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.

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.

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 activity of the N-terminal substrate recognition domains in proteasomal ATPases.

The proteasome forms the core of the protein quality control system in archaea and eukaryotes and also occurs in one bacterial lineage, the Actinobacteria. Access to its proteolytic compartment is controlled by AAA ATPases, whose N-terminal domains (N domains) are thought to mediate substrate recognition. The N domains of an archaeal proteasomal ATPase, Archaeoglobus fulgidus PAN, and of its actinobacterial homolog, Rhodococcus erythropolis ARC, form hexameric rings, whose subunits consist of an N-terminal coiled coil and a C-terminal OB domain. In ARC-N, the OB domains are duplicated and form separate rings. PAN-N and ARC-N can act as chaperones, preventing the aggregation of heterologous proteins in vitro, and this activity is preserved in various chimeras, even when these include coiled coils and OB domains from unrelated proteins. The structures suggest a molecular mechanism for substrate processing based on concerted radial motions of the coiled coils relative to the OB rings.

Exploiting tertiary structure through local folds for crystallographic phasing.

We describe an algorithm for phasing protein crystal X-ray diffraction data that identifies, retrieves, refines and exploits general tertiary structural information from small fragments available in the Protein Data Bank. The algorithm successfully phased, through unspecific molecular replacement combined with density modification, all-helical, mixed alpha-beta, and all-beta protein structures. The method is available as a software implementation: Borges.

Crystal structure and functional mechanism of a human antimicrobial membrane channel.

Multicellular organisms fight bacterial and fungal infections by producing peptide-derived broad-spectrum antibiotics. These host-defense peptides compromise the integrity of microbial cell membranes and thus evade pathways by which bacteria develop rapid antibiotic resistance. Although more than 1,700 host-defense peptides have been identified, the structural and mechanistic basis of their action remains speculative. This impedes the desired rational development of these agents into next-generation antibiotics. We present the X-ray crystal structure as well as solid-state NMR spectroscopy, electrophysiology, and MD simulations of human dermcidin in membranes that reveal the antibiotic mechanism of this major human antimicrobial, found to suppress Staphylococcus aureus growth on the epidermal surface. Dermcidin forms an architecture of high-conductance transmembrane channels, composed of zinc-connected trimers of antiparallel helix pairs. Molecular dynamics simulations elucidate the unusual membrane permeation pathway for ions and show adjustment of the pore to various membranes. Our study unravels the comprehensive mechanism for the membrane-disruptive action of this mammalian host-defense peptide at atomistic level. The results may form a foundation for the structure-based design of peptide antibiotics.

Structural basis and target-specific modulation of ADP sensing by the Synechococcus elongatus PII signaling protein.

PII signaling proteins comprise one of the most versatile signaling devices in nature and have a highly conserved structure. In cyanobacteria, PipX and N-acetyl-L-glutamate kinase are receptors of PII signaling, and these interactions are modulated by ADP, ATP, and 2-oxoglutarate. These effector molecules bind interdependently to three anti-cooperative binding sites on the trimeric PII protein and thereby affect its structure. Here we used the PII protein from Synechococcus elongatus PCC 7942 to reveal the structural basis of anti-cooperative ADP binding. Furthermore, we clarified the mutual influence of PII-receptor interaction and sensing of the ATP/ADP ratio. The crystal structures of two forms of trimeric PII, one with one ADP bound and the other with all three ADP-binding sites occupied, revealed significant differences in the ADP binding mode: at one site (S1) ADP is tightly bound through side-chain and main-chain interactions, whereas at the other two sites (S2 and S3) the ADP molecules are only bound by main-chain interactions. In the presence of the PII-receptor PipX, the affinity of ADP to the first binding site S1 strongly increases, whereas the affinity for ATP decreases due to PipX favoring the S1 conformation of PII-ADP. In consequence, the PII-PipX interaction is highly sensitive to subtle fluctuations in the ATP/ADP ratio. By contrast, the PII-N-acetyl-L-glutamate kinase interaction, which is negatively affected by ADP, is insensitive to these fluctuations. Modulation of the metabolite-sensing properties of PII by its receptors allows PII to differentially perceive signals in a target-specific manner and to perform multitasking signal transduction.

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.

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.

The 3D structures of VDAC represent a native conformation.

The most abundant protein of the mitochondrial outer membrane is the voltage-dependent anion channel (VDAC), which facilitates the exchange of ions and molecules between mitochondria and cytosol and is regulated by interactions with other proteins and small molecules. VDAC has been studied extensively for more than three decades, and last year three independent investigations revealed a structure of VDAC-1 exhibiting 19 transmembrane beta-strands, constituting a unique structural class of beta-barrel membrane proteins. Here, we provide a historical perspective on VDAC research and give an overview of the experimental design used to obtain these structures. Furthermore, we validate the protein refolding approach and summarize the biochemical and biophysical evidence that links the 19-stranded structure to the native form of VDAC.

Axial helix rotation as a mechanism for signal regulation inferred from the crystallographic analysis of the E. coli serine chemoreceptor.

Bacterial chemotaxis receptors are elongated homodimeric coiled-coil bundles, which transduce signals generated in an N-terminal sensor domain across 15-20nm to a conserved C-terminal signaling subdomain. This signal transduction regulates the activity of associated kinases, altering the behavior of the flagellar motor and hence cell motility. Signaling is in turn modulated by selective methylation and demethylation of specific glutamate and glutamine residues in an adaptation subdomain. We have determined the structure of a chimeric protein, consisting of the HAMP domain from Archaeoglobus fulgidus Af1503 and the methyl-accepting domain of Escherichia coli Tsr. It shows a 21nm coiled coil that alternates between two coiled-coil packing modes: canonical knobs-into-holes and complementary x-da, a variant form related to the canonical one by axial rotation of the helices. Comparison of the obtained structure to the Thermotoga maritima chemoreceptor TM1143 reveals that they adopt different axial rotation states in their adaptation subdomains. This conformational change is presumably induced by the upstream HAMP domain and may modulate the affinity of the chemoreceptor to the methylation-demethylation system. The presented findings extend the cogwheel model for signal transmission to chemoreceptors.

Challenging the state of the art in protein structure prediction: Highlights of experimental target structures for the 10th Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10.

For the last two decades, CASP has assessed the state of the art in techniques for protein structure prediction and identified areas which required further development. CASP would not have been possible without the prediction targets provided by the experimental structural biology community. In the latest experiment, CASP10, more than 100 structures were suggested as prediction targets, some of which appeared to be extraordinarily difficult for modeling. In this article, authors of some of the most challenging targets discuss which specific scientific question motivated the experimental structure determination of the target protein, which structural features were especially interesting from a structural or functional perspective, and to what extent these features were correctly reproduced in the predictions submitted to CASP10. Specifically, the following targets will be presented: the acid-gated urea channel, a difficult to predict transmembrane protein from the important human pathogen Helicobacter pylori; the structure of human interleukin (IL)-34, a recently discovered helical cytokine; the structure of a functionally uncharacterized enzyme OrfY from Thermoproteus tenax formed by a gene duplication and a novel fold; an ORFan domain of mimivirus sulfhydryl oxidase R596; the fiber protein gene product 17 from bacteriophage T7; the bacteriophage CBA-120 tailspike protein; a virus coat protein from metagenomic samples of the marine environment; and finally, an unprecedented class of structure prediction targets based on engineered disulfide-rich small proteins.

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 and function of the PorB porin from disseminating Neisseria gonorrhoeae.

The outer membrane of Gram-negative bacteria contains a large number of channel-forming proteins, porins, for the uptake of small nutrient molecules. Neisseria gonorrhoeae PorBIA (PorB of serotype A) are associated with disseminating diseases and mediate a rapid bacterial invasion into host cells in a phosphate-sensitive manner. To gain insights into this structure-function relationship we analysed PorBIA by X-ray crystallography in the presence of phosphate and ATP. The structure of PorBIA in the complex solved at a resolution of 3.3 Å (1 Å=0.1 nm) displays a surplus of positive charges inside the channel. ATP ligand-binding in the channel is co-ordinated by the positively charged residues of the channel interior. These residues ligate the aromatic, sugar and pyrophosphate moieties of the ligand. Two phosphate ions were observed in the structure, one of which clamped by two arginine residues (Arg92 and Arg124) localized at the extraplasmic channel exit. A short ß-bulge in ß2-strand together with the long L3 loop narrow the barrel diameter significantly and further support substrate specificity through hydrogen bond interactions. Interestingly the structure also comprised a small peptide as a remnant of a periplasmic protein which physically links porin molecules to the peptidoglycan network. To test the importance of Arg92 on bacterial invasion the residue was mutated. In vivo assays of bacteria carrying a R92S mutation confirmed the importance of this residue for host-cell invasion. Furthermore systematic sequence and structure comparisons of PorBIA from Neisseriaceae indicated Arg92 to be unique in disseminating N. gonorrhoeae thereby possibly distinguishing invasion-promoting porins from other neisserial porins.

Structural and mechanistic studies of pesticin, a bacterial homolog of phage lysozymes.

Yersinia pestis produces and secretes a toxin named pesticin that kills related bacteria of the same niche. Uptake of the bacteriocin is required for activity in the periplasm leading to hydrolysis of peptidoglycan. To understand the uptake mechanism and to investigate the function of pesticin, we combined crystal structures of the wild type enzyme, active site mutants, and a chimera protein with in vivo and in vitro activity assays. Wild type pesticin comprises an elongated N-terminal translocation domain, the intermediate receptor binding domain, and a C-terminal activity domain with structural analogy to lysozyme homologs. The full-length protein is toxic to bacteria when taken up to the target site via the outer or the inner membrane. Uptake studies of deletion mutants in the translocation domain demonstrate their critical size for import. To further test the plasticity of pesticin during uptake into bacterial cells, the activity domain was replaced by T4 lysozyme. Surprisingly, this replacement resulted in an active chimera protein that is not inhibited by the immunity protein Pim. Activity of pesticin and the chimera protein was blocked through introduction of disulfide bonds, which suggests unfolding as the prerequisite to gain access to the periplasm. Pesticin, a muramidase, was characterized by active site mutations demonstrating a similar but not identical residue pattern in comparison with T4 lysozyme.

Dps biomineralizing proteins: multifunctional architects of nature.

Dps proteins are the structural relatives of bacterioferritins and ferritins ubiquitously present in the bacterial and archaeal kingdoms. The ball-shaped enzymes play important roles in the detoxification of ROS (reactive oxygen species), in iron scavenging to prevent Fenton reactions and in the mechanical protection of DNA. Detoxification of ROS and iron chaperoning represent the most archetypical functions of dodecameric Dps enzymes. Recent crystallographic studies of these dodecameric complexes have unravelled species-dependent mechanisms of iron uptake into the hollow spheres. Subsequent functions in iron oxidation at ferroxidase centres are highly conserved among bacteria. Final nucleation of iron as iron oxide nanoparticles has been demonstrated to originate at acidic residues located on the inner surface. Some Dps enzymes are also implicated in newly observed catalytic functions related to the formation of molecules playing roles in bacterium-host cell communication. Most recently, Dps complexes are attracting attention in semiconductor science as biomimetic tools for the technical production of the smallest metal-based quantum nanodots used in nanotechnological approaches, such as memory storage or solar cell development.

Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease.

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC-ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.

Mechanism of 2-oxoglutarate signaling by the Synechococcus elongatus PII signal transduction protein.

P(II) proteins control key processes of nitrogen metabolism in bacteria, archaea, and plants in response to the central metabolites ATP, ADP, and 2-oxoglutarate (2-OG), signaling cellular energy and carbon and nitrogen abundance. This metabolic information is integrated by P(II) and transmitted to regulatory targets (key enzymes, transporters, and transcription factors), modulating their activity. In oxygenic phototrophs, the controlling enzyme of arginine synthesis, N-acetyl-glutamate kinase (NAGK), is a major P(II) target, whose activity responds to 2-OG via P(II). Here we show structures of the Synechococcus elongatus P(II) protein in complex with ATP, Mg(2+), and 2-OG, which clarify how 2-OG affects P(II)-NAGK interaction. P(II) trimers with all three sites fully occupied were obtained as well as structures with one or two 2-OG molecules per P(II) trimer. These structures identify the site of 2-OG located in the vicinity between the subunit clefts and the base of the T loop. The 2-OG is bound to a Mg(2+) ion, which is coordinated by three phosphates of ATP, and by ionic interactions with the highly conserved residues K58 and Q39 together with B- and T-loop backbone interactions. These interactions impose a unique T-loop conformation that affects the interactions with the P(II) target. Structures of P(II) trimers with one or two bound 2-OG molecules reveal the basis for anticooperative 2-OG binding and shed light on the intersubunit signaling mechanism by which P(II) senses effectors in a wide range of concentrations.

The crystal structure of the dimeric colicin M immunity protein displays a 3D domain swap.

Bacteriocins are proteins secreted by many bacterial cells to kill related bacteria of the same niche. To avoid their own suicide through reuptake of secreted bacteriocins, these bacteria protect themselves by co-expression of immunity proteins in the compartment of colicin destination. In Escherichia coli the colicin M (Cma) is inactivated by the interaction with the Cma immunity protein (Cmi). We have crystallized and solved the structure of Cmi at a resolution of 1.95Å by the recently developed ab initio phasing program ARCIMBOLDO. The monomeric structure of the mature 10kDa protein comprises a long N-terminal α-helix and a four-stranded C-terminal ß-sheet. Dimerization of this fold is mediated by an extended interface of hydrogen bond interactions between the α-helix and the four-stranded ß-sheet of the symmetry related molecule. Two intermolecular disulfide bridges covalently connect this dimer to further lock this complex. The Cmi protein resembles an example of a 3D domain swapping being stalled through physical linkage. The dimer is a highly charged complex with a significant surplus of negative charges presumably responsible for interactions with Cma. Dimerization of Cmi was also demonstrated to occur in vivo. Although the Cmi-Cma complex is unique among bacteria, the general fold of Cmi is representative for a class of YebF-like proteins which are known to be secreted into the external medium by some Gram-negative bacteria.

Structural basis of outer membrane protein biogenesis in bacteria.

In Escherichia coli, a multicomponent BAM (ß-barrel assembly machinery) complex is responsible for recognition and assembly of outer membrane ß-barrel proteins. The functionality of BAM in protein biogenesis is mainly orchestrated through the presence of two essential components, BamA and BamD. Here, we present crystal structures of four lipoproteins (BamB-E). Monomeric BamB and BamD proteins display scaffold architectures typically implied in transient protein interactions. BamB is a ß-propeller protein comprising eight WD40 repeats. BamD shows an elongated fold on the basis of five tetratricopeptide repeats, three of which form the scaffold for protein recognition. The rod-shaped BamC protein has evolved through the gene duplication of two conserved domains known to mediate protein interactions in structurally related complexes. By contrast, the dimeric BamE is formed through a domain swap and indicates fold similarity to the ß-lactamase inhibitor protein family, possibly integrating cell wall stability in BAM function. Structural and biochemical data show evidence for the specific recognition of amphipathic sequences through the tetratricopeptide repeat architecture of BamD. Collectively, our data advance the understanding of the BAM complex and highlight the functional importance of BamD in amphipathic outer membrane ß-barrel protein motif recognition and protein delivery.