RE Coil: An Antimicrobial Peptide Regulator

Autor: Roscoe Platt, Maxim G. Ryadnov, Galina V. Mukamolova, Ayman Hawrani, James Spencer
Rok vydání: 2009
Předmět:
Zdroj: Angewandte Chemie. 121:9856-9859
ISSN: 0044-8249
DOI: 10.1002/ange.200904780
Popis: Antimicrobial peptides (AMPs) are universal effector molecules of the innate immune system. The peptides provide local antimicrobial responses that target microbial membranes. They kill microorganisms by attacking various components at cellular surfaces and in the cytoplasm through selfpromoted uptake. The peptides can also exhibit immunomodulatory activity and inhibit and “monitor” bacterial growth. However, the specific mechanisms that enable such control over microbial invasion remain unknown. With a growing demand for new treatments that would circumvent bacterial cross-resistance, the elucidation of such mechanisms at the molecular level is of considerable interest in applied chemistry. Herein we propose a molecular rationale for the regulation of antimicrobial intervention on the basis of an a-helical antimicrobial peptide regulator. The antimicrobial activity of AMPs is attributed to their ability to fold upon contact with microbial surfaces. The adopted conformations, a helices or b sheets, appear to play an auxiliary role in affording amphipathic structures—threedimensional shapes with hydrophobic and cationic amino acid residues that form separate clusters. This behavior enables AMPs to assemble within anionic microbial membranes into membrane-disrupting pores or channels. What has yet to be shown is whether the ability of AMPs to assemble and form such structures may be associated with other more specific functions. We hypothesize that AMPs form these structures to regulate antimicrobial activity. To investigate this hypothesis, we designed an antimicrobial regulator in the form of an a-helical system comprising two individually unfolded complementary peptides: an antimicrobial sequence rich in arginine residues (R coil) and its binding partner based on oppositely charged glutamate residues (E coil; Figure 1). The antimicrobial component binds to, and disrupts, microbial membranes, whereas the complementary sequence cannot bind membranes and exhibits no antibacterial effects. Together the two peptides fold into an a-helical coiled coil (RE coil), whereby the antimicrobial component becomes inactivated (Figure 1). To construct the regulator, we adapted original parameters introduced by others for the design of amphipathic and membrane-active sequences and built upon our own experience in engineering coiled-coil systems. The peptide sequences are composed of heptad repeats typical of a helices, PHPPHPP, in which P is polar or small (e.g. alanine) and H is hydrophobic. In both peptides, isoleucine and leucine residues were used in alternate hydrophobic positions to provide the stoichiometric pairing of R coil with E coil. In R coil, the polar sites were created from basic arginine residues and neutral glutamine and alanine residues to give an RI(Q/A)RLR(Q/A) repeat. E coil was constructed from EI(Q/A)(A/Q)LEE repeats to mirror R coil electrostatically, with polar faces formed by acidic glutamate residues and neutral glutamine and alanine residues. Upon binding microbial membranes, R coil folds into an amphipathic a helix (Figure 2a; see also Figure S1 in the Supporting Information). When mixed with E coil, R coil is converted into the membrane-inactive RE coil formed through the interfacial burial of hydrophobic residues and bridging electrostatic interactions (Figure 1, Figure 2b; see also the Supporting Information). To probe the regulator, we first monitored its folding cycle in solution by circular dichroism (CD) spectroscopy. Neither of the individual peptides folded in aqueous buffers at micromolar concentrations (see Figure S2a in the Supporting Information). In the presence of anionic phospholipid vesicles, the composition of which mimics that of bacterial membranes, R coil underwent a coil–helix transition. No Figure 1. Idealized representation of the RE-coil mechanism. I) R coil in a random coil conformation folds into helices upon interacting with membranes. II) E coil is a random coil until it binds R coil with the formation of a coiled coil. III) R coil bound by E coil loses its membrane activity and is removed from the cycle.
Databáze: OpenAIRE
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