Two-dimensional1H NMR study of recombinant insect defensin A in water: Resonance assignments, secondary structure and global folding

Abstract

A 500 MHz 2D 1H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities, 3JNH-alpha H coupling constants as well as 1H/2H exchange rates and delta delta/delta T temperature coefficients of NH protons strongly support the existence of an alpha-helix (residues 14-24) and of an antiparallel beta-sheet (residues 27-40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from 3JNH-alpha H coupling constants, (iii) 12 hydrogen bonds mostly deduced from 1H/2H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a beta-sheet is linked to an alpha-helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.

A novel insect defensin mediates the inducible antibacterial activity in larvae of the dragonfly Aeschna cyanea (Paleoptera, Odonata)

“…where it links the N-terminal loop to the /{ sheet. In Aeschnu, the relative positions of the other cysteine residues are compatible with the formation of a cysteine- The scqucnces of Aescltna defensin and Phormia defensin A are displayed with elements of the sccondary structure of Phormiu defensin as determined by NMR studies [8].…”

“…Four groups of inducible antibacterial peptides or polypeptides have been isolated and totally or partially characterized, which are: the cecropins, 4-kDa cationic pcptides devoid of cysteines, which form two amphipathic tl helices ( [3], reviewed in [4]); the insect defensins, 4-kDa cationic peptides which contain six cysteine residues engaged in three intramolecular disulfide bridges and characterized by an amphipathic CI helix linked via two disulfide bridges to an antiparallel j? sheet [5][6][7][8]; three as yet incompletely characterized, small (2 -4-kDa) cationic proline-rich peptides or peptide families which are the apidaecins 191, abaecin [lo] and the drosocins (Bulet P. and Dimarcq J. L., unpublished results); several distinct polypeptides ranging in size over 8 -27 kDa, mostly cationic and frcquently rich in Correspondence to J. A. Hoffmann, Laboratoirc de Biologie Generalc, UniversitC Louis Pasteur, URA CNRS 1490, Bases xnoleculaires de la rtponsc immunitaire des insectes, 12 rue de l'universite, F-67000 Strasbourg, France…”

Penaeidins, a New Family of Antimicrobial Peptides Isolated from the Shrimp Penaeus vannamei (Decapoda)

“…However, the cysteine motif in penaeidins has no significant homology with those found in any of the molecules mentioned above. For example, the cysteine stabilized ␣␤ motif characteristic of insect and plant defensins (47)(48)(49) as well as some scorpion toxins (50), which stabilizes an ␣-helix on a ␤-sheet through two disulfide bridges (48), is not found in the penaeidins. Moreover, as the penaeidin disulfide bridge positions are still unknown, we cannot predict the three-dimensional structure by homology with any of the antimicrobial peptides whose structures have been characterized to date.…”

Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae

“…A number of different in vitro activities have been attributed to this class of peptide including inhibition of in vitro protein synthesis [8,9], inhibition of ~-amylases [10] and antimicrobial activity [5][6][7]. Based on the inhibitory activity of at least some members of this peptide family to plant pathogenic fungi, their expression patterns in planta [11,12] and their three-dimensional structures, which show homology to that of insect defensins [15], we have proposed the name plant defensin to describe this family [16].…”