Structure–function relationships in histidine-rich antimicrobial peptides from Atlantic cod

Abstract

Gad-1 and Gad-2 are antimicrobial peptide (AMP) sequences encoded by paralogous genes. They are rich in histidine, which suggests that their activity might be pH-dependent. We examined their structure-function relationships with a view to learning how to improve AMP therapeutic ratios. Activity assays with Gram-negative bacteria and cancer cell lines demonstrate that Gad-2 is substantially more active at slightly acidic pH than it is at neutral pH. By contrast, the activity of Gad-1 at lower pH is similar to its activity at pH7. Circular dichroism spectra indicate that the greater functional plasticity of Gad-2 correlates with a greater structural plasticity; Gad-2's percent helicity varies dramatically with altered pH and lipid environment. Interestingly, Gad-2's highest levels of helicity do not correspond to the conditions where it is most active. High resolution solution NMR structures were determined in SDS micelles at pH5, conditions that induce an intermediate level of helicity in the peptides. Gad-1 is more helical than Gad-2, with both peptides exhibiting the greatest helical tendencies in their central region and lowest helicity in their N-termini. The high resolution structures suggest that maximum activity relies on the appropriate balance between an N-terminal region with mixed hydrophobic/hydrophilic structure features and an amphipathic central and C-terminal region. Taken together with previous studies, our results suggest that to improve the therapeutic ratio of AMPs, consideration should be given to including sequential histidine-pairs, keeping the overall charge of the peptide modest, and retaining a degree of structural plasticity and imperfect amphipathicity.

pH Dependent Antimicrobial Peptides and Proteins, Their Mechanisms of Action and Potential as Therapeutic Agents

“…In some cases though, histidine, aspartic acid and glutamic acid residues appear to play multiple roles in promoting the activity of their parent antimicrobial molecules. For example, the N-terminal regions of gad-1 and gad-2 include a number of sequential histidine pairs that appear to be important to their ability for lipid targeting and interaction, channel formation and thereby the disruption of microbial membranes at low pH [29,30,94,99,100]. …”

“…Low pH was found to enhance the activity of both peptides against Escherichia coli , which appeared to involve membrane interaction, but interestingly, although gad-1 and gad-2 were predominantly α-helical at neutral pH, acid conditions led to a large decrease in the levels of α-helicity possessed by these peptides [29]. These results contrast with most α-helical AMPs where an enhanced capacity for membranolysis and antimicrobial activity is generally associated with increased levels of this secondary structure [96,97].…”

“…These results contrast with most α-helical AMPs where an enhanced capacity for membranolysis and antimicrobial activity is generally associated with increased levels of this secondary structure [96,97]. It was proposed that gad-1 and gad-2 each possessed a structural plasticity, which facilitated an appropriate balance between amphiphilic and mixed hydrophobic/hydrophilic structural features that promoted maximal levels of membrane interaction and antibacterial activity [29]. Gad-1 and gad-2 were found to be histidine rich AMPs and the enhanced capacity of these peptides for membranolysis and antimicrobial activity at low pH appeared to involve these residues [29].…”

“…It was proposed that gad-1 and gad-2 each possessed a structural plasticity, which facilitated an appropriate balance between amphiphilic and mixed hydrophobic/hydrophilic structural features that promoted maximal levels of membrane interaction and antibacterial activity [29]. Gad-1 and gad-2 were found to be histidine rich AMPs and the enhanced capacity of these peptides for membranolysis and antimicrobial activity at low pH appeared to involve these residues [29]. It is well established that histidine (pKa 6.5) is uncharged at physiological pH but fully positively charged at low pH, thereby enhancing the potential of AMPs for interaction with anionic membranes under acid conditions [46,98].…”

“…It is well established that histidine (pKa 6.5) is uncharged at physiological pH but fully positively charged at low pH, thereby enhancing the potential of AMPs for interaction with anionic membranes under acid conditions [46,98]. However, there also appeared to be a complex relationship between the level of histidine residues possessed by these AMPs and their pH dependent capacity for membrane interaction [29,30]. In response, molecular dynamic simulation studies were undertaken and predicted that the number of sequential histidine pairs contained by gad-1 and gad-2 were important to their ability for membrane disruption [30].…”