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An 1H NMR determination of the three-dimensional structures of mirror-image forms of a Leu-5 variant of the trypsin inhibitor from Ecballium elaterium (EETI-II).

Nielsen KJ, Alewood D, Andrews J, Kent SB, Craik DJ
Protein Sci 3 291-302 (1994)
Cited: 16 times
EuropePMC logo PMID: 8003965

Abstract

The 3-dimensional structures of mirror-image forms of a Leu-5 variant of the trypsin inhibitor Ecballium elaterium (EETI-II) have been determined by 1H NMR spectroscopy and simulated annealing calculations incorporating NOE-derived distance constraints. Spectra were assigned using 2-dimensional NMR methods at 400 MHz, and internuclear distances were determined from NOESY experiments. Three-bond spin-spin couplings between C alpha H and amide protons, amide exchange rates, and the temperature dependence of amide chemical shifts were also measured. The structure consists largely of loops and turns, with a short region of beta-sheet. The Leu-5 substitution produces a substantial reduction in affinity for trypsin relative to native EETI-II, which contains an Ile at this position. The global structure of the Leu-5 analogue studied here is similar to that reported for native EETI-II (Heitz A, Chiche L, Le-Nguyen D, Castro B, 1989, Biochemistry 28:2392-2398) and to X-ray and NMR structures of the related proteinase inhibitor CMTI-I (Bode W et al., 1989, FEBS Lett 242:285-292; Holak TA et al., 1989a, J Mol Biol 210:649-654; Holak TA, Gondol D, Otlewski J, Wilusz T, 1989b, J Mol Biol 210:635-648; Holak TA, Habazettl J, Oschkinat H, Otlewski J, 1991, J Am Chem Soc 113:3196-3198). The region near the scissile bond is the most disordered part of the structure, based on geometric superimposition of 40 calculated structures. This disorder most likely reflects additional motion being present in this region relative to the rest of the protein. This motional disorder is increased in the Leu-5 analogue relative to the native form and may be responsible for its reduced trypsin binding. A second form of the protein synthesized with all (D) amino acids was also studied by NMR and found to have a spectrum identical with that of the (L) form. This is consistent with the (D) form being a mirror image of the (L) form and not distinguishable by NMR in an achiral solvent (i.e., H2O). The (D) form has no activity against trypsin, as would be expected for a mirror-image form.

Reviews citing this publication (2)

  1. Cyclotides as grafting frameworks for protein engineering and drug design applications. Poth AG, Chan LY, Craik DJ. Biopolymers 100 480-491 (2013)
  2. Chemical protein synthesis. Wilken J, Kent SB. Curr. Opin. Biotechnol. 9 412-426 (1998)

Articles citing this publication (14)

  1. A common structural motif incorporating a cystine knot and a triple-stranded beta-sheet in toxic and inhibitory polypeptides. Pallaghy PK, Nielsen KJ, Craik DJ, Norton RS. Protein Sci. 3 1833-1839 (1994)
  2. Amide proton temperature coefficients as hydrogen bond indicators in proteins. Cierpicki T, Otlewski J. J. Biomol. NMR 21 249-261 (2001)
  3. A biomimetic strategy in the synthesis and fragmentation of cyclic protein. Tam JP, Lu YA. Protein Sci. 7 1583-1592 (1998)
  4. Solution structure by NMR of circulin A: a macrocyclic knotted peptide having anti-HIV activity. Daly NL, Koltay A, Gustafson KR, Boyd MR, Casas-Finet JR, Craik DJ. J. Mol. Biol. 285 333-345 (1999)
  5. Substitutions at the P(1) position in BPTI strongly affect the association energy with serine proteinases. Grzesiak A, Helland R, Smalås AO, Krowarsch D, Dadlez M, Otlewski J. J. Mol. Biol. 301 205-217 (2000)
  6. Identification of epitope-like consensus motifs using mRNA display. Baggio R, Burgstaller P, Hale SP, Putney AR, Lane M, Lipovsek D, Wright MC, Roberts RW, Liu R, Szostak JW, Wagner RW. J. Mol. Recognit. 15 126-134 (2002)
  7. Proton nuclear magnetic resonance studies on huwentoxin-I from the venom of the spider Selenocosmia huwena: 2. Three-dimensional structure in solution. Qu Y, Liang S, Ding J, Liu X, Zhang R, Gu X. J Protein Chem 16 565-574 (1997)
  8. Variability of the canonical loop conformations in serine proteinases inhibitors and other proteins. Apostoluk W, Otlewski J. Proteins 32 459-474 (1998)
  9. Structure of single-disulfide variants of bovine pancreatic trypsin inhibitor (BPTI) as probed by their binding to bovine beta-trypsin. Krokoszynska I, Dadlez M, Otlewski J. J. Mol. Biol. 275 503-513 (1998)
  10. Comparison of the solution conformations of a human immunodeficiency virus peptidomimetic and its retro-inverso isomer using 1H NMR spectroscopy. Higgins KA, Bicknell W, Keah HH, Hearn MT. J Pept Res 50 421-435 (1997)
  11. Barnase fusion as a tool to determine the crystal structure of the small disulfide-rich protein McoEeTI. Niemann HH, Schmoldt HU, Wentzel A, Kolmar H, Heinz DW. J. Mol. Biol. 356 1-8 (2006)
  12. Synthesis, cloning and expression in Escherichia coli of a gene coding for the Met8-->Leu CMTI I--a representative of the squash inhibitors of serine proteinases. Bolewska K, Krowarsch D, Otlewski J, Jaroszewski L, Bierzynski A. FEBS Lett. 377 172-174 (1995)
  13. Heterochiral Knottin Protein: Folding and Solution Structure. Mong SK, Cochran FV, Yu H, Graziano Z, Lin YS, Cochran JR, Pentelute BL. Biochemistry 56 5720-5725 (2017)
  14. Partially folded bovine pancreatic trypsin inhibitor analogues attain fully native structures when co-crystallized with S195A rat trypsin. Getun IV, Brown CK, Tulla-Puche J, Ohlendorf D, Woodward C, Barany G. J. Mol. Biol. 375 812-823 (2008)


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