Combined X-ray and NMR Analysis of the Stability of the Cyclotide Cystine Knot Fold That Underpins Its Insecticidal Activity and Potential Use as a Drug Scaffold
2009; Elsevier BV; Volume: 284; Issue: 16 Linguagem: Inglês
10.1074/jbc.m900021200
ISSN1083-351X
AutoresConan K. Wang, Shuhong Hu, Jennifer L. Martin, Tove Sjögren, János Hajdu, Lars Bohlin, Per Claeson, Ulf Göransson, K. Johan Rosengren, Jun Tang, Ning‐Hua Tan, David J. Craik,
Tópico(s)Phytoplasmas and Hemiptera pathogens
ResumoCyclotides are a family of plant defense proteins that are highly resistant to adverse chemical, thermal, and enzymatic treatment. Here, we present the first crystal structure of a cyclotide, varv F, from the European field pansy, Viola arvensis, determined at a resolution of 1.8 Å. The solution state NMR structure was also determined and, combined with measurements of biophysical parameters for several cyclotides, provided an insight into the structural features that account for the remarkable stability of the cyclotide family. The x-ray data confirm the cystine knot topology and the circular backbone, and delineate a conserved network of hydrogen bonds that contribute to the stability of the cyclotide fold. The structural role of a highly conserved Glu residue that has been shown to regulate cyclotide function was also determined, verifying its involvement in a stabilizing hydrogen bond network. We also demonstrate that varv F binds to dodecylphosphocholine micelles, defining the binding orientation and showing that its structure remains unchanged upon binding, further demonstrating that the cyclotide fold is rigid. This study provides a biological insight into the mechanism by which cyclotides maintain their native activity in the unfavorable environment of predator insect guts. It also provides a structural basis for explaining how a cluster of residues important for bioactivity may be involved in self-association interactions in membranes. As well as being important for their bioactivity, the structural rigidity of cyclotides makes them very suitable as a stable template for peptide-based drug design. Cyclotides are a family of plant defense proteins that are highly resistant to adverse chemical, thermal, and enzymatic treatment. Here, we present the first crystal structure of a cyclotide, varv F, from the European field pansy, Viola arvensis, determined at a resolution of 1.8 Å. The solution state NMR structure was also determined and, combined with measurements of biophysical parameters for several cyclotides, provided an insight into the structural features that account for the remarkable stability of the cyclotide family. The x-ray data confirm the cystine knot topology and the circular backbone, and delineate a conserved network of hydrogen bonds that contribute to the stability of the cyclotide fold. The structural role of a highly conserved Glu residue that has been shown to regulate cyclotide function was also determined, verifying its involvement in a stabilizing hydrogen bond network. We also demonstrate that varv F binds to dodecylphosphocholine micelles, defining the binding orientation and showing that its structure remains unchanged upon binding, further demonstrating that the cyclotide fold is rigid. This study provides a biological insight into the mechanism by which cyclotides maintain their native activity in the unfavorable environment of predator insect guts. It also provides a structural basis for explaining how a cluster of residues important for bioactivity may be involved in self-association interactions in membranes. As well as being important for their bioactivity, the structural rigidity of cyclotides makes them very suitable as a stable template for peptide-based drug design. Cyclotides are an intriguing family of plant-derived proteins (1Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol... 1999; 294: 1327-1336Google Scholar, 2Craik D.J. Daly N.L. Mulvenna J. Plan M.R. Trabi M. Curr. Protein Pept. Sci... 2004; 5: 297-315Google Scholar) that act in plant defense (3Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A... 2001; 98: 10614-10619Google Scholar, 4Jennings C.V. Rosengren K.J. Daly N.L. Plan M. Stevens J. Scanlon M.J. Waine C. Norman D.G. Anderson M.A. Craik D.J. Biochemistry.. 2005; 44: 851-860Google Scholar) and display a range of interesting biological activities, including uterotonic (5Gran L. Lloydia.. 1973; 36: 174-178Google Scholar), human immunodeficiency virus inhibitory (6Gustafson K.R. McKee T.C. Bokesch H.R. Curr. Protein Pept. Sci... 2004; 5: 331-340Google Scholar), antimicrobial (7Tam J. Lu Y. Yang J. Chiu K. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 8913-8918Google Scholar), cancer cell toxicity (8Svangård E. Göransson U. Hocaoglu Z. Gullbo J. Larsson R. Claeson P. Bohlin L. J. Nat. Prod... 2004; 67: 144-147Google Scholar), and neurotensin antagonistic activities (9Witherup K. Bogusky M. Anderson P. Ramjit H. Ransom R. Wood T. Sardana M. J. Nat. Prod... 1994; 57: 1619-1625Google Scholar). Their natural function as plant defense agents was deduced from findings that cyclotides effectively inhibited the growth of two common cotton pests, Helicoverpa punctigera and Helicoverpa armigera, when the larvae of these pests were fed a cyclotide-containing diet (4Jennings C.V. Rosengren K.J. Daly N.L. Plan M. Stevens J. Scanlon M.J. Waine C. Norman D.G. Anderson M.A. Craik D.J. Biochemistry.. 2005; 44: 851-860Google Scholar). The ability of cyclotides to deliver their activity in the harsh environment of predator insect guts relies on them being exceptionally stable. Indeed, cyclotides have been shown to be resistant to harsh thermal, chemical, and enzymatic conditions in vitro (10Daly N.L. Gustafson K.R. Craik D.J. FEBS Lett... 2004; 574: 69-72Google Scholar, 11Colgrave M.L. Craik D.J. Biochemistry.. 2004; 43: 5965-5975Google Scholar). Their remarkable stability also makes cyclotides potentially valuable scaffolds for pharmaceutical or agrochemical applications (12Craik D.J. Simonsen S. Daly N.L. Curr. Opin. Drug Discovery Dev... 2002; 5: 251-260Google Scholar). Cyclotides are the largest family of naturally occurring head-to-tail cyclized proteins, but other examples of circular proteins have been discovered in recent years in bacteria, plants, and animals (13Trabi M. Craik D.J. Trends Biochem. Sci... 2002; 27: 132-138Google Scholar, 14Craik D.J. Science.. 2006; 311: 1563-1564Google Scholar). The cyclotides are distinguished from other circular proteins in that their cysteine residues form three disulfide bonds that are arranged in a cystine knot motif (1Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol... 1999; 294: 1327-1336Google Scholar). Positioned in the protein core, one of the disulfide bonds (CysIII–CysVI) threads through an embedded ring formed by the other two disulfide bonds (CysI–CysIV and CysII–CysV) and their connecting backbone sequences (15Craik D.J. Daly N.L. Waine C. Toxicon.. 2001; 39: 43-60Google Scholar). Recent studies have shown that an intact cystine knot is required to maintain the remarkable stability of cyclotides (10Daly N.L. Gustafson K.R. Craik D.J. FEBS Lett... 2004; 574: 69-72Google Scholar, 11Colgrave M.L. Craik D.J. Biochemistry.. 2004; 43: 5965-5975Google Scholar), highlighting the importance of a structural understanding of the cystine knot topology. The sequences between successive cysteine residues are referred to as loops and are considered to "display" the residues that define cyclotide bioactivities. Loops 1 and 4 are highly conserved in size and sequence across all cyclotides, reflecting their structural involvement in the cystine knot. By contrast, a variety of residues are displayed within the other loops, leading to the description of cyclotides as a natural combinatorial template (16Craik D.J. Cemazar M. Daly N.L. Curr. Opin. Drug Discovery Dev... 2007; 10: 176-184Google Scholar), based around a cyclic cystine knot core. Current estimates suggest that there are at least 9000 cyclotides in the Violaceae (violet) plant family (17Simonsen S.M. Sando L. Ireland D.C. Colgrave M.L. Bharathi R. Goransson U. Craik D.J. Plant Cell.. 2005; 17: 3176-3189Google Scholar) and >50,000 in the Rubiaceae (coffee) family (18Gruber C.W. Elliott A.G. Ireland D.C. Delprete P.G. Dessein S. Goransson U. Trabi M. Wang C.K. Kinghorn A.B. Robbrecht E. Craik D.J. Plant Cell.. 2008; 20: 2471-2483Google Scholar), making them a very large group of plant proteins. The cyclotides have been divided into two main subfamilies, Möbius and bracelet (1Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol... 1999; 294: 1327-1336Google Scholar), depending on the presence or absence, respectively, of a cis-Pro peptide bond in loop 5. Varv F, a previously uncharacterized cyclotide, belongs to the Möbius subfamily according to its sequence so it was of interest to see if the proposed cis-Pro peptide bond, which is the key structural feature used to classify the cyclotides, is present in its three-dimensional structure. Until now, structural studies on cyclotides have been performed using nuclear magnetic resonance (NMR), a technique well suited to the study of small proteins (19Craik D.J. Daly N.L. Mol. Biosyst... 2007; 3: 257-265Google Scholar). Although NMR has provided valuable insight into the nature of cyclotides, some structural features were initially difficult to unambiguously define. For example, there has been some debate over the disulfide bond connectivity. Apart from the knotted arrangement (CysI–CysIV, CysII–CysV, and CysIII–CysVI), which we initially suggested (20Saether O. Craik D.J. Campbell I.D. Sletten K. Juul J. Norman D.G. Biochemistry.. 1995; 34: 4147-4158Google Scholar), a laddered arrangement was later proposed (CysI–CysVI, CysII–CysV, and CysIII–CysIV) based on an examination of local NOEs between Cys residues (21Skjeldal L. Gran L. Sletten K. Volkman B. Arch. Biochem. Biophys... 2002; 399: 142-148Google Scholar). Although the connectivity has been resolved on the basis of an analysis of high field NMR data relating to the side chain angles of the cysteine residues (22Rosengren K.J. Daly N.L. Plan M.R. Waine C. Craik D.J. J. Biol. Chem... 2003; 278: 8606-8616Google Scholar), and by a chemical analysis of partially reduced species (23Göransson U. Craik D.J. J. Biol. Chem... 2003; 278: 48188-48196Google Scholar, 24Nair S.S. Romanuka J. Billeter M. Skjeldal L. Emmett M.R. Nilsson C.L. Marshall A.G. Biochim. Biophys. Acta.. 2006; 1764: 1568-1576Google Scholar), a crystal structure would provide conclusive evidence of the disulfide bond topology. Another important structural feature of cyclotides is their hydrogen bond network, which is thought to stabilize the cyclic cystine knot framework. In particular, a highly conserved glutamic acid residue in loop 1, which has been shown to regulate cyclotide function in mutational studies (25Herrmann A. Svangård E. Claeson P. Gullbo J. Bohlin L. Göransson U. Cell Mol. Life Sci... 2006; 63: 235-245Google Scholar, 26Simonsen S.M. Sando L. Rosengren K.J. Wang C.K. Colgrave M.L. Daly N.L. Craik D.J. J. Biol. Chem... 2008; 283: 9805-9813Google Scholar), is believed to be structurally important due to an interaction with the backbone amides of two residues in loop 3 (22Rosengren K.J. Daly N.L. Plan M.R. Waine C. Craik D.J. J. Biol. Chem... 2003; 278: 8606-8616Google Scholar). Again, a crystal structure would be useful for defining this interaction. Generally, analysis of hydrogen bonding patterns in proteins using NMR relies on D2O exchange experiments or the temperature dependence of amide proton chemical shifts, quantified by so-called temperature coefficients (ΔδNH/ΔT). The sensitivity of backbone amide protons to temperature means that temperature coefficients correlate with the hydrogen bonding properties of the amides, with hydrogen-bonded amides generally showing smaller temperature coefficients than non-hydrogen bonded amides. In the current study we show that a comparison of temperature coefficients, slow exchange parameters, and direct determination of hydrogen bonds from a range of cyclotide structures (crystal and solution) can be used to derive general conclusions about the role of hydrogen bonds in stabilizing the generic cyclotide framework. In this study, we structurally characterized varv F (27Göransson U. Luijendijk T. Johansson S. Bohlin L. Claeson P. J. Nat. Prod... 1999; 62: 283-286Google Scholar), a member of the Möbius subfamily of cyclotides that has been reported to have strong cytotoxic activity (28Lindholm P. Göransson U. Johansson S. Claeson P. Gullbo J. Larsson R. Bohlin L. Backlund A. Mol. Cancer Ther... 2002; 1: 365-369Google Scholar). The crystal structure that is presented here is the first crystal structure of a cyclotide. Comparison of the crystal structure with the NMR structure also determined in this study confirms the unique features of the cyclotide fold, including the cystine knot topology and β-sheet structure. We investigate the hydrogen bond network, believed to contribute to the stability of the cyclotides, based on an analysis of amide temperature coefficients, D2O exchange experiments, and the predicted hydrogen bonds from the crystal and NMR structures. We also determined the structural characteristics of micelle-bound varv F because cyclotides are believed to act through membrane interactions and so relating the solution and crystal structures to a structure representing the membrane-bound state provides an insight into the mode of action of cyclotides. Furthermore, a comprehensive understanding of the structural features that stabilize the cyclotide framework is important because the stability of cyclotides is key to their natural activity and also contributes to their potential as scaffolds of bioengineered drugs. Varv F Isolation, Crystallization, and Data Collection—Varv F was isolated from Viola arvensis as described in Göransson et al. (27Göransson U. Luijendijk T. Johansson S. Bohlin L. Claeson P. J. Nat. Prod... 1999; 62: 283-286Google Scholar). Briefly, the dried aerial plant part was defatted with dichloromethane and then extracted with 50% aqueous ethanol. The aqueous extract was filtered through polyamide to remove tannins, followed by removal of low molecular weight substances by size exclusion chromatography on Sephadex G-10. Polysaccharides and buffer salts were then removed by reverse phase solid phase extraction. The resulting cyclotide-enriched fraction was separated with a Sephadex LH-20 column, and eluted with 30% methanol, 0.05% trifluoroacetic acid in water. Under these conditions this column separates cyclotides according to their content of aromatic amino acid residues. Varv F, which is distinguished from the majority of cyclotides by its content of two aromatic residues, could then be easily collected. Final purification was done using reverse phase (RP) 2The abbreviations used are: RP, reverse phase; HPLC, high performance liquid chromatography; PDB, Protein data bank; DPC, dodecylphosphocholine; Bicine, N,N-bis(2-hydroxyethyl)glycine.-high performance liquid chromatography (HPLC). Crystal Screens 1 and 2 (Hampton Research, Aliso Viejo, CA) were used to screen for crystallization conditions using the hanging drop method. For the screen, varv F was dissolved in water at a concentration of 0.5 mm.1 μl of that solution was suspended in 1 μl of each reservoir solution, over reservoir/wells containing 500 μl of the 96 different buffers in the Crystal Screens. After 5 months at 20 °C, a single crystal was formed in buffer condition number 47 of Crystal Screen 2 containing 2.0 m magnesium chloride hexahydrate, 0.1 m Bicine, pH 9.0. The crystal belongs to space group I4(1)32 with unit cell dimensions: a = b = c = 84.08 Å. There is one molecule in the asymmetric unit with a solvent content of 70% (29Matthews B.W. J. Mol. Biol... 1968; 33: 491-497Google Scholar). Prior to data collection, crystals were removed from the crystallization drop and then flash-frozen in liquid nitrogen. Data to 1.8 Å resolution were collected at 100 K using monochromatic x-rays at a wavelength of 1.052 Å on beamline I711 at MAXlab (Lund, Sweden). The data were processed using programs DENZO and SCALEPACK (30Minor W. XDISPLAYF.Purdue University, West Lafayette, IN. 1993; Google Scholar, 31Otwinowski Z. Proceedings of the CCP4 Study Weekend: Data Collections and Processing.(Sawyer, L., Isaacs, N., and Bailey, S., eds) SERC Daresbury Laboratory, Warrington, UK. 1993; Google Scholar). Crystal Structure Determination and Refinement—The structure was solved by molecular replacement using the program PHASER (32Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr... 2001; 57: 1373-1382Google Scholar, 33Storoni L.C. McCoy A.J. Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr... 2004; 60: 432-438Google Scholar). The search model was the high resolution NMR structure of kalata B1 (Protein Data Bandk code 1NB1) with three Cys residues (CysIV, CysV, and CysVI) replaced by alanine. The final structure was generated after several rounds of model building in O (34Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A Found. Crystallogr... 1991; 47: 110-119Google Scholar), and simulated annealing and positional and individual B-factor refinement in CNS (35Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr... 1998; 54: 905-921Google Scholar). The 2Fo - Fc and Fo - Fc electron density maps clearly revealed three disulfide bonds formed by CysI and CysIV, CysII and CysV, and CysIII and CysVI. Water molecules were included where the difference electron density showed a peak above 3σ and the modeled water made stereochemically reasonable hydrogen bonds. The quality and geometry of the model were evaluated by PROCHECK. A Ramachandran plot showed that 85.7% of the residues were in the core allowed region and 14.3% were in the additionally allowed region. As will be discussed later, the x-ray structure of varv F was in excellent agreement with its NMR structure, suggesting that the determined conformation is not affected by solution conditions. The crystal structure has been assigned PDB code 3E4H. Isolation of varv F for NMR Spectroscopy—Extraction of varv F from the aerial parts of V. arvensis was carried out using our previously established procedure (3Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A... 2001; 98: 10614-10619Google Scholar). Fresh plant material (collected from the Northeastern region of Victoria, Australia) was ground in a kitchen blender and left overnight in 1:1 dichloromethane:methanol. The extract was filtered, and distilled water was added to promote the separation of the aqueous partition, which was collected and the residual methanol was evaporated in vacuo. Several steps of RP-HPLC were then employed to purify the protein. Preparative RP-HPLC was performed on a Waters 600 Controller system equipped with a Waters 484 Tuneable Absorbance Detector. The aqueous extract was loaded onto a Phenomenex Jupiter C18 column (250 × 22 mm, 5 μm, 300 Å) and eluted at a flow rate of 8 ml/min with a 1% buffer B (90% HPLC grade acetonitrile in H2O, 0.09% trifluoroacetic acid) per min of gradient. Semi-preparative RP-HPLC was performed on an Agilent 1100 series system with variable wavelength detector and a Phenomenex Jupiter C18 column (250 × 10 mm, 5 μm, 300 Å). Analytical RP-HPLC was performed using a Phenomenex Jupiter C18 column (250 × 4.6 mm, 5 μm, 300 Å). Masses were analyzed on a Micromass LCT mass spectrometer equipped with an electrospray ionization source. NMR Spectroscopy and Structure Calculations—Varv F was dissolved in 90% H2O, 10% D2O at pH ∼6 giving a final concentration of 0.4 mm. A Bruker ARX 500, Bruker ARX 600, or Bruker DMX 750 was used to record spectra with sample temperatures within the range of 288–328 K. All spectra were recorded in phase-sensitive mode by using time-proportional phase incrementation (36Marion D. Wüthrich K. Biochem. Biophys. Res. Commun... 1983; 113: 967-974Google Scholar). Two-dimensional experiments obtained included a TOCSY (37Braunschweiler L. Ernst R.R. J. Magn. Reson... 1983; 53: 521-528Google Scholar) with 80 ms mixing time, NOESY (38Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys... 1979; 71: 4546-4553Google Scholar) with 200 ms mixing time, DQF-COSY (39Rance M. Sorensen O.W. Bodenhausen G. Wagner G. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun... 1983; 27: 157-162Google Scholar), and E-COSY (40Griesinger C. Sorensen O.W. Ernst R.R. J. Magn. Reson... 1987; 75: 474-492Google Scholar) in 100% D2O. Water suppression for TOCSY and NOESY experiments was achieved by using a modified WATERGATE sequence (41Piotto M. Saudek V. Sklenar V. J. Biomol. NMR.. 1992; 2: 661-665Google Scholar), whereas lower power irradiation during the relaxation delay was used in the DQF-COSY experiment. Spectra were acquired with 4096 data points in the F2 and 512 increments in the F1 dimension. The F1 and F2 dimensions were multiplied by a sine-squared function prior to Fourier transformation. Chemical shifts were internally referenced to sodium 2,2-dimethyl-2-silapentane-5-sulfonate. Distance restraints were derived from cross-peaks in NOESY spectra recorded with a mixing time of 200 ms at 288 and 298 K. Spectra were analyzed with the program SPARKY (42Goddard T.D. Kneller D.G. SPARKY 3.University of California, San Francisco, CA. 2005; Google Scholar). Backbone dihedral angle restraints were derived from 3JHNHα coupling constants measured from line shape analysis of antiphase cross-peak splitting in the DQF-COSY spectrum, whereas χ1 dihedral angles were derived from 3JHαHβ coupling constants from the E-COSY spectrum together with NOE intensities. After initial structure calculations were performed using CYANA (43Güntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol... 1997; 273: 283-298Google Scholar), hydrogen bond restraints for slowly exchanging amides were added. In total, 279 distance restraints, comprising 65 sequential, 63 non-sequential restraints, and 151 intra-residue restraints, were determined from NOESY spectra; 29 dihedral angle restraints (15 ϕ and 14 χ1) were derived based on coupling constants from DQF-COSY and E-COSY spectra, and slowly exchanging amides detected >16 h after dissolution of the sample in D2O were used to derive upper limit distance restraints for nine hydrogen bonds. Final sets of 50 structures were calculated using a torsion angle-simulated annealing protocol within CNS (35Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr... 1998; 54: 905-921Google Scholar) and were further refined in a water shell (44Linge J.P. Nilges M. J. Biomol. NMR.. 1999; 13: 51-59Google Scholar). The structures were analyzed with MOLMOL (45Koradi R. Billeter M. Wüthrich K. J. Mol. Graph... 1996; 14: 51-55Google Scholar), PROMOTIF (46Hutchinson E.G. Thornton J.M. Protein Sci... 1996; 5: 212-220Google Scholar), and PROCHECK (47Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr... 1993; 26: 283-291Google Scholar). For the 20 lowest energy structures, 81.8% of the residues were in the most favored and 18.2% in the additionally allowed regions of the Ramachandran plot. The NMR structure of varv F has been deposited in the PDB (code 2K7G). Hydrogen Bond Analysis—To measure the temperature dependence of the amide chemical shifts (ΔδNH/ΔT), one-dimensional and TOCSY spectra were recorded at increasing temperatures in steps of 10 K from 280 to 330 K or from 278 to 328 K. The chemical shift movements were documented and fitted to a linear function. Temperature coefficients for the cyclotides were measured at two pH values, namely 3.3 and 6.4 for kalata B1, 3.2 and 6.0 for kalata B2, 2.8 and 6.0 for varv F, 2.9 and 5.6 for cycloviolacin O1, and 2.0 and 5.5 for kalata B5. Amide protons detected in TOCSY spectra after a minimum of 16 h following dissolution in D2O were classified as slowly exchanging. TOCSY spectra of kalata B1, kalata B2, varv F, cycloviolacin O1 (all at pH ∼ 4), and kalata B5 (pH ∼ 5.5) were acquired after 16–72 h. Previously reported slowly exchanging amides were used for kalata B1 (20Saether O. Craik D.J. Campbell I.D. Sletten K. Juul J. Norman D.G. Biochemistry.. 1995; 34: 4147-4158Google Scholar) and kalata B2 (4Jennings C.V. Rosengren K.J. Daly N.L. Plan M. Stevens J. Scanlon M.J. Waine C. Norman D.G. Anderson M.A. Craik D.J. Biochemistry.. 2005; 44: 851-860Google Scholar). Hydrogen bond donor and acceptor pairs were determined using an in-house program. Each amide was classified as a hydrogen bond donor if for at least half of the corresponding ensemble of structures, they were able to satisfy empirical criteria (i.e. the distance between the hydrogen atom connected to the donor and the acceptor is less than 2.4 Å, and the angle subtended by the line from the hydrogen atom of the donor to the donor and the line from the hydrogen atom to the acceptor is greater than 90° (48McDonald I.K. Thornton J.M. J. Mol. Biol... 1994; 238: 777-793Google Scholar)). For a few structures within the ensembles, certain amides, which normally belong to conserved hydrogen bonded positions, were not formally classified as hydrogen bonded but the failure to recognize these hydrogen bonds was found to be related to the stringency of the empirical criteria. For example, in kalata B1, the amide of Val-4 was not predicted to be a hydrogen bond donor using the criteria mentioned above, but after relaxing the distance restraint for a hydrogen bond to 2.75 Å, it was classified as being in a hydrogen bond. Similarly for Cys-19 of kalata B2 and Cys-20 of kalata B5, slight modification of the strict hydrogen bond criteria resulted in the respective amides being classified as hydrogen bond donors, consistent with the hydrogen bonding properties of the corresponding amides in the other cyclotides studied. These observations were taken into account when the temperature coefficients and slow exchanging nature of the amides were analyzed with respect to their hydrogen bonding classification. In kalata B5, the amide of Gly-16 did not have a predicted acceptor in more than half of the ensemble of structures, but had a number of possible bonding partners, including Glu-6, Cys-13, and Ile-14, and was classified as being involved in a hydrogen bond. Cyclotide Binding to DPC Micelles—Titration of varv F (1.9 mm) in 10% D2O with dodecylphosphocholine (DPC) was performed at 30 °C, pH 2.9. The one-dimensional NMR spectrum was acquired and the diffusion rate (49Altieri A.S. Hinton D.P. Byrd R.A. J. Am. Chem. Soc... 1995; 117: 7566-7567Google Scholar) of the cyclotide-DPC complex was measured at each titration point, ranging from a DPC to cyclotide ratio of ∼2:1 to 60:1. Under the condition of fast exchange between the unbound and bound states, the chemical shift of an affected proton (δobs) depends on the chemical shift of the bound state (δbound), the chemical shift of the free state (δfree), and on the bound peptide to free peptide according to the following equation. δobs=δfree[νF]free+δbound[νF]bound[νF]0(Eq.1) [νF]0=[νF]free+[νF]bound(Eq.2) The Langmuir isotherm shown in the following equation was used for analysis (50Shenkarev Z.O. Nadezhdin K.D. Sobol V.A. Sobol A.G. Skjeldal L. Arseniev A.S. FEBS J... 2006; 273: 2658-2672Google Scholar) and provided values for Ka, the affinity constant, of the peptide for the DPC micelle, and N, the number of DPC molecules that form the site of the peptide binding. Exp(ΔG0RT)=1Ka=[νF]free([DPC]−N[νF]bound)N[νF]bound(Eq.3) Modeling of Cyclotide-DPC Complex—Titration of varv F/DPC samples with 5- and 16-doxylstearate was performed at 50 °C. NOESY spectra (100 ms mixing time) were measured at 0 and 8 mm doxylstearate at pH 5. The paramagnetic attenuation induced by doxylstearate was qualitatively characterized by calculating the relative cross-peak intensity. RCIi=100%IiprobeIi0(Eq.4) Here Iiprobe and I0probe are the intraresidual HN-Hα cross-peak intensities (Hδ2–Hδ3 for prolines) in the spectra of samples with and without a paramagnetic probe. A geometric model of the varv F-micelle complex was built based on an approach described previously for kalata B1 (51Dubovskii P.V. Dementieva D.V. Bocharov E.V. Utkin Y.N. Arseniev A.S. J. Mol. Biol... 2001; 305: 137-149Google Scholar), i.e. by constraining selected protons to be either inside or outside of a spherical micelle based on their relative broadening by the spin label. Using the 5-doxylstearate data, the HN and Hα atoms for which the relative intensity of their cross-peaks was <20% were restricted to be inside the micelle and other HN and Hα atoms were restricted to be outside the micelle. The Crystal Structure of varv F—Like many small disulfide-rich proteins, cyclotides are difficult to crystallize. However, after many trials on a range of cyclotides from plants in Rubiaceae and Violaceae, we obtained a crystal of varv F, a member of the Möbius subfamily of cyclotides. The crystal diffracted to 1.8 Å resolution and belongs to space group I4(1)32 with unit cell dimensions: a = b = c = 84.08 Å. Crystallographic statistics for data collection and refinement are shown in Table 1. The data are 99.9% complete to 1.8 Å, and the overall merging Rsym is 6.1%. The crystal structure was determined by molecular replacement using the NMR structure of kalata B1 (PDB code 1NB1) as the search model, refined to a final Rfactor of 22.4% with an Rfree of 24.4% at 1.8-Å resolution. The mean coordinate error of the structure is estimated to be 0.22 Å, based on the Luzzati plot analysis (52Luzzati D. Ann. Inst. Pasteur (Paris).. 1953; 85: 277-281Google Scholar).Table 1Data collection and refinement statistics for the crystal structure of varv F Data collection Space group I4(1)32 Cell dimensions a, b, c (Å) 84.08, 84.08, 84.08 α, β, γ (°) 90, 90, 90 Resolution (Å) 30-1.80 (1.86-1.80)aHighest resolution shell is shown in parentheses No. observations 178,276 No. unique reflections 4,978 RmergebRmerge = Σhkl(Σi(|Ihkl,i – 〈Ihkl〉|))/Σhkl,i〈Ihkl〉, where Ihkl,i i
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