The role of bacterial transport systems in the removal of host antimicrobial peptides in Gram-negative bacteria.

Antibiotic resistance is a global issue that threatens our progress in healthcare and life expectancy. In recent years, antimicrobial peptides (AMPs) have been considered as promising alternatives to the classic antibiotics. AMPs are potentially superior due to their lower rate of resistance development, since they primarily target the bacterial membrane ('Achilles' heel' of the bacteria). However, bacteria have developed mechanisms of AMP resistance, including the removal of AMPs to the extracellular space by efflux pumps such as the MtrCDE or AcrAB-TolC systems, and the internalization of AMPs to the cytoplasm by the Sap transporter, followed by proteolytic digestion. In this review, we focus on AMP transport as a resistance mechanism compiling all the experimental evidence for the involvement of efflux in AMP resistance in Gram-negative bacteria and combine this information with the analysis of the structures of the efflux systems involved. Finally, we expose some open questions with the aim of arousing the interest of the scientific community towards the AMPs-efflux pumps interactions. All the collected information broadens our understanding of AMP removal by efflux pumps and gives some clues to assist the rational design of AMP-derivatives as inhibitors of the efflux pumps.

Supramolecular lipid nanoparticles as delivery carriers for non-invasive cancer theranostics.

Nanotheranostics is an emerging frontier of personalized medicine research particularly for cancer, which is the second leading cause of death. Supramolecular aspects in theranostics are quite allured to achieve more regulation and controlled features. Supramolecular nanotheranostics architecture is focused on engineering of modular supramolecular assemblies benefitting from their mutable and stimuli-responsive properties which confer an ultimate potential for the fabrication of unified innovative nanomedicines with controlled features. Amalgamation of supramolecular approaches to nano-based features further equip the potential of designing novel approaches to overcome limitations seen by the conventional theranostic strategies, for curing even the lethal diseases and endowing personalized therapeutics with optimistic prognosis, endorsing their clinical translation. Among many potential nanocarriers for theranostics, lipid nanoparticles (LNPs) have shown various promising advances in theranostics and their formulation can be tailored for several applications. Despite the great advancement in cancer nanotheranostics, there are still many challenges that need to be highlighted to fill the literature gap. For this purpose, herein, we have presented a systematic overview on the subject and proposed LNPs as the potential material to manage cancer via non-invasive approaches by highlighting the use of supramolecular approaches to make them robust for cancer theranostics. We have concluded the review by entailing the future perspectives of lipid nanotheranostics towards clinical translation.

Cellular Uptake and Intracellular Phosphorylation of GS-441524: Implications for Its Effectiveness against COVID-19.

GS-441524 is an adenosine analog and the parent nucleoside of the prodrug remdesivir, which has received emergency approval for treatment of COVID-19. Recently, GS-441524 has been proposed to be effective in the treatment of COVID-19, perhaps even being superior to remdesivir for treatment of this disease. Evaluation of the clinical effectiveness of GS-441524 requires understanding of its uptake and intracellular conversion to GS-441524 triphosphate, the active antiviral substance. We here discuss the potential impact of these pharmacokinetic steps of GS-441524 on the formation of its active antiviral substance and effectiveness for treatment of COVID-19. Available protein expression data suggest that several adenosine transporters are expressed at only low levels in the epithelial cells lining the alveoli in the lungs, i.e., the alveolar cells or pneumocytes from healthy lungs. This may limit uptake of GS-441524. Importantly, cellular uptake of GS-441524 may be reduced during hypoxia and inflammation due to decreased expression of adenosine transporters. Similarly, hypoxia and inflammation may lead to reduced expression of adenosine kinase, which is believed to convert GS-441524 to GS-441524 monophosphate, the perceived rate-limiting step in the intracellular formation of GS-441524 triphosphate. Moreover, increases in extracellular and intracellular levels of adenosine, which may occur during critical illnesses, has the potential to competitively decrease cellular uptake and phosphorylation of GS-441524. Taken together, tissue hypoxia and severe inflammation in COVID-19 may lead to reduced uptake and phosphorylation of GS-441524 with lowered therapeutic effectiveness as a potential outcome. Hypoxia may be particularly critical to the ability of GS-441524 to eliminate SARS-CoV-2 from tissues with low basal expression of adenosine transporters, such as alveolar cells. This knowledge may also be relevant to treatments with other antiviral adenosine analogs and anticancer adenosine analogs as well.

Time-Resolved Studies of Ytterbium Distribution at Interfacial Surfaces of Ferritin-like Dps Protein Demonstrate Metal Uptake and Storage Pathways.

Cage-shaped protein (CSP) complexes are frequently used in bionanotechnology, and they have a variety of different architectures and sizes. The smallest cage-shaped protein, Dps (DNA binding protein from starved cells), can naturally form iron oxide biominerals in a multistep process of ion attraction, translocation, oxidation, and nucleation. The structural basis of this biomineralization mechanism is still unclear. The aim of this paper is to further develop understanding of this topic. Time-resolved metal translocation of Yb3+ ions has been investigated on Dps surfaces using X-ray crystallography. The results reveal that the soak time of protein crystals with Yb3+ ions strongly affects metal positions during metal translocation, in particular, around and inside the ion translocation pore. We have trapped a dynamic state with ongoing translocation events and compared this to a static state, which is reached when the cavity of Dps is entirely filled by metal ions and translocation is therefore blocked. By comparison with La3+ and Co2+ datasets, the time-dependence together with the coordination sphere chemistry primarily determine metal-protein interactions. Our data can allow structure-based protein engineering to generate CSPs for the production of tailored nanoparticles.

Structural Plasticity of LL-37 Indicates Elaborate Functional Adaptation Mechanisms to Bacterial Target Structures.

The human cathelicidin LL-37 is a multifunctional peptide of the human innate immune system. Among the various functions of LL-37, its antimicrobial activity is important in controlling the microorganisms of the human body. The target molecules of LL-37 in bacteria include membrane lipids, lipopolysaccharides (LPS), lipoteichoic acid (LTA), proteins, DNA and RNA. In this mini-review, we summarize the entity of LL-37 structural data determined over the last 15 years and specifically discuss features implicated in the interactions with lipid-like molecules. For this purpose, we discuss partial and full-length structures of LL-37 determined in the presence of membrane-mimicking detergents. This constantly growing structural database is now composed of monomers, dimers, tetramers, and fiber-like structures. The diversity of these structures underlines an unexpected plasticity and highlights the conformational and oligomeric adaptability of LL-37 necessary to target different molecular scaffolds. The recent co-crystal structures of LL-37 in complex with detergents are particularly useful to understand how these molecules mimic lipids and LPS to induce oligomerization and fibrillation. Defined detergent binding sites provide deep insights into a new class of peptide scaffolds, widening our view on the fascinating world of the LL-37 structural factotum. Together, the new structures in their evolutionary context allow for the assignment of functionally conserved residues in oligomerization and target interactions. Conserved phenylalanine and arginine residues primarily mediate those interactions with lipids and LPS. The interactions with macromolecules such as proteins or DNA remain largely unexplored and open a field for future studies aimed at structures of LL-37 complexes. These complexes will then allow for the structure-based rational design of LL-37-derived peptides with improved antibiotic properties.

Polymerase delta-interacting protein 38 (PDIP38) modulates the stability and activity of the mitochondrial AAA+ protease CLPXP.

Over a decade ago Polymerase δ interacting protein of 38 kDa (PDIP38) was proposed to play a role in DNA repair. Since this time, both the physiological function and subcellular location of PDIP38 has remained ambiguous and our present understanding of PDIP38 function has been hampered by a lack of detailed biochemical and structural studies. Here we show, that human PDIP38 is directed to the mitochondrion in a membrane potential dependent manner, where it resides in the matrix compartment, together with its partner protein CLPX. Our structural analysis revealed that PDIP38 is composed of two conserved domains separated by an α/ß linker region. The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like ß-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-terminal Zinc binding domain of CLPX. In contrast, the C-terminal (DUF525) domain forms an immunoglobin-like ß-sandwich fold, which contains a highly conserved putative substrate binding pocket. Importantly, PDIP38 modulates the substrate specificity of CLPX and protects CLPX from LONM-mediated degradation, which stabilises the cellular levels of CLPX. Collectively, our findings shed new light on the mechanism and function of mitochondrial PDIP38, demonstrating that PDIP38 is a bona fide adaptor protein for the mitochondrial protease, CLPXP.

The structure of the antimicrobial human cathelicidin LL-37 shows oligomerization and channel formation in the presence of membrane mimics.

The human cathelicidin LL-37 serves a critical role in the innate immune system defending bacterial infections. LL-37 can interact with molecules of the cell wall and perforate cytoplasmic membranes resulting in bacterial cell death. To test the interactions of LL-37 and bacterial cell wall components we crystallized LL-37 in the presence of detergents and obtained the structure of a narrow tetrameric channel with a strongly charged core. The formation of a tetramer was further studied by cross-linking in the presence of detergents and lipids. Using planar lipid membranes a small but defined conductivity of this channel could be demonstrated. Molecular dynamic simulations underline the stability of this channel in membranes and demonstrate pathways for the passage of water molecules. Time lapse studies of E. coli cells treated with LL-37 show membrane discontinuities in the outer membrane followed by cell wall damage and cell death. Collectively, our results open a venue to the understanding of a novel AMP killing mechanism and allows the rational design of LL-37 derivatives with enhanced bactericidal activity.

Insight into the RssB-Mediated Recognition and Delivery of σs to the AAA+ Protease, ClpXP.

In Escherichia coli, SigmaS (σS) is the master regulator of the general stress response. The cellular levels of σS are controlled by transcription, translation and protein stability. The turnover of σS, by the AAA+ protease (ClpXP), is tightly regulated by a dedicated adaptor protein, termed RssB (Regulator of Sigma S protein B)-which is an atypical member of the response regulator (RR) family. Currently however, the molecular mechanism of σS recognition and delivery by RssB is only poorly understood. Here we describe the crystal structures of both RssB domains (RssBN and RssBC) and the SAXS analysis of full-length RssB (both free and in complex with σS). Together with our biochemical analysis we propose a model for the recognition and delivery of σS by this essential adaptor protein. Similar to most bacterial RRs, the N-terminal domain of RssB (RssBN) comprises a typical mixed (ßα)5-fold. Although phosphorylation of RssBN (at Asp58) is essential for high affinity binding of σS, much of the direct binding to σS occurs via the C-terminal effector domain of RssB (RssBC). In contrast to most RRs the effector domain of RssB forms a ß-sandwich fold composed of two sheets surrounded by α-helical protrusions and as such, shares structural homology with serine/threonine phosphatases that exhibit a PPM/PP2C fold. Our biochemical data demonstrate that this domain plays a key role in both substrate interaction and docking to the zinc binding domain (ZBD) of ClpX. We propose that RssB docking to the ZBD of ClpX overlaps with the docking site of another regulator of RssB, the anti-adaptor IraD. Hence, we speculate that docking to ClpX may trigger release of its substrate through activation of a "closed" state (as seen in the RssB-IraD complex), thereby coupling adaptor docking (to ClpX) with substrate release. This competitive docking to RssB would prevent futile interaction of ClpX with the IraD-RssB complex (which lacks a substrate). Finally, substrate recognition by RssB appears to be regulated by a key residue (Arg117) within the α5 helix of the N-terminal domain. Importantly, this residue is not directly involved in σS interaction, as σS binding to the R117A mutant can be restored by phosphorylation. Likewise, R117A retains the ability to interact with and activate ClpX for degradation of σS, both in the presence and absence of acetyl phosphate. Therefore, we propose that this region of RssB (the α5 helix) plays a critical role in driving interaction with σS at a distal site.

Metal Positions and Translocation Pathways of the Dodecameric Ferritin-like Protein Dps.

Iron storage in biology is carried out by cage-shaped proteins of the ferritin superfamily, one of which is the dodecameric protein Dps. In Dps, four distinct steps lead to the formation of metal nanoparticles: attraction of ion-aquo complexes to the protein matrix, passage of these complexes through translocation pores, oxidation of these complexes at ferroxidase centers, and, ultimately, nanoparticle formation. In this study, we investigated Dps from Listeria innocua to structurally characterize these steps for Co2+, Zn2+, and La3+ ions. The structures reveal that differences in their ion coordination chemistry determine alternative metal ion-binding sites on the areas of the surface surrounding the translocation pore that captures nine La3+, three Co2+, or three Zn2+ ions as aquo clusters and passes them on for translocation. Inside these pores, ion-selective conformational changes at key residues occur before a gating residue to actively move ions through the constriction zone. Ions upstream of the Asp130 gate residue are typically hydrated, while ions downstream directly interact with the protein matrix. Inside the cavity, ions move along negatively charged residues to the ferroxidase center, where seven main residues adapt to the three different ions by dynamically changing their conformations. In total, we observed more than 20 metal-binding sites per Dps monomer, which clearly highlights the metal-binding capacity of this protein family. Collectively, our results provide a detailed structural description of the preparative steps for amino acid-assisted biomineralization in Dps proteins, demonstrating unexpected protein matrix plasticity.

Structure and function of the Ts2631 endolysin of Thermus scotoductus phage vB_Tsc2631 with unique N-terminal extension used for peptidoglycan binding.

To escape from hosts after completing their life cycle, bacteriophages often use endolysins, which degrade bacterial peptidoglycan. While mesophilic phages have been extensively studied, their thermophilic counterparts are not well characterized. Here, we present a detailed analysis of the structure and function of Ts2631 endolysin from thermophilic phage vB_Tsc2631, which is a zinc-dependent amidase. The active site of Ts2631 consists of His30, Tyr58, His131 and Cys139, which are involved in Zn2+ coordination and catalysis. We found that the active site residues are necessary for lysis yet not crucial for peptidoglycan binding. To elucidate residues involved in the enzyme interaction with peptidoglycan, we tested single-residue substitution variants and identified Tyr60 and Lys70 as essential residues. Moreover, substitution of Cys80, abrogating disulfide bridge formation, inactivates Ts2631, as do substitutions of His31, Thr32 and Asn85 residues. The endolysin contains a positively charged N-terminal extension of 20 residues that can protrude from the remainder of the enzyme and is crucial for peptidoglycan binding. We show that the deletion of 20 residues from the N-terminus abolished the bacteriolytic activity of the enzyme. Because Ts2631 exhibits intrinsic antibacterial activity and unusual thermal stability, it is perfectly suited as a scaffold for the development of antimicrobial agents.

Ten Years of High Resolution Structural Research on the Voltage Dependent Anion Channel (VDAC)-Recent Developments and Future Directions.

Mitochondria are evolutionarily related to Gram-negative bacteria and both comprise two membrane systems with strongly differing protein composition. The major protein in the outer membrane of mitochondria is the voltage-dependent anion channel (VDAC), which mediates signal transmission across the outer membrane but also the exchange of metabolites, most importantly ADP and ATP. More than 30 years after its discovery three identical high-resolution structures were determined in 2008. These structures show a 19-stranded anti-parallel beta-barrel with an N-terminal helix located inside. An odd number of beta-strands is also shared by Tom40, another member of the VDAC superfamily. This indicates that this superfamily is evolutionarily relatively young and that it has emerged in the context of mitochondrial evolution. New structural information obtained during the last decade on Tom40 can be used to cross-validate the structure of VDAC and vice versa. Interpretation of biochemical and biophysical studies on both protein channels now rests on a solid basis of structural data. Over the past 10 years, complementary structural and functional information on proteins of the VDAC superfamily has been collected from in-organello, in-vitro, and in silico studies. Most of these findings have confirmed the validity of the original structures. This short article briefly reviews the most important advances on the structure and function of VDAC superfamily members collected during the last decade and summarizes how they enhanced our understanding of the channel.

Structural remodeling and oligomerization of human cathelicidin on membranes suggest fibril-like structures as active species.

Antimicrobial peptides as part of the mammalian innate immune system target and remove major bacterial pathogens, often through irreversible damage of their cellular membranes. To explore the mechanism by which the important cathelicidin peptide LL-37 of the human innate immune system interacts with membranes, we performed biochemical, biophysical and structural studies. The crystal structure of LL-37 displays dimers of anti-parallel helices and the formation of amphipathic surfaces. Peptide-detergent interactions introduce remodeling of this structure after occupation of defined hydrophobic sites at the dimer interface. Furthermore, hydrophobic nests are shaped between dimer structures providing another scaffold enclosing detergents. Both scaffolds underline the potential of LL-37 to form defined peptide-lipid complexes in vivo. After adopting the activated peptide conformation LL-37 can polymerize and selectively extract bacterial lipids whereby the membrane is destabilized. The supramolecular fibril-like architectures formed in crystals can be reproduced in a peptide-lipid system after nanogold-labelled LL-37 interacted with lipid vesicles as followed by electron microscopy. We suggest that these supramolecular structures represent the LL-37-membrane active state. Collectively, our study provides new insights into the fascinating plasticity of LL-37 demonstrated at atomic resolution and opens the venue for LL-37-based molecules as novel antibiotics.

The Human Antimicrobial Peptides Dermcidin and LL-37 Show Novel Distinct Pathways in Membrane Interactions.

Mammals protect themselves from inflammation triggered by microorganisms through secretion of antimicrobial peptides (AMPs). One mechanism by which AMPs kill bacterial cells is perforating their membranes. Membrane interactions and pore formation were investigated for α-helical AMPs leading to the formulation of three basic mechanistic models: the barrel stave, toroidal, and carpet model. One major drawback of these models is their simplicity. They do not reflect the real in vitro and in vivo conditions. To challenge and refine these models using a structure-based approach we set out to investigate how human cathelicidin (LL-37) and dermcidin (DCD) interact with membranes. Both peptides are α-helical and their structures have been solved at atomic resolution. DCD assembles in solution into a hexameric pre-channel complex before the actual membrane targeting and integration step can occur, and the complex follows a deviation of the barrel stave model. LL-37 interacts with lipids and shows the formation of oligomers generating fibril-like supramolecular structures on membranes. LL-37 further assembles into transmembrane pores with yet unknown structure expressing a deviation of the toroidal pore model. Both of their specific targeting mechanisms will be discussed in the context of the "old" models propagated in the literature.

Ferroxidase-Mediated Iron Oxide Biomineralization: Novel Pathways to Multifunctional Nanoparticles.

Iron oxide biomineralization occurs in all living organisms and typically involves protein compartments ranging from 5 to 100nm in size. The smallest iron-oxo particles are formed inside dodecameric Dps protein cages, while the structurally related ferritin compartments consist of twice as many identical protein subunits. The largest known compartments are encapsulins, icosahedra made of up to 180 protein subunits that harbor additional ferritin-like proteins in their interior. The formation of iron-oxo particles in all these compartments requires a series of steps including recruitment of iron, translocation, oxidation, nucleation, and storage, that are mediated by ferroxidase centers. Thus, compartmentalized iron oxide biomineralization yields uniform nanoparticles strictly determined by the sizes of the compartments, allowing customization for highly diverse nanotechnological applications.

Structure Determination of the BAM Complex Accessory Lipoproteins BamB-E.

Outer membrane protein biogenesis is a fundamental and essential process in all Gram-negative bacteria. The key players conducting this process are organized in the ß-barrel assembly machinery (BAM) complex. This complex has recently attracted a lot of attention due to its importance in cell wall generation, maintenance, and the fascinating yet partially unknown mechanism. The currently best studied example is the BAM complex from E. coli which comprises five proteins, BamA-BamE, two of which, BamA and BamD, are essential for cell survival. Four of the complex proteins, BamB-BamE, are lipoproteins and are attached to the outer membrane via N-terminal lipid anchors. Two of them, BamB and BamD, comprise protein folds known to mediate protein-protein interactions through WD40 and TPR domains, respectively. Structures of BamB to BamE have been determined using X-ray crystallography, NMR and SAXS techniques. Details on protein preparation, crystallization, data acquisition, and determination of structures are given here along with the brief summary of the currently available structural Bam protein repertoire.

Acidic pH and divalent cation sensing by PhoQ are dispensable for systemic salmonellae virulence.

Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain (PD) that promotes virulence by detecting the macrophage phagosome. PhoQ activity is repressed by divalent cations and induced in environments of acidic pH, limited divalent cations, and cationic antimicrobial peptides (CAMP). Previously, it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence. We defined conformational changes produced in the PhoQ PD on exposure to acidic pH that indicate structural flexibility is induced in α-helices 4 and 5, suggesting this region contributes to pH sensing. Therefore, we engineered a disulfide bond between W104C and A128C in the PhoQ PD that restrains conformational flexibility in α-helices 4 and 5. PhoQ(W104C-A128C) is responsive to CAMP, but is inhibited for activation by acidic pH and divalent cation limitation. phoQ(W104C-A128C) Salmonella enterica Typhimurium is virulent in mice, indicating that acidic pH and divalent cation sensing by PhoQ are dispensable for virulence.

Characterization of the insertase BamA in three different membrane mimetics by solution NMR spectroscopy.

The insertase BamA is the central protein of the Bam complex responsible for outer membrane protein biogenesis in Gram-negative bacteria. BamA features a 16-stranded transmembrane ß-barrel and five periplasmic POTRA domains, with a total molecular weight of 88 kDa. Whereas the structure of BamA has recently been determined by X-ray crystallography, its functional mechanism is not well understood. This mechanism comprises the insertion of substrates from a dynamic, chaperone-bound state into the bacterial outer membrane, and NMR spectroscopy is thus a method of choice for its elucidation. Here, we report solution NMR studies of different BamA constructs in three different membrane mimetic systems: LDAO micelles, DMPC:DiC7PC bicelles and MSP1D1:DMPC nanodiscs. The impact of biochemical parameters on the spectral quality was investigated, including the total protein concentration and the detergent:protein ratio. The barrel of BamA is folded in micelles, bicelles and nanodiscs, but the N-terminal POTRA5 domain is flexibly unfolded in the absence of POTRA4. Measurements of backbone dynamics show that the variable insertion region of BamA, located in the extracellular lid loop L6, features high local flexibility. Our work establishes biochemical preparation schemes for BamA, which will serve as a platform for structural and functional studies of BamA and its role within the Bam complex by solution NMR spectroscopy.

Structure of BamA, an essential factor in outer membrane protein biogenesis.

Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the ß-barrel assembly machinery (BAM) for OMP recognition, folding and assembly. In Escherichia coli this function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of the E. coli BamA ß-barrel and P5 domain was determined at 3 Šresolution. These data add information beyond that provided in the recently published crystal structures of BamA from Haemophilus ducreyi and Neisseria gonorrhoeae and are a valuable basis for the interpretation of pertinent functional studies. In an `open' conformation, E. coli BamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer. E. coli BamA is characterized by a discontinuous ß-barrel with impaired ß1-ß16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer.

Structure of the atypical bacteriocin pectocin M2 implies a novel mechanism of protein uptake.

The colicin-like bacteriocins are potent protein antibiotics that have evolved to efficiently cross the outer membrane of Gram-negative bacteria by parasitizing nutrient uptake systems. We have structurally characterized the colicin M-like bacteriocin, pectocin M2, which is active against strains of Pectobacterium spp. This unusual bacteriocin lacks the intrinsically unstructured translocation domain that usually mediates translocation of these bacteriocins across the outer membrane, containing only a single globular ferredoxin domain connected to its cytotoxic domain by a flexible α-helix, which allows it to adopt two distinct conformations in solution. The ferredoxin domain of pectocin M2 is homologous to plant ferredoxins and allows pectocin M2 to parasitize a system utilized by Pectobacterium to obtain iron during infection of plants. Furthermore, we identify a novel ferredoxin-containing bacteriocin pectocin P, which possesses a cytotoxic domain homologous to lysozyme, illustrating that the ferredoxin domain acts as a generic delivery module for cytotoxic domains in Pectobacterium.