Interactions of TonB-dependent transporter FoxA with siderophores and antibiotics that affect binding, uptake, and signal transduction.

The outer membrane of gram-negative bacteria prevents many antibiotics from reaching intracellular targets. However, some antimicrobials can take advantage of iron import transporters to cross this barrier. We showed previously that the thiopeptide antibiotic thiocillin exploits the nocardamine xenosiderophore transporter, FoxA, of the opportunistic pathogen Pseudomonas aeruginosa for uptake. Here, we show that FoxA also transports the xenosiderophore bisucaberin and describe at 2.5 Å resolution the crystal structure of bisucaberin bound to FoxA. Bisucaberin is distinct from other siderophores because it forms a 3:2 rather than 1:1 siderophore-iron complex. Mutations in a single extracellular loop of FoxA differentially affected nocardamine, thiocillin, and bisucaberin binding, uptake, and signal transduction. These results show that in addition to modulating ligand binding, the extracellular loops of siderophore transporters are of fundamental importance for controlling ligand uptake and its regulatory consequences, which have implications for the development of siderophore-antibiotic conjugates to treat difficult infections.

Structural insights into a novel family of integral membrane siderophore reductases.

Gram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent transporters. However, the subsequent route through the inner membrane differs across many bacterial species and siderophore chemistries and is not understood in detail. Here, we report the crystal structure of the inner membrane protein FoxB (from Pseudomonas aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a fold with two tightly bound heme molecules. In combination with in vitro reduction assays and in vivo iron uptake studies, these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fe-siderophore uptake.

Computational models in the service of X-ray and cryo-electron microscopy structure determination.

Critical assessment of structure prediction (CASP) conducts community experiments to determine the state of the art in computing protein structure from amino acid sequence. The process relies on the experimental community providing information about not yet public or about to be solved structures, for use as targets. For some targets, the experimental structure is not solved in time for use in CASP. Calculated structure accuracy improved dramatically in this round, implying that models should now be much more useful for resolving many sorts of experimental difficulties. To test this, selected models for seven unsolved targets were provided to the experimental groups. These models were from the AlphaFold2 group, who overall submitted the most accurate predictions in CASP14. Four targets were solved with the aid of the models, and, additionally, the structure of an already solved target was improved. An a posteriori analysis showed that, in some cases, models from other groups would also be effective. This paper provides accounts of the successful application of models to structure determination, including molecular replacement for X-ray crystallography, backbone tracing and sequence positioning in a cryo-electron microscopy structure, and correction of local features. The results suggest that, in future, there will be greatly increased synergy between computational and experimental approaches to structure determination.

Comparison of lipidic carrier systems for integral membrane proteins - MsbA as case study.

Membrane protein research suffers from the drawback that detergents, which are commonly used to solubilize integral membrane proteins (IMPs), often lead to protein instability and reduced activity. Recently, lipid nanodiscs (NDs) and saposin-lipoprotein particles (Salipro) have emerged as alternative carrier systems that keep membrane proteins in a native-like lipidic solution environment and are suitable for biophysical and structural studies. Here, we systematically compare nanodiscs and Salipros with respect to long-term stability as well as activity and stability of the incorporated membrane protein using the ABC transporter MsbA as model system. Our results show that both systems are suitable for activity measurements as well as structural studies in solution. Based on our results we suggest screening of different lipids with respect to activity and stability of the incorporated IMP before performing structural studies.

A microfluidic flow-focusing device for low sample consumption serial synchrotron crystallography experiments in liquid flow.

Serial synchrotron crystallography allows low X-ray dose, room-temperature crystal structures of proteins to be determined from a population of microcrystals. Protein production and crystallization is a non-trivial procedure and it is essential to have X-ray-compatible sample environments that keep sample consumption low and the crystals in their native environment. This article presents a fast and optimized manufacturing route to metal-polyimide microfluidic flow-focusing devices which allow for the collection of X-ray diffraction data in flow. The flow-focusing conditions allow for sample consumption to be significantly decreased, while also opening up the possibility of more complex experiments such as rapid mixing for time-resolved serial crystallography. This high-repetition-rate experiment allows for full datasets to be obtained quickly (∼1 h) from crystal slurries in liquid flow. The X-ray compatible microfluidic chips are easily manufacturable, reliable and durable and require sample-flow rates on the order of only 30 µl h-1.

Lipid-like Peptides can Stabilize Integral Membrane Proteins for Biophysical and Structural Studies.

A crucial bottleneck in membrane protein structural biology is the difficulty in identifying a detergent that can maintain the stability and functionality of integral membrane proteins (IMPs). Detergents are poor membrane mimics, and their common use in membrane protein crystallography may be one reason for the challenges in obtaining high-resolution crystal structures of many IMP families. Lipid-like peptides (LLPs) have detergent-like properties and have been proposed as alternatives for the solubilization of G protein-coupled receptors and other membrane proteins. Here, we systematically analyzed the stabilizing effect of LLPs on integral membrane proteins of different families. We found that LLPs could significantly stabilize detergent-solubilized IMPs in vitro. This stabilizing effect depended on the chemical nature of the LLP and the intrinsic stability of a particular IMP in the detergent. Our results suggest that screening a subset of LLPs is sufficient to stabilize a particular IMP, which can have a substantial impact on the crystallization and quality of the crystal.

Structural and biophysical analysis of nuclease protein antibiotics.

Protein antibiotics (bacteriocins) are a large and diverse family of multidomain toxins that kill specific Gram-negative bacteria during intraspecies competition for resources. Our understanding of the mechanism of import of such potent toxins has increased significantly in recent years, especially with the reporting of several structures of bacteriocin domains. Less well understood is the structural biochemistry of intact bacteriocins and how these compare across bacterial species. Here, we focus on endonuclease (DNase) bacteriocins that target the genomes of Escherichia coli and Pseudomonas aeruginosa, known as E-type colicins and S-type pyocins, respectively, bound to their specific immunity (Im) proteins. First, we report the 3.2 Šstructure of the DNase colicin ColE9 in complex with its ultra-high affinity Im protein, Im9. In contrast with Im3, which when bound to the ribonuclease domain of the homologous colicin ColE3 makes contact with the translocation (T) domain of the toxin, we find that Im9 makes no such contact and only interactions with the ColE9 cytotoxic domain are observed. Second, we report small-angle X-ray scattering data for two S-type DNase pyocins, S2 and AP41, into which are fitted recently determined X-ray structures for isolated domains. We find that DNase pyocins and colicins are both highly elongated molecules, even though the order of their constituent domains differs. We discuss the implications of these architectural similarities and differences in the context of the translocation mechanism of protein antibiotics through the cell envelope of Gram-negative bacteria.

Discovery, characterization and in vivo activity of pyocin SD2, a protein antibiotic from Pseudomonas aeruginosa.

Increasing rates of antibiotic resistance among Gram-negative pathogens such as Pseudomonas aeruginosa means alternative approaches to antibiotic development are urgently required. Pyocins, produced by P. aeruginosa for intraspecies competition, are highly potent protein antibiotics known to actively translocate across the outer membrane of P. aeruginosa. Understanding and exploiting the mechanisms by which pyocins target, penetrate and kill P. aeruginosa is a promising approach to antibiotic development. In this work we show the therapeutic potential of a newly identified tRNase pyocin, pyocin SD2, by demonstrating its activity in vivo in a murine model of P. aeruginosa lung infection. In addition, we propose a mechanism of cell targeting and translocation for pyocin SD2 across the P. aeruginosa outer membrane. Pyocin SD2 is concentrated at the cell surface, via binding to the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide (LPS), from where it can efficiently locate its outer membrane receptor FpvAI. This strategy of utilizing both the CPA and a protein receptor for cell targeting is common among pyocins as we show that pyocins S2, S5 and SD3 also bind to the CPA. Additional data indicate a key role for an unstructured N-terminal region of pyocin SD2 in the subsequent translocation of the pyocin into the cell. These results greatly improve our understanding of how pyocins target and translocate across the outer membrane of P. aeruginosa. This knowledge could be useful for the development of novel anti-pseudomonal therapeutics and will also support the development of pyocin SD2 as a therapeutic in its own right.

Lectin-like bacteriocins from Pseudomonas spp. utilise D-rhamnose containing lipopolysaccharide as a cellular receptor.

Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of D-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing D-rhamnose and not D-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins.

Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment.

Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

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.

Stopped-flow-time-resolved SAXS for studies of ligand-driven protein dimerization.

Protein function is highly dependent on conformational changes and association or dissociation into numerous oligomeric states. Stopped-flow approaches are suitable for probing transient kinetics in proteins, and combining this approach with small-angle X-ray scattering offers an excellent probe into the structural kinetics of protein function. In this chapter we describe in detail the methodological aspects of our recent investigation of ATP-driven dimerization of nucleotide-binding domains from the bacterial transporter MsbA using stopped-flow small-angle X-ray scattering experiments. Despite extensive studies into the structure and function of MsbA, the structural-temporal insights into the conformational rearrangements and transient intermediates along the pathway in this transporter are missing. In our stopped-flow experiments we observe the rapid formation of a transient protein dimer and subsequent dimer decay over hundreds of seconds. Thus, this approach can be used to detect kinetic parameters associated with conformational changes over a wide range of time-scales for soluble and membrane proteins.

Studying integral membrane protein by SANS using stealth reconstitution systems.

Structural studies of integral membrane proteins (IMPs) are challenging as many of them require a lipid environment for full activity and stability. Reconstitution of IMPs into carrier systems such as nanodiscs or Salipro that mimic the native lipidic environment allow structural studies of membrane proteins in solution. The difficulty with this approach when applied to scattering techniques is the contribution of the carrier system to the scattering intensity and the subsequent challenging data analysis. Recently, so-called stealth carrier systems have been developed and applied to small-angle neutron scattering (SANS) studies of integral membrane proteins that become invisible to neutrons due to specific deuteration and solvent contrast-variation. In this chapter, we describe in detail how the well-studied ATP-binding cassette (ABC) transporter protein MsbA can be reconstituted into stealth nanodiscs and subsequently be studied by SANS. This approach allows for a direct observation of the scattering signal from MsbA without the contribution of the surrounding carrier system and enables detection of different conformational states. The protocols can also be adapted to other stealth carrier systems (such as stealth Salipro).

Cryo-EM structure of MsbA in saposin-lipid nanoparticles (Salipro) provides insights into nucleotide coordination.

The ATP-binding cassette transporter MsbA is a lipid flippase, translocating lipid A, glycolipids, and lipopolysaccharides from the inner to the outer leaflet of the inner membrane of Gram-negative bacteria. It has been used as a model system for time-resolved structural studies as several MsbA structures in different states and reconstitution systems (detergent/nanodiscs/peptidiscs) are available. However, due to the limited resolution of the available structures, detailed structural information on the bound nucleotides has remained elusive. Here, we have reconstituted MsbA in saposin A-lipoprotein nanoparticles (Salipro) and determined the structure of ADP-vanadate-bound MsbA by single-particle cryo-electron microscopy to 3.5 Å resolution. This procedure has resulted in significantly improved resolution and enabled us to model all side chains and visualise detailed ADP-vanadate interactions in the nucleotide-binding domains. The approach may be applicable to other dynamic membrane proteins.

Nocardamine-Dependent Iron Uptake in Pseudomonas aeruginosa: Exclusive Involvement of the FoxA Outer Membrane Transporter.

Iron is a key nutrient for almost all living organisms. Paradoxically, it is poorly soluble and consequently poorly bioavailable. Bacteria have thus developed multiple strategies to access this metal. One of the most common consists of the use of siderophores, small compounds that chelate ferric iron with very high affinity. Many bacteria are able to produce their own siderophores or use those produced by other microorganisms (exosiderophores) in a piracy strategy. Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, and is also able to use a large panel of exosiderophores. We investigated the ability of P. aeruginosa to use nocardamine (NOCA) and ferrioxamine B (DFOB) as exosiderophores under iron-limited planktonic growth conditions. Proteomic and RT-qPCR approaches showed induction of the transcription and expression of the outer membrane transporter FoxA in the presence of NOCA or DFOB in the bacterial environment. Expression of the proteins of the heme- or pyoverdine- and pyochelin-dependent iron uptake pathways was not affected by the presence of these two tris-hydroxamate siderophores. 55Fe uptake assays using foxA mutants showed ferri-NOCA to be exclusively transported by FoxA, whereas ferri-DFOB was transported by FoxA and at least one other unidentified transporter. The crystal structure of FoxA complexed with NOCA-Fe revealed very similar siderophore binding sites between NOCA-Fe and DFOB-Fe. We discuss iron uptake by hydroxamate exosiderophores in P. aeruginosa cells in light of these results.

Structural Kinetics of MsbA Investigated by Stopped-Flow Time-Resolved Small-Angle X-Ray Scattering.

Recent structures of full-length ATP-binding cassette (ABC) transporter MsbA in different states indicate large conformational changes during the reaction cycle that involve transient dimerization of its nucleotide-binding domains (NBDs). However, a detailed molecular understanding of the structural changes and associated kinetics of MsbA upon ATP binding and hydrolysis is still missing. Here, we employed time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing, to investigate the kinetics and accompanying structural changes of NBD dimerization (upon ATP binding) and subsequent dissociation (upon ATP hydrolysis) in the context of isolated NBDs as well as full-length MsbA in lipid nanodiscs. Our data allowed us to structurally characterize the major states involved in the process and determine time constants for NBD dimerization and dissociation. In the full-length protein, these structural transitions occur on much faster time scales, indicating close-proximity effects and structural coupling of the transmembrane domains with the NBDs.

Ternary structure of the outer membrane transporter FoxA with resolved signalling domain provides insights into TonB-mediated siderophore uptake.

Many microbes and fungi acquire the essential ion Fe3+ through the synthesis and secretion of high-affinity chelators termed siderophores. In Gram-negative bacteria, these ferric-siderophore complexes are actively taken up using highly specific TonB-dependent transporters (TBDTs) located in the outer bacterial membrane (OM). However, the detailed mechanism of how the inner-membrane protein TonB connects to the transporters in the OM as well as the interplay between siderophore- and TonB-binding to the transporter is still poorly understood. Here, we present three crystal structures of the TBDT FoxA from Pseudomonas aeruginosa (containing a signalling domain) in complex with the siderophore ferrioxamine B and TonB and combine them with a detailed analysis of binding constants. The structures show that both siderophore and TonB-binding is required to form a translocation-competent state of the FoxA transporter in a two-step TonB-binding mechanism. The complex structure also indicates how TonB-binding influences the orientation of the signalling domain.

High-throughput stability screening for detergent-solubilized membrane proteins.

Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to study the structure of an IMP, detergent solubilization from the membrane is usually the first step in the workflow. Here, we establish a simple, high-throughput screening method to identify optimal detergent conditions for membrane protein stabilization. We apply differential scanning fluorimetry in combination with scattering upon thermal denaturation to study the unfolding of integral membrane proteins. Nine different prokaryotic and eukaryotic membrane proteins were used as test cases to benchmark our detergent screening method. Our results show that it is possible to measure the stability and solubility of IMPs by diluting them from their initial solubilization condition into different detergents. We were able to identify groups of detergents with characteristic stabilization and destabilization effects for selected targets. We further show that fos-choline and PEG family detergents may lead to membrane protein destabilization and unfolding. Finally, we determined thenmodynamic parameters that are important indicators of IMP stability. The described protocol allows the identification of conditions that are suitable for downstream handling of membrane proteins during purification.

Conformation-specific detection of calmodulin binding using the unnatural amino acid p-azido-phenylalanine (AzF) as an IR-sensor.

Calmodulin (CaM) is a very conserved, ubiquitous, eukaryotic protein that binds four Ca2+ ions with high affinity. It acts as a calcium sensor by translating Ca2+ signals into cellular processes such as metabolism, inflammation, immune response, memory, and muscle contraction. Calcium binding to CaM leads to conformational changes that enable Ca2+/CaM to recognize and bind various target proteins with high affinity. The binding mode and binding partners of CaM are very diverse, and a consensus binding sequence is lacking. Here, we describe an elegant system that allows conformation-specific detection of CaM-binding to its binding partners. We incorporate the unnatural amino acid p-azido-phenylalanine (AzF) in different positions of CaM and follow its unique spectral signature by infrared (IR)-spectroscopy of the azido stretching vibration. Our results suggest that the AzF vibrational probe is sensitive to the chemical environment in different CaM/CaM-binding domain (CaMBD) complexes, which allows differentiating between different binding motifs according to the spectral characteristics of the azido stretching mode. We corroborate our results with a crystal structure of AzF-labelled CaM (CaM108AzF) in complex with a binding peptide from calmodulin-dependent protein kinase IIα identifying the structural basis for the observed IR frequency shifts.

Photocage-initiated time-resolved solution X-ray scattering investigation of protein dimerization.

This work demonstrates a new method for investigating time-resolved structural changes in protein conformation and oligomerization via photocage-initiated time-resolved X-ray solution scattering by observing the ATP-driven dimerization of the MsbA nucleotide-binding domain. Photocaged small molecules allow the observation of single-turnover reactions of non-naturally photoactivatable proteins. The kinetics of the reaction can be derived from changes in X-ray scattering associated with ATP-binding and subsequent dimerization. This method can be expanded to any small-molecule-driven protein reaction with conformational changes traceable by X-ray scattering where the small molecule can be photocaged.