Alkaloids of anuran skin: antimicrobial function?

A variety of alkaloids, most of which occur or are structurally related to alkaloids that occur in skin glands of dendrobatid poison frogs, were assayed for antimicrobial activity against the Gram-positive bacterium Bacillus subtilis, the Gram-negative bacterium Escherichia coli and the fungus Candida albicans. Certain pyrrolidines, piperidines and decahydroquinolines, perhydro-histrionicotoxin, and a synthetic pumiliotoxin were active against B. subtilis. Only 2-n-nonylpiperidine was active against E. coli. One pyrrolidine, two piperidines, two decahydroquinolines, and the synthetic pumiliotoxin were active against the fungus C. albicans. The results suggest that certain of the skin alkaloids of poison frogs, in addition to being noxious to predators, may also benefit the frog through protection against skin infections.     

Solution NMR studies of Aβ monomer dynamics

Aβ is widely recognized as a key molecule in Alzheimer's disease, causing neurotoxicity through Aβ aggregates such as Aβ oligomers and fibrils. Aβ40 and Aβ42, composed of 40 and 42 residues, respectively, are the major Aβ species in human brain. Aβ42 aggregates much faster than Aβ40 but the mechanism of such difference in aggregation propensity is poorly understood. Using NMR spin relaxation, we have shown that Aβ40 and Aβ42 monomers have different dynamics in both backbone and sidechain on the ps-ns time scale. Aβ42 is more rigid in C-terminus in both backbone and sidechain while Aβ40 has more rigid methyl groups in the central hydrophobic cluster (CHC: Aβ17-21). These observations are consistent with differences in the major conformations of Aβ40 and Aβ42 monomers derived from replica exchange MD (REMD). To further demonstrate the relevance of dynamics in aggregation mechanism, a perturbation was introduced to Aβ42 in the form of M35 oxidation. After M35 side chain oxidation to sulfoxide, Aβ42 experiences Aβ40-like changes in dynamics. At the same time, M35 oxidation causes dramatic reduction in Aβ42 aggregation rate. These data have thus established an important role for protein dynamics in the mechanism of Aβ aggregation.     

Solution NMR studies of polytopic α-helical membrane proteins

NMR spectroscopy has established itself as one of the main techniques for the structural study of integral membrane proteins. Remarkably, over the last few years, substantial progress has been achieved in the structure determination of increasingly complex polytopical α-helical membrane proteins, with their size approaching ～100kDa. Such advances are the result of significant improvements in NMR methodology, sample preparation and powerful selective isotope labelling schemes. We review the requirements facilitating such work based on the more recent solution NMR studies of α-helical proteins. While the majority of such studies still use detergent-solubilized proteins, alternative more native-like lipid-based media are emerging. Recent interaction, dynamics and conformational studies are discussed that cast a promising light on the future role of NMR in this important and exciting area.     

Linking microbial community structure to β-glucosidic function in soil aggregates

To link microbial community 16S structure to a measured function in a natural soil, we have scaled both DNA and β-glucosidase assays down to a volume of soil that may approach a unique microbial community. β-Glucosidase activity was assayed in 450 individual aggregates, which were then sorted into classes of high or low activities, from which groups of 10 or 11 aggregates were identified and grouped for DNA extraction and pyrosequencing. Tandem assays of ATP were conducted for each aggregate in order to normalize these small groups of aggregates for biomass size. In spite of there being no significant differences in the richness or diversity of the microbial communities associated with high β-glucosidase activities compared with the communities associated with low β-glucosidase communities, several analyses of variance clearly show that the communities of these two groups differ. The separation of these groups is partially driven by the differential abundances of members of the Chitinophagaceae family. It may be observed that functional differences in otherwise similar soil aggregates can be largely attributed to differences in resource availability, rather than to the presence or absence of particular taxonomic groups.     

Cell-penetrating peptides and antimicrobial peptides: how different are they?

Some cationic peptides, referred to as CPPs (cell-penetrating peptides), have the ability to translocate across biological membranes in a non-disruptive way and to overcome the impermeable nature of the cell membrane. They have been successfully used for drug delivery into mammalian cells; however, there is no consensus about the mechanism of cellular uptake. Both endocytic and non-endocytic pathways are supported by experimental evidence. The observation that some AMPs (antimicrobial peptides) can enter host cells without damaging their cytoplasmic membrane, as well as kill pathogenic agents, has also attracted attention. The capacity to translocate across the cell membrane has been reported for some of these AMPs. Like CPPs, AMPs are short and cationic sequences with a high affinity for membranes. Similarities between CPPs and AMPs prompted us to question if these two classes of peptides really belong to unrelated families. In this Review, a critical comparison of the mechanisms that underlie cellular uptake is undertaken. A reflection and a new perspective about CPPs and AMPs are presented.     

Acetylcholinesterase: how is structure related to function?

In accordance with its biological role, termination of neurotransmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine, acetylcholinesterase is one of nature's most efficient enzymes. Solution of its three-dimensional structure revealed that its active site is located at the bottom of a deep and narrow gorge. Such an architecture was unanticipated in view of its high turnover number. The present review examines how the highly specialized structure of acetylcholinesterase, with its sequestered active site, contributes to its catalytic efficacy, and discusses how the traffic of substrate and products to and from the active site is controlled.     

Role of helicity of α-helical antimicrobial peptides to improve specificity

A major barrier to the use of antimicrobial peptides as antibiotics is the toxicity or ability to lyse eukaryotic cells. In this study, a 26-residue amphipathic α-helical antimicrobial peptide A12L/A20L (Ac-KWKSFLKTFKSLK KTVLHTLLKAISS-amide) was used as the framework to design a series of D- and L-diastereomeric peptides and study the relationships of helicity and biological activities of α-helical antimicrobial peptides. Peptide helicity was measured by circular dichroism spectroscopy and demonstrated to correlate with the hydrophobicity of peptides and the numbers of D-amino acid substitutions. Therapeutic index was used to evaluate the selectivity of peptides against prokaryotic cells. By introducing D-amino acids to replace the original L-amino acids on the non-polar face or the polar face of the helix, the hemolytic activity of peptide analogs have been significantly reduced. Compared to the parent peptide, the therapeutic indices were improved of 44-fold and 22-fold against Gram-negative and Grampositive bacteria, respectively. In addition, D- and L-diastereomeric peptides exhibited lower interaction with zwitterionic eukaryotic membrane and showed the significant membrane damaging effect to bacterial cells. Helicity was proved to play a crucial role on peptide specificity and biological activities. By simply replacing the hydrophobic or the hydrophilic amino acid residues on the non-polar or the polar face of these amphipathic derivatives of the parent peptide with D-amino acids, we demonstrated that this method could have excellent potential for the rational design of antimicrobial peptides with enhanced specificity.     

From protein structure to biochemical function?

Here we describe various methods currently under development aimed at identifying a proteins function from its three-dimensional structure. We are combining a number of these methods to create a pipeline of applications, called ProFunc, which will take a given 3D structure, run all the applications on it and compile and summarise the results obtained. The aim is to provide a best guess as to the proteins function from the evidence provided by the different methods. Here we present three examples, using structures solved by the Midwest Center for Structural Genomics consortium, illustrating the strengths and weaknesses of current approaches.     

Cationic membrane peptides: atomic-level insight of structure–activity relationships from solid-state NMR

Many membrane-active peptides, such as cationic cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs), conduct their biological functions by interacting with the cell membrane. The interactions of charged residues with lipids and water facilitate membrane insertion, translocation or disruption of these highly hydrophobic species. In this review, we will summarize high-resolution structural and dynamic findings towards the understanding of the structure–activity relationship of lipid membrane-bound CPPs and AMPs, as examples of the current development of solid-state NMR (SSNMR) techniques for studying membrane peptides. We will present the most recent atomic-resolution structure of the guanidinium-phosphate complex, as constrained from experimentally measured site-specific distances. These SSNMR results will be valuable specifically for understanding the intracellular translocation pathway of CPPs and antimicrobial mechanism of AMPs, and more generally broaden our insight into how cationic macromolecules interact with and cross the lipid membrane.     

Complete backbone and DENQ side chain NMR assignments in proteins from a single experiment: implications to structure–function studies

Resonance assignment is the first and the most crucial step in all nuclear magnetic resonance (NMR) investigations on structure–function relationships in biological macromolecules. Often, the assignment exercise has to be repeated several times when specific interactions with ligands, substrates etc., have to be elucidated for understanding the functional mechanisms. While the protein backbone serves to provide a scaffold, the side chains interact directly with the ligands. Such investigations will be greatly facilitated, if there are rapid methods for obtaining exhaustive information with minimum of NMR experimentation. In this context, we present here a pulse sequence which exploits the recently introduced technique of parallel detection of multiple nuclei, e.g. ¹H and ¹³C, and results in two 3D-data sets simultaneously. These yield complete backbone resonance assignment (¹H^N, ¹⁵N, ¹³CO, ¹Hα/¹³Cα, and ¹Hβ/¹³Cβ chemical shifts) and side chain assignment of D, E, N and Q residues. Such an exhaustive assignment has the potential of yielding accurate 3D structures using one or more of several algorithms which calculate structures of the molecules very reliably on the basis of NMR chemical shifts alone. The side chain assignments of D, E, N, and Q will be extremely valuable for interaction studies with different ligands; D and E side chains are known to be involved in majority of catalytic activities. Utility of this experiment has been demonstrated with Ca²⁺ bound M-crystallin, which contains largely D, E, N and Q residues at the metal binding sites.     

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

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 pH 7. 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 pH 5, 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.     

How do bacteria resist human antimicrobial peptides?

Cationic antimicrobial peptides (CAMPs), such as defensins, cathelicidins and thrombocidins, are an important human defense mechanism, protecting skin and epithelia against invading microorganisms and assisting neutrophils and platelets. Staphylococcus aureus, Salmonella enterica and other bacterial pathogens have evolved countermeasures to limit the effectiveness of CAMPs, including the repulsion of CAMPs by reducing the net negative charge of the bacterial cell envelope through covalent modification of anionic molecules (e.g. teichoic acids, phospholipids and lipid A); expelling CAMPs through energy-dependent pumps; altering membrane fluidity; and cleaving CAMPs with proteases. Mutants susceptible to CAMPs are more efficiently inactivated by phagocytes and are virulence-attenuated, indicating that CAMP resistance plays a key role in bacterial infections.     

How reliable are molecular dynamics simulations of membrane active antimicrobial peptides?

Membrane-active antimicrobial peptides (AMPs) are challenging to study experimentally, but relatively easy to investigate using molecular dynamics (MD) computer simulations. For this reason, a large number of MD studies of AMPs have been reported over recent years. Yet relatively little effort has focused on the validity of such simulations. Are these results reliable, and do they agree with what is known experimentally? And how much meaningful information can be obtained? To answer these questions, we demonstrate here some of the requirements and limitations of running MD simulations for several common AMPs: PGLa, melittin, maculatin and BP100. The two most important findings are: (a) simulation results depend strongly on force field parameters, making experimental verification of the simulations obligatory, and (b) slow orientational and conformational fluctuations mean that much longer sampling timescales (multi-μs) are needed if quantitative agreement between simulation averages and experimental data is to be achieved. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Membrane-disruptive abilities of β-hairpin antimicrobial peptides correlate with conformation and activity: A P and H NMR study

The membrane interaction and solution conformation of two mutants of the β-hairpin antimicrobial peptide, protegrin-1 (PG-1), are investigated to understand the structural determinants of antimicrobial potency. One mutant, [A_6,8,13,15] PG-1, does not have the two disulfide bonds in wild-type PG-1, while the other, [Δ_4,18 G₁₀] PG-1, has only half the number of cationic residues. ³¹P solid-state NMR lineshapes of uniaxially aligned membranes indicate that the membrane disorder induced by the three peptides decreases in the order of PG-1>[Δ_4,18 G₁₀] PG-1?[A_6,8,13,15] PG-1. Solution NMR studies of the two mutant peptides indicate that [Δ_4,18 G₁₀] PG-1 preserves the β-hairpin fold of the wild-type peptide while [A_6,8,13,15] PG-1 adopts a random coil conformation. These NMR results correlate well with the known activities of these peptides. Thus, for this class of peptides, the presence of a β-hairpin fold is more essential than the number of cationic charges for antimicrobial activity. This study indicates that ³¹P NMR lineshapes of uniaxially aligned membranes are well correlated with antimicrobial activity, and can be used as a diagnostic tool to understand the peptide-lipid interactions of these antimicrobial peptides.     

Binding of amphipathic α-helical antimicrobial peptides to lipid membranes: Lessons from temporins B and L

Temporins constitute a family of amphipathic α-helical antimicrobial peptides (AMP) and contain some of the shortest cytotoxic peptides, comprised of only 10-14 residues. General characteristics of temporins parallel those of other AMP, both in terms of structural features and biophysical properties relating to their interactions with membrane lipids, with selective lipid-binding properties believed to underlie the discrimination between target vs host cells. Lipid-binding properties also contribute to the cytotoxicity AMP, causing permeabilization of their target cell membranes. The latter functional property of AMP involves highly interdependent acidic phospholipid-induced conformational changes, aggregation, and formation of toxic oligomers in the membrane. These oligomers are subsequently converted to amyloid-type fibers, as demonstrated for e.g. temporins B and L in our laboratory, and more recently for dermaseptins by Auvynet et al. Amyloid state represents the generic minimum in the folding/aggregation free energy landscape, and for AMP its formation most likely serves to detoxify the peptides, in keeping with the current consensus on mature amyloid being inert and non-toxic. The above scenario is supported by sequence analyses of temporins as well as other amphipathic α-helical AMP belonging to diverse families. Accordingly, sequence comparison identifies ‘conformational switches’, domains with equal probabilities for adopting random coil, α-helical and β-sheet structures. These regions were further predicted also to aggregate and assemble into amyloid β-sheets. Taken together, the lipid-binding properties and structural characterization lend support to the notion that the mechanism of membrane permeabilization by temporins B and L and perhaps of most AMP could be very similar, if not identical, to that of the paradigm amyloid forming cytotoxic peptides, responsible for degenerative cell loss in e.g. prion, Alzheimer's and Parkinson's disease, and type 2 diabetes.     

The antihypertensive effect of peptides: a novel alternative to drugs?

Many types of bioactive peptides that inhibit angiotensin I, angiotensin I converting enzyme (ACE) and Ang II type 1 receptor (AT1) in the cardiovascular system contribute to the prevention and treatment of hypertension. These inhibitory peptides are derived from many food proteins or artificial synthetic products. Further research examining the bioavailability of ACE inhibitory peptides will lead to the development of more effective ACE inhibitory peptides and foods. Our research also demonstrates that ACE inhibitory peptide LAP may lower blood pressure with no adverse effects.     

Molecular dynamics simulations of helical antimicrobial peptides in SDS micelles: what do point mutations achieve?

We report long time scale simulations of the 18-residue helical antimicrobial peptide ovispirin-1 and its analogs novispirin-G10 and novispirin-T7 in SDS micelles. The SDS micelle serves as an economical and effective model for a cellular membrane. Ovispirin, which is initially placed along a micelle diameter, diffuses out to the water-SDS interface and stabilizes to an interface-bound steady state in 16.35 ns of simulation. The final conformation, orientation, and the structure of ovispirin are in good agreement with the experimentally observed properties of the peptide in presence of lipid bilayers. The simulation succeeds in capturing subtle differences of the membrane-bound peptide structure as predicted by solid state NMR. The novispirins also undergo identical diffusion patterns and similar final conformations. Although the final interface-bound states are similar, the simulations illuminate the structural and binding properties of the mutant peptides which make them less toxic compared to ovispirin. Based on previous data and the current simulations, we propose that introduction of a bend/hinge at the center of helical antimicrobial peptides (containing a specific C-terminal motif), without disrupting the helicity of the peptides might attenuate host-cell toxicity as well as improve membrane binding properties to bacterial cellular envelopes.