Abstract
alpha-Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu(15) residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide.
Highlights
Broad spectrum endogenous antimicrobial peptides, including defensins, contribute to the innate immune response [1]
We present the corrected high resolution solution structure of Crp4 and the structure of the mutant (E15D)Crp4 in which the conserved salt bridge has been removed by effectively shortening the side chain of the Glu residue but otherwise making no change to the charge state of the native peptide
The role of this salt bridge in human neutrophil ␣-defensin 2 (HNP2) was investigated by site-directed mutagenesis, which showed that salt bridge disruption or removal did not diminish HNP2 antibacterial activity or HNP2 precursor folding in vitro [17]
Summary
Cryptdin; HNP, human neutrophil ␣-defensin; RK, rabbit kidney defensin; PIPES, 1,4-piperazinediethanesulfonic acid; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; MMP, matrix metalloproteinase; TOCSY, total correlation spectroscopy. We present the corrected high resolution solution structure of Crp and the structure of the mutant (E15D)Crp in which the conserved salt bridge has been removed by effectively shortening the side chain of the Glu residue but otherwise making no change to the charge state of the native peptide. The final ␣-defensin canonical feature is the occurrence of Arg and Glu, respectively, at positions 7 and 15 in mouse Crp, which are predicted to form a conserved salt bridge [17]. The role of this salt bridge in HNP2 was investigated by site-directed mutagenesis, which showed that salt bridge disruption or removal did not diminish HNP2 antibacterial activity or HNP2 precursor folding in vitro [17].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
