Overexpression and structural study of the cathelicidin motif of the protegrin-3 precursor.
Numerous precursors of antibacterial peptides with unrelated sequences share a similar prosequence of 96-101 residues, referred to as the cathelicidin motif. The structure of this widespread motif has not yet been reported. The cathelicidin motif of protegrin-3 (ProS) was overexpressed in Escherichia coli as a His-tagged protein to facilitate its purification. The His tag was then removed by thrombin cleavage. In addition, the complete proprotegrin-3 (ProS-PG-3) (120 residues) was overexpressed in baculovirus-infected insect cells. As it contained the antibacterial peptide protegrin-3 in its C-terminal part, ProS-PG-3 contained four disulfide bonds. At neutral pH, ProS and ProS-PG-3 adopted two slowly exchanging conformations that existed in a ratio of 55/45. This ratio was progressively modified at acidic pH to reach a 90/10 value at pH 3.0, suggesting that electrostatic interactions are involved in such a conformational change. Therefore, the structural study of the main conformer was undertaken at pH 3.0 by circular dichroism, mass spectrometry, and homo- and heteronuclear NMR. In parallel, a model for the ProS structure was built from the X-ray structure of the chicken cystatin. ProS and the chicken cystatin share two conserved disulfide bonds as well as a high conservation of hydrophobic residues. The ProS model features the conservation of a hydrophobic core made of the interface between the N-terminal helix and the wrapping beta-sheet. Although the full assignment of the main conformer of ProS could not be obtained, available NMR data validated the presence of the N-terminal helix and of a four-stranded beta-sheet, in agreement with the cystatin fold. Moreover, we clearly demonstrated that ProS and ProS-PG-3 share the same global structure, suggesting that the presence of the highly constrained beta-hairpin of protegrin does not significantly modify the structure of the cathelicidin motif of the protegrin precursor.
Sanchez
Jean Frédéric
JF
Centre de Biochimie Structurale, UMR 5048 CNRS-UM1/UMR 554 INSERM-UM1, Université Montpellier 1, Faculté de Pharmacie, 15 avenue Charles Flahault, 34093 Montpellier Cedex 5, France.
Wojcik
Franck
F
Yang
Yin-Shan
YS
Strub
Marie-Paule
MP
Strub
Jean Marc
JM
Van Dorsselaer
Alain
A
Martin
Marianne
M
Lehrer
Robert
R
Ganz
Tomas
T
Chavanieu
Alain
A
Calas
Bernard
B
Aumelas
André
A
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides; D016367:Baculoviridae / Q000235:genetics; D001798:Blood Proteins / Q000737:chemistry*; D002645:Chickens; D002942:Circular Dichroism; D003460:Crystallization; D018360:Crystallography, X-Ray; D015891:Cystatins / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D015891:Cystatins; D011498:Protein Precursors; D011506:Proteins; D011994:Recombinant Proteins; C121626:cathelicidin 3 protein, Equus caballus; C094881:protegrin-3