Roles of inter- and intramolecular tryptophan interactions in membrane-active proteins revealed by racemic protein crystallography

Abstract

Tryptophan is frequently found on the surface of membrane-associated proteins that interact with the lipid membrane. However, because of their multifaceted interactions, it is difficult to pinpoint the structure-activity relationship of each tryptophan residue. Here, we describe the use of racemic protein crystallography to probe dedicated tryptophan interactions of a model tryptophan-rich bacteriocin aureocin A53 (AucA) by inclusion and/or exclusion of potential ligands. In the presence of tetrahedral anions that are isosteric to the head group of phospholipids, distinct tryptophan H-bond networks were revealed. H-bond donation by W40 was critical for antibacterial activity, as its substitution by 1-methyltryptophan resulted in substantial loss of activity against bacterial clinical isolates. Meanwhile, exclusion of tetrahedral ions revealed that W3 partakes in formation of a dimeric interface, thus suggesting that AucA is dimeric in solution and dissociated to interact with the phosphate head group in the presence of the lipid membrane. Based on these findings, we could predict the tryptophan residue responsible for activity as well as the oligomeric state of a distant homologue lacticin Q (48%).

Optimizing Bothropstoxin-I-Derived Peptides: Exploring the Antibacterial Potential of p-BthW

“…The suggested alteration for this peptide was based on the relationship between tryptophan and antimicrobial activity. This amino acid plays a crucial role in proteins and peptides interacting with membranes. , In addition, several studies show a relationship between this amino acid and increased antimicrobial activity without increasing hemolytic activity. − …”

“…This amino acid plays a crucial role in proteins and peptides interacting with membranes. 13 , 14 In addition, several studies show a relationship between this amino acid and increased antimicrobial activity without increasing hemolytic activity. 14 − 17 …”

“…This amino acid is crucial for proteins and peptides to interact with membranes. 13,14 This simple exchange vastly increased the activity against Gram-negative bacteria, highlighting the importance of the structure−activity relationship.…”

“…An image providing the location of p-BthTX-I in the protein Bothropstoxin-I is provided in Figure , as well as the alignment between the previously studied peptide and the new analogue p-BthW. This amino acid is crucial for proteins and peptides to interact with membranes. , This simple exchange vastly increased the activity against Gram-negative bacteria, highlighting the importance of the structure–activity relationship.…”

“…This amino acid plays a crucial role in proteins and peptides interacting with membranes. 13,14 In addition, several studies show a relationship between this amino acid and increased antimicrobial activity without increasing hemolytic activity. 14−17 The initial assessment of the newly synthesized peptide was its comparison with its previously evaluated analogue for antibacterial activity and cytotoxicity.…”