Beyond NMR spectra of antimicrobial peptides: dynamical images at atomic resolution and functional insights

Abstract

There is a considerable current interest in understanding the function of antimicrobial peptides for the development of potent novel antibiotic compounds with a very high selectivity. Since their interaction with the cell membrane is the major driving force for their function, solid-state NMR spectroscopy is the unique method of choice to study these insoluble, non-crystalline, membrane-peptide complexes. Here I discuss solid-state NMR studies of antimicrobial peptides that have reported high-resolution structure, dynamics, orientation, and oligomeric states of antimicrobial peptides in a membrane environment, and also address important questions about the mechanism of action at atomic-level resolution. Increasing number of solid-state NMR applications to antimicrobial peptides are expected in the near future, as these compounds are promising candidates to overcome ever-increasing antibiotic resistance problem and are well suited for the development and applications of solid-state NMR techniques.

Figure 1. Biological pathways representing biochemical and functional properties of cationic antimicrobial peptides

Understanding the events in the mechanism of an AMP is a complex problem and a major challenge to existing biophysical and biochemical tools. There are a number features worth mentioning. Step 1 could be the key in escaping from enzymatic degradation and it depends on the concentration of peptide and ions, and pH. Fibril formation of an AMP, reported for LL-37, could be important in the stabilization of AMPs so that they can be available when required to fight against infection. Steps 2 and 3 could be considerably different. Step 2 involves a combination of the formation of a minimum energy amphipathic structure, enthalpy-driven membrane binding and folding processes. On the other hand, step 3 is a combination of refolding of the oligomeric structure, enthalpy-driven binding and folding processes. In addition, steps 2, 3 and 4 highly depend on the membrane composition. AMPs could cause cell death by any one of the mechanisms and/or by just gently permeabilizing to alter the voltage across the membrane. There are many exciting questions still need to be addressed. What roles fibrils play in the function of an AMP? Are fibrils of an AMP toxic? Are there any similarities between AMP fibrils and amyloid fibrils? Are the size and structure of AMP oligomers in solution and membrane different? To what extent the oligomeric size, structure, orientation, and depth of insertion in membrane depend on the membrane composition, peptide concentration and temperature? While the structure and organization of lipids vary, how different are the various types of membrane disrupting mechanisms energetically? Are some of these structures kinetically trapped? What causes one type of mechanism to change to another for a given peptide? It is well established that the conformation, dynamics, and phase of lipids depend on the temperature. Does freezing the model membrane containing an AMP for characterization using solid-state NMR experiments alter the peptide structure, peptide-peptide interactions, peptide-lipid interaction, peptide-water-lipid interactions, membrane orientation, oligomeric size, pore geometry, etc. How does an AMP pass through the outer membrane of a Gram-negative bacteria? What type of interactions and structures expected when an AMP enters the cell wall? How similar/different are the structural measurements obtained from model membranes and living cells? These questions are difficult to be addressed satisfactorily as the amino acid composition of an AMP also plays a role and complicates the efforts in seeking for a general answer. Nevertheless, solid-state NMR spectroscopy is the only technique that can provide accurate answers to most of these questions, and is highly valuable in providing insights at each and every stage of this biochemical pathway as mentioned in the text. In addition, solid-state NMR measurements combined with results from calorimetric experiments, fluorescence experiments, and molecular dynamics simulations could be more valuable to satisfactorily address this complex biological problem. It should be noted that some AMPs have been shown to function by interacting with receptor proteins in membranes or by permeating the cell membrane and interfering with RNA function, which are not covered in this review.

Figure 2. A summary of advantages and concerns of various NMR methods and model membranes used to study antimicrobial peptides

A combination of different types of NMR approaches and suitably selected model membranes can address biological questions with regard to the function of AMPs as discussed in the text.