin acts by inhibiting b-glucan synthesis and thereby cell wall construction in sensitive strains of yeast. The structural similarity of MiAMP1 and WmKT, which originate from plant and fungal phyla respectively, may re¯ect a similar mode of action. # 1999 Academic Press

*Corresponding author

Keywords: NMR spectroscopy; plant antimicrobial protein; Greek key b-barrel; yeast killer toxin; b-barrelins

Introduction Disease resistance in plants depends on numerous defence mechanisms, including the production of antimicrobial proteins (AMPs). At present, a

Abbreviations used: AMP, antimicrobial peptide/ protein; NOE, nuclear Overhauser enhancement; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; TOCSY, total correlated spectroscopy; DQF-COSY, double quantum ®ltered correlated spectroscopy, NOESY, NOE spectroscopy, HSQC, heteronuclear single quantum ®ltered correlated spectroscopy; SAR, structure-activity relationship; CSI, chemical shift index; RMSD, rootmean-square deviation. E-mail address of the corresponding author: [email protected] 0022-2836/99/430629±10 $30.00/0

large number of low molecular mass proteins (<100 amino acid residues) with potent antimicrobial activity have been identi®ed in a wide range of plants (Broekaert et al., 1997). These antimicrobial proteins have been grouped, on the basis of their primary sequences, into several different families including lipid-transfer proteins, thionins, plant defensins and hevein and knottin-type proteins (Broekaert et al., 1997). While all have antimicrobial activity, structurally the groups are quite different. Thionins have a compact L-shape made up of two a-helices forming the vertical arm and a two-stranded b-sheet forming the horizontal arm. Plant defensins consist of a triple-stranded b-sheet and an a-helix which lies parallel with the sheet. Lipid-transfer proteins are bundles comprised of four a-helices joined by loops. Heveins are made up of a triple-stranded b-sheet and a short a-helix # 1999 Academic Press

630 which connects the second strand to the third. Finally, knottins, as their name implies, have a knot-like structure characterised by a triplestranded b-sheet and a long loop connecting the ®rst strand to the second (Broekaert et al., 1997). In recent years, much attention has been focused on the use of these proteins in the design of novel fungicides and as a source of genes for engineering disease-resistant transgenic plants (Rao, 1995; Terras et al., 1995; Carmona et al., 1993; Epple et al., 1997). A recent screen for antimicrobial proteins in taxonomically distinct plants native to Australia resulted in the identi®cation, puri®cation and cloning of a novel plant antimicrobial protein. This protein, subsequently named MiAMP1, was isolated from the kernel of the macadamia nut and had broad-range antimicrobial activity in vitro (Marcus et al., 1997). MiAMP1 has a molecular mass of 8.1 kDa, is highly basic (pI 10.1) and consists of 76 amino acid residues including six disulphide-linked cysteine residues. A study of the antimicrobial activity of MiAMP1 demonstrated that it inhibited several major taxonomic groups of microbes in vitro, including many plant pathogens. In contrast, MiAMP1 appeared to have no effect on the viability of plant and animal cell lines. This lack of toxicity towards plant and animal cells is particularly important if this protein is to be considered a suitable candidate for the control of pathogens by expression in transgenic food crops and for the design of new fungicides (Marcus et al., 1997). MiAMP1 exhibits no sequence homology to known proteins and therefore potentially represents a new structural class of plant defence proteins. Despite this lack of sequence homology, MiAMP1 has some general features in common with other antimicrobial proteins. For example, its highly basic nature suggests a mode of action involving contact with a negatively charged surface such as the plasma membrane. Also, the activity of MiAMP1 against most microbes is reduced in the presence of increased salt concentrations, further suggesting that electrostatic interactions may be important in its mode of action. However, the observation that increasing salt concentrations do not affect its inhibition of all sensitive microbial strains indicates there is diversity in its antimicrobial activity. Potentially exciting applications of MiAMP1 are associated with its use as a template for the design of new fungicides and the engineering of disease resistance in plants through expression of its transgenes. However, until now, neither the structure nor mode of action of MiAMP1 have been determined. Here, we describe the solution structure of MiAMP1 and show that it adopts a Greek key bbarrel unique amongst plant antimicrobial proteins.

NMR Structure of MiAMP1

Results Spectral assignments NMR spectra were recorded at pH 5.0 and temperatures from 283 to 313 K. Peak assignments were made from DQF-COSY, TOCSY and NOESY spectra acquired on unlabelled protein at 283 and 308 K. Spectra acquired at lower pH (2.3) were essentially unchanged, suggesting that there was no pH-dependent conformational change. In the early stages of the assignment, some ambiguities were resolved from a HSQC spectrum of the recombinant 15N-labelled sample at 308 K. The NMR data at 283 and 308 K and pH 5.0 were used for structure calculations. MiAMP1 contains two proline residues within its sequence: Pro9 and Pro66. Assignment of a strong dai !ai‡1 NOE for Gly8-Pro9 and a strong dai !di‡1 NOE for Asn65Pro66 in the 2H2O NOESY spectra con®rmed the amide bonds preceding Pro9 and Pro67 are in the cis and trans conformations, respectively. Analysis of spectra acquired at 5 K increments between 283 K and 308 K showed that certain peaks associated with residues 5-7, 14-17 and 27-30 became broader as the temperature was increased. An extreme example of this is the resonances associated with Gln28, which broadened to such a degree they were not evident in spectra recorded above 298 K. The broadening appears to be associated with a conformational process in the vicinity of the residues noted above. At higher temperatures, the resonances were observed to be in intermediate exchange whereas at lower temperatures, peak narrowing was detected, indicating slow exchange. The three-dimensional structure described below provided an insight into the nature of the conformational change. Secondary structure and disulphide connectivity The observed short and medium-range NOEs, chemical shifts, NH exchange data and 3JNH-aH coupling constants are summarised in Figure 1. From this information and from long-range NOEs it was possible to identify the secondary structure of MiAMP1 as consisting of two antiparallel bsheets, each made up of four b-strands. A schematic diagram of the secondary structure is shown in Figure 2. From the arrangement of the b-strands it was concluded that MiAMP1 consists of two identical Greek key motifs belonging to the class (3,1)c (Hutchinson & Thornton, 1993) (Figure 3). MiAMP1 contains six disulphide-linked cysteine residues. One of the disulphide linkages, Cys11 ! Cys64, was determined through trypsin digestion and characterisation of the resulting fragments by mass spectrometry. The other two could not be readily detected by this method and instead were determined from the NMR data and subsequent structure calculations. The three possible combinations were (Cys21 ! Cys76

NMR Structure of MiAMP1

631

Figure 1. Summary of the data used for residue assignments and secondary structure identi®cation. Black bars represent NOEs present in the 250 ms NOESY spectrum (308 K, pH 5.0). The thickness of the bars represents the strengths of the NOEs. The chemical shift index: four or more ‡1 and three or more ÿ1 indicate extended and helical structure, respectively. Open circles represent slowly exchanging amide protons still present after 24 hours. Half-®lled circles represent medium-exchanging amide protons still present after ®ve hours. Large (>9.5 Hz) and small (<5 Hz) 3 JNH-aH coupling constants are represented by black and white squares, respectively. Arrows represent the regions of b-strands found in MiAMP1.

and Cys23 ! Cys49), (Cys21 ! Cys49 and Cys23 ! Cys76) or (Cys21 ! Cys23 and Cys49 ! Cys76). Through the assignment of NOEs between the bH atoms of Cys21 and Cys76, and Cys23 and Cys49, it was deduced that the ®rst of the three combinations was most likely. This was subsequently con®rmed through calculations of the three-dimensional structure described below. Three-dimensional structure determination NOESY spectra were used to derive a set of 1571 distance restraints consisting of 602 intra-residual, 330 sequential, 107 medium-range and 532 longrange distance restraints. An additional 43 dihedral angle restraints were obtained from the DQF-

COSY spectrum and, together with the distance restraints, were used to calculate a set of 50 structures. In preliminary calculations, ambiguous distance restraints (Folmer et al., 1997) were used to test the three possible combinations of disulphide bond connectivities. Analysis of distances within the resultant structures indicated that Cys21 ! Cys76 and Cys23 ! Cys49 were the correct connectivities. To con®rm that the incorporation of ambiguous distance restraints had resulted in the correct choice of disulphide bridges, three sets of structures were calculated with the three possible combinations added in as covalent bonds. Analysis of the energies and geometries of these sets of structures unequivocally proved that Cys21 ! Cys76 and Cys23 ! Cys49 were the cor-

632

NMR Structure of MiAMP1

Figure 3. Topology of the two Greek key motifs of MiAMP1. (a) The two individual motifs. Dots represent the position of the disulphide-linked cysteine residues with broken lines representing the linkages. (b) The motifs combine to form the b-barrel.

RMSD values (Figure 4) and the backbone overlay shown in Figure 5, these structures are precisely de®ned. The average pairwise RMSDs for the backbone atoms (N, C, Ca) and heavy atoms over the Ê and 1.58 A Ê , respectively entire molecule are 0.84 A (Table 1). In addition, 99.8 % of the backbone angles fall into the allowed regions of the Ramachandran plot (Table 1) (Laskowski et al., 1993). Description of the three-dimensional structure Figure 2. A schematic diagram of the two antiparallel b-sheets (labelled Sheet 1 and Sheet 2). Arrows represent the observation of NOEs between protons. Broken lines represent hydrogen bonds.

rect connections. Therefore, the ®nal round of structure calculations included these connections along with the chemically deduced one (Cys11 ! Cys64). From a generated set of 50 structures, 20 were selected, on the basis of lowest energies, to represent the solution structure of MiAMP1. The statistical data from the analysis of these 20 structures are presented in Table 1. As is evident from the

It is clear from the structure of MiAMP1 (Figure 6) that the major structural elements are eight b-strands arranged into two identical Greek key motifs. The motifs are organised in such a way that three strands of one motif and one strand of the other make up the two antiparallel b-sheets (Figure 3). In turn, the orientation of the sheets with respect to each other results in a very stable structure referred to as a Greek key b-barrel. The eight strands encompass residues 2-6, 15-19, 22-25, 31-35, 41-44, 53-57, 61-63 and 71-74. Two classic bbulges are found at the ends of two of the strands and comprise residues 43,54 and 55, and 44,70 and 71. The strands are joined by a series of turns and loops including three b-turns encompassing residues 8-11, 36-39 and 47-50. Two loops are anchored to the sheets by one disulphide bond

633

NMR Structure of MiAMP1 Table 1. Structural and energetic statistics for a family of 20 MiAMP1 structures A. Mean RMSD from experimental distance restraints Ê) NOE (A Dihedral (deg.) B. Mean RSMD from idealised covalent geometry Ê) Bonds (A Angles (deg.) Impropers (deg.) C. Restraint violations Ê Number of NOE violations >0.2 A Ê) Maximum violation (A Number of Dihedral Angle violations >2  Maximum violation (deg.) D. Mean energies (kJ molÿ1)a ENOE Ecdih Evdw Ebond Eimproper Eangle ETotal E. Pairwise RMSD Ê) Backbone atoms 1-76 (N, Ca, C) (A Ê) Heavy-atoms (A Ê) Backbone atoms-b-strands (N, Ca, C) (A Ê) Heavy-atoms (A F. Ramachandran plot Residues in most favoured regions (%) Residues in additional allowed regions (%) Residues in generously allowed regions (%) Residues in disallowed regions (%)

0.011  0.001 0.32  0.09 0.0069  0.0001 2.18  0.02 0.15  0.01 0 0.186 0 1.987 5.7  1.1 0.3  0.1 ÿ400  5 8.9  0.3 1.4  0.2 134  3 ÿ250  4 0.84  0.10 1.58  0.12 0.79  0.09 1.58  0.12 63.9 33.1 2.8 0.2

a Force constants for the calculation of square-well potentials for the NOE and dihedral angle Ê 2 and 200 kcal molÿ1 radÿ2, respectively. restraints were 50 kcal molÿ1 A

each. The other disulphide bond links the C-terminus to an adjacent b-strand.

Discussion The broad-range antimicrobial activity of MiAMP1 creates interest in determining relationships between structure and activity. The ultimate aim of such studies is the design of functional antimicrobial molecular mimics as fungicides or the engineering of disease-resistant transgenic plants using genes encoding for MiAMP1 or its variants. We have solved the solution structure of MiAMP1 as a ®rst step in delineating these structure-activity relationships (SARs). Amongst the antimicrobial plant proteins characterised to date, MiAMP1 is unique in both its sequence and structure, and thus represents a new class of plant AMP. We propose that this new class of plant AMP be named the bbarrelins after the Greek key b-barrel structure. The three-dimensional structure of MiAMP1 is made up of eight b-strands arranged into two Greek key motifs. These motifs ®t together in such a way as to form a b-barrel with a tightly packed hydrophobic core. The b-barrel of MiAMP1 has an Ê, a elliptical cross-section with a long axis of 28 A Ê , and a height of 24 A Ê . Apart short axis of 13 A from the strands, the structure of MiAMP1 also incorporates two loops which fold over each of the b-sheets and are braced in this position by one disulphide bond each. Interestingly, as can be seen in Figure 3, all of the disulphide bonds link the

two motifs together and may therefore serve to further stabilise the structure. While the sequence and structure of MiAMP1 are unique, if one aligns the two Greek key motifs consisting of residues 1-38 and 39-76 there is weak sequence homology between the two motifs (Figure 7). This homology at the amino acid level is more clearly evident in the three-dimensional structure which has a 2-fold axis of symmetry. Indeed, it was not until the three-dimensional structure was solved, revealing the strong structural homology of the two Greek key motifs, that the weak sequence homology between the N and Cterminal halves of the protein was evident. The degree of sequence homology is signi®cantly weaker than is seen in the well known examples of Greek key motifs in bg crystallins (Brandon & Tooze, 1991). Despite the structural homology between the two motifs of MiAMP1, there is an interesting difference between them. It was noted earlier that peaks arising from residues 5-7, 14-17 and 27-30 broaden with increasing temperature. As can be seen from the representation of the surface of MiAMP1 in Figure 8(a), all of these residues are located close to each other in one motif and form part of the surface of MiAMP1. This suggests that the broadening re¯ects a slow conformational exchange in this region. Since most residues are in b-sheet regions and the structure is very well de®ned, it is unlikely the broadening is the result of a conformational change of the strands. Instead,

634

NMR Structure of MiAMP1

Ê ) of the Figure 4. The RMSD (A backbone atoms to the average structure versus residue number.

one or more of the residues near the sheet may undergo exchange between two conformations, in turn producing two exchanging environments for nearby residues. His27 is positioned within the broadened region and is directly next to Gln28 which experiences the greatest perturbation to its linewidths. In addition, the side-chain of His27 is not well de®ned as a result of a lack of medium to long-range NOEs between its side-chain protons and other protons within the protein. Inspection of the structures shows that the side-chain of this residue can readily ¯ip between alternative conformations and it appears that such an interchange is responsible for the observed exchange broadening of the cluster of proximate residues. There is no histidine residue in the corresponding position of the second Greek key motif and there is no observed broadening in this motif.

A surface charge analysis of MiAMP1 shown in Figure 8(b), reveals that MiAMP1 has an amphipathic structure with a clustering of most of the charges on one face. Many plant and animal AMPs have amphipathic structures which, together with a preponderance of basic charges, is believed to aid the interaction of the proteins with negatively charged membrane surfaces (Rao, 1995; Powell et al., 1995; Zhong et al., 1995). Thus, it appears likely that MiAMP1's mode of action may involve interaction with membrane surfaces. A comparison of Figure 8(a) and (b) reveals that the region of highest charge density lies on the same face as the region of spectral broadening. The functional signi®cance of this (if any) remains unknown. The Greek key b-barrel structure is common to a diverse range of proteins. A search using DALI (Holm & Sander, 1993) showed that several functionally distinct proteins have structures similar to

Figure 5. Stereoview of the backbone superimposition of 20 of the lowest energy structures.

635

NMR Structure of MiAMP1

Figure 6. The lowest energy structure of MiAMP1. The b-strands of sheet 1 and sheet 2 are represented by red and green arrows, respectively. Disulphide bonds are coloured yellow and are shown in the ball-and-stick representation. The structures were drawn using the program MOLMOL (Koradi et al., 1996).

MiAMP1. These included members of the bg-crystallin family, protein S, a bacterial spore coat protein and WmKT, a yeast killer toxin from Williopsis mrakii (Antuch et al., 1996). Most signi®cant, was the striking similarity of the structure of MiAMP1, a plant protein, to the yeast protein WmKT. WmKT (formerly known as the HM-1 toxin) is an 88 residue, highly basic AMP which is secreted into the extracellular matrix by some strains of W. mrakii (formerly known as Hansenula mrakii) to prevent the growth of other yeasts (Antuch et al., 1996; Yamamoto et al., 1986). Its antifungal activity results from its ability to inhibit b-glucan synthesis in sensitive strains of yeast which, in turn, affects cell wall construction (Yamamoto et al., 1986; Takasuka et al., 1995). Figure 9 shows a comparison of the structures of WmKT and MiAMP1. The arrangement of bstrands within the Greek keys and subsequent arrangement of the Greek keys to form the b-barrel is identical with that of MiAMP1. In addition, like MiAMP1, WmKT possesses two loops which are tethered to the b-sheets via disulphide bonds. It is interesting to hypothesise that the similar structures and activities of MiAMP1 and WmKT, which originate from plant and fungal phyla, respectively, may also re¯ect a similar mode of action.

In conclusion, this study has determined the structure of MiAMP1 to be a Greek key b-barrel. The unique sequence and structure of MiAMP1 places it in a new class of plant antimicrobial proteins which we have named the b-barrelins. With the structure of MiAMP1 solved, preliminary insights into its SARs have already been gained. In particular, an analysis of the surface properties of MiAMP1 suggests its activity may, in part, be the result of an ability to interact with negatively charged membrane surfaces. Also, the striking similarity of MiAMP1 to WmKT in both structure and activity raises the possibility that the mode of action of MiAMP1 may be similar to that of WmKT. Future work will concentrate on further de®ning MiAMP1õÂs mode of action through the design, expression and analysis of mutants.

Materials and Methods Materials Native MiAMP1 was extracted and puri®ed as described by Marcus et al., 1997, while recombinant unlabelled and 15N-labelled peptides were expressed as described by Harrison et al., 1999. Samples of MiAMP1 prepared for NMR spectroscopy contained 1.5 mM of the peptide dissolved in the following solvent systems:

Figure 7. The alignment of the two Greek key motifs of MiAMP1. Motif 1 and motif 2 consist of residues 1-38 and 39-76, respectively. Homologous residues identi®ed using the program MacVector (Oxford Molecular, USA) are highlighted in bold and include 13 % sequence identity and 18 % sequence similarity. The disulphide connectivities which are in symmetrical positions are represented with continuous lines. The non-symmetrical disulphide bond is represented with a broken line. Shaded arrows represent strands belonging to sheet 1 whereas white arrows represent strands belonging to sheet 2.

636

Figure 8. (a) Two 180  transposed views of the surface of MiAMP1. Motif 1 (residues 1-38) and motif 2 (residues 39-76) are coloured yellow and white, respectively. The orange area represents the region of the molecule undergoing slow exchange. This area is all within motif 1. (b) The surface electrostatic potential of MiAMP1. The charged residues are largely clustered on the same face of the molecule as the residues undergoing slow conformational exchange. The structures were drawn using the program MOLMOL (Koradi et al., 1996).

90 % H2O:10 % 2H2O at pH 2.3 and 5.0, and 100 % 2H2O at pH 5.0. NMR spectroscopy NMR spectra were recorded on a Bruker DMX 750 MHz spectrometer at temperatures of 308 K (90 %

NMR Structure of MiAMP1 H2O:10 % 2H2O at pH 2.3 and 5.0) and at 5 K increments between 283 K and 308 K (90 % H2O:10 % 2H2O at pH 5.0). Two-dimensional homonuclear spectra were recorded in phase-sensitive mode using time proportional phase incrementation for quadrature detection in the f1-dimension (Marion & WuÈthrich, 1983). The 2D experiments included TOCSY (BrauÈnschweiler & Ernst, 1983) using a MLEV-17 spin-lock sequence (Bax & Davis, 1985) with a mixing time of 80 ms, NOESY (Jeener et al., 1979) with mixing times of 80, 100, 150, 200, 250 and 300 ms and DQF-COSY (Rance et al., 1983). For the DQFCOSY experiment, the water proton signal was suppressed by low power irradiation during the relaxation delay (1.8 seconds). Solvent suppression in the TOCSY and NOESY experiments was achieved using a modi®ed WATERGATE sequence (Piotto et al., 1992) in which two gradient pulses of 1 ms duration and 6 G cmÿ1 strength were applied on either side of the binomial pulse. The 2D spectra were collected over 4096 data points in the f2dimension and 512 increments in the f1-dimension over a spectral width corresponding to 13 ppm. TOCSY spectra were acquired with 16 scans per increment, DQF-COSY spectra with 32 scans per increment and NOESY spectra with either 64 or 80 scans per increment. Slowly exchanging NH protons were detected by the acquisition of a series of 1D and TOCSY spectra following the addition of 100 % 2H2O to a sample of MiAMP1 at 308 K (pH 5.0). The 3JNH-aH coupling constants were measured from a high-resolution (8 K  1 K) DQF-COSY spectrum using lineshape ®tting with the program AURELIA (Bruker). A 1H-15N HSQC spectrum (Palmer et al., 1991; Kay et al., 1992; Schleucher et al., 1994) was recorded at 308 K, on a 1.5 mM sample containing 15N-labelled peptide in 90 % H2O:10 % 2H2O at pH 5.0, to assist with the assignment of some ambiguous spin systems. Spectral widths in the 1H and 15N dimensions were 9765 and 3041 Hz, respectively. A total of 4096 data points were acquired with 200 f1 increments and eight scans per increment. The data were processed and analysed on a Silicon Graphics SGI 4D/30 computer using the UXNMR software package in conjunction with X-EASY (Eccles et al., 1991). The f1-dimension was zero-®lled to 2048 real data points with the f1 and f2-dimensions being multiplied by

Figure 9. A comparison of the structures of (a) WmKT and (b) MiAMP1. The b-strands are represented by arrows. Disulphide bonds are shown in ball-and-stick representation.

NMR Structure of MiAMP1 a sine-squared function shifted by 90  prior to Fourier transformation. Polynomial baseline correction was used in selected regions to improve the appearance of the spectrum. Spectra were referenced to internal DSS. Structure calculations Peak volumes in the 100 ms NOESY spectra (90 % H2O:10 % 2H2O, 308 K and 283 K, and 100 % 2H2O at pH 5.0, 308 K) were measured in XEASY. These were classi®ed as strong, medium, weak or very weak correÊ, sponding to upper bounds of 2.7, 3.5, 5.0 or 6.0 A respectively (Clore et al., 1986a). The upper limits of methylene, methyl and aromatic protons were adjusted using standard pseudoatom corrections (WuÈthrich et al., 1983). Backbone dihedral angle restraints were derived from 3JNH-aH coupling constants, with f restrained to ÿ120(40) for 3JNH-aH > 9 Hz and ÿ60(30) for 3 JNH-aH < 5 Hz. These measurements were made from lineshape analysis of antiphase cross-peak splitting in the DQF-COSY spectrum. Additional dihedral restraints of ÿ100(80) were added where it was clear the intraresidual ai ! NHi NOE was weaker than the ai ÿ 1 ! NHi NOE (Clubb et al., 1994). Three-dimensional structures were calculated using simulated annealing and energy minimisation protocols within X-PLOR (BruÈnger, 1992). An ab initio simulated annealing protocol incorporating ambiguous distance restraints and ¯oating point chirality was used to generate a set of 50 structures starting from template structures with randomised j and f angles and extended side-chains (Nilges et al., 1988; Folmer et al., 1997). This protocol consists of 50 ps of molecular dynamics at 2000 K followed by 15 ps of cooling to 100 K. The structures were then subjected to 2000 cycles of energy minimisation using the conjugate gradient Powell algorithm under the in¯uence of the CHARMm force®eld (Clore et al., 1986b; Brooks et al., 1983). The 2H2O exchange data and analysis of the structures allowed the addition of 31 hydrogen bond restraints into the structure calculations. Protein Data Bank accession number The coordinates for the 20 re®ned, lowest energy conformers have been deposited in the Protein Data Bank under the accession code 1C01.

Acknowledgements D.J.C. is an Australian Research Council Senior Fellow. This work was supported by The University of Queensland Travelling Scholarship (A.M.M.) and a Sugar Research and Development Corporation Postgraduate Scholarship (S.J.H.).

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638

NMR Structure of MiAMP1

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Edited by P. E. Wright (Received 11 June 1999; received in revised form 27 August 1999; accepted 27 August 1999)