Interaction Mode of the Novel Monobactam AIC499 Targeting Penicillin Binding Protein 3 of Gram-Negative Bacteria.

Novel antimicrobial strategies are urgently required because of the rising threat of multi drug resistant bacterial strains and the infections caused by them. Among the available target structures, the so-called penicillin binding proteins are of particular interest, owing to their good accessibility in the periplasmic space, and the lack of homologous proteins in humans, reducing the risk of side effects of potential drugs. In this report, we focus on the interaction of the innovative ß-lactam antibiotic AIC499 with penicillin binding protein 3 (PBP3) from Escherichia coli and Pseudomonas aeruginosa. This recently developed monobactam displays broad antimicrobial activity, against Gram-negative strains, and improved resistance to most classes of ß-lactamases. By analyzing crystal structures of the respective complexes, we were able to explore the binding mode of AIC499 to its target proteins. In addition, the apo structures determined for PBP3, from P. aeruginosa and the catalytic transpeptidase domain of the E. coli orthologue, provide new insights into the dynamics of these proteins and the impact of drug binding.

Discovery of Pyrrolidine-2,3-diones as Novel Inhibitors of P. aeruginosa PBP3.

The alarming threat of the spread of multidrug resistant bacteria currently leaves clinicians with very limited options to combat infections, especially those from Gram-negative bacteria. Hence, innovative strategies to deliver the next generation of antibacterials are urgently needed. Penicillin binding proteins (PBPs) are proven targets inhibited by ß-lactam antibiotics. To discover novel, non-ß-lactam inhibitors against PBP3 of Pseudomonas aeruginosa, we optimised a fluorescence assay based on a well-known thioester artificial substrate and performed a target screening using a focused protease-targeted library of 2455 compounds, which led to the identification of pyrrolidine-2,3-dione as a potential scaffold to inhibit the PBP3 target. Further chemical optimisation using a one-pot three-component reaction protocol delivered compounds with excellent target inhibition, initial antibacterial activities against P. aeruginosa and no apparent cytotoxicity. Our investigation revealed the key structural features; for instance, 3-hydroxyl group (R2) and a heteroaryl group (R1) appended to the N-pyrroldine-2,3-dione via methylene linker required for target inhibition. Overall, the discovery of the pyrrolidine-2,3-dione class of inhibitors of PBP3 brings opportunities to target multidrug-resistant bacterial strains and calls for further optimisation to improve antibacterial activity against P. aeruginosa.

Correction: Freischem et al. Interaction Mode of the Novel Monobactam AIC499 Targeting Penicillin Binding Protein 3 of Gram-Negative Bacteria. Biomolecules 2021, 11, 1057.

In the original article [...].

Structure of the SLy1 SAM homodimer reveals a new interface for SAM domain self-association.

Sterile alpha motif (SAM) domains are protein interaction modules that are involved in a diverse range of biological functions such as transcriptional and translational regulation, cellular signalling, and regulation of developmental processes. SH3 domain-containing protein expressed in lymphocytes 1 (SLy1) is involved in immune regulation and contains a SAM domain of unknown function. In this report, the structure of the SLy1 SAM domain was solved and revealed that this SAM domain forms a symmetric homodimer through a novel interface. The interface consists primarily of the two long C-terminal helices, α5 and α5', of the domains packing against each other. The dimerization is characterized by a dissociation constant in the lower micromolar range. A SLy1 SAM domain construct with an extended N-terminus containing five additional amino acids of the SLy1 sequence further increases the stability of the homodimer, making the SLy1 SAM dimer two orders of magnitude more stable than previously studied SAM homodimers, suggesting that the SLy1 SAM dimerization is of functional significance. The SLy1 SAM homodimer contains an exposed mid-loop surface on each monomer, which may provide a scaffold for mediating interactions with other SAM domain-containing proteins via a typical mid-loop-end-helix interface.

Proline Restricts Loop I Conformation of the High Affinity WW Domain from Human Nedd4-1 to a Ligand Binding-Competent Type I ß-Turn.

Sequence alignment of the four WW domains from human Nedd4-1 (neuronal precursor cell expressed developmentally down-regulated gene 4-1) reveals that the highest sequence diversity exists in loop I. Three residues in this type I ß-turn interact with the PPxY motif of the human epithelial Na+ channel (hENaC) subunits, indicating that peptide affinity is defined by the loop I sequence. The third WW domain (WW3*) has the highest ligand affinity and unlike the other three hNedd4-1 WW domains or other WW domains studied contains the highly statistically preferred proline at the ( i + 1) position found in ß-turns. In this report, molecular dynamics simulations and experimental data were combined to characterize loop I stability and dynamics. Exchange of the proline to the equivalent residue in WW4 (Thr) results in the presence of a predominantly open seven residue Ω loop rather than the type I ß-turn conformation for the wild-type apo-WW3*. In the presence of the ligand, the structure of the mutated loop I is locked into a type I ß-turn. Thus, proline in loop I ensures a stable peptide binding-competent ß-turn conformation, indicating that amino acid sequence modulates local flexibility to tune binding preferences and stability of dynamic interaction motifs.

Structure and interaction of Corynebacterium pseudotuberculosis cold shock protein A with Y-box single-stranded DNA fragment.

Cold shock proteins (Csps) function to preserve cell viability at low temperatures by binding to nucleic acids and consequently control gene expression. The mesophilic bacterium Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis in animals, and infection in livestock is a considerable economic burden worldwide. In this report, the structure of cold shock protein A from Cp (Cp-CspA) and biochemical analysis of its temperature-dependent interaction with a Y-box ssDNA motif is presented. The Cp-CspA structure contains five ß-strands making up a ß-barrel fold with 11 hydrophobic core residues and two salt bridges that confers it with a melting temperature of ~ 54 °C that is similar to mesophilic Bs-CspB. Chemical shift perturbations analysis revealed that residues in the nucleic acid-binding motifs (RNP 1 and 2) and loop 3 are involved in binding to the Y-box fragment either by direct interaction or by conformational rearrangements remote from the binding region. Fluorescence quenching experiments of Cp-CspA showed that the dissociation constants for Y-box ssDNA binding is nanomolar and the binding affinity decreased as the temperature increased, indicating that the interaction is enthalpically driven and the hydrogen bonds and van der Waals forces are important contributions for complex stabilization. The Y31 of Cp-CspA is a particular occurrence among Csps from mesophilic bacteria that provide a possible explanation for the higher binding affinity to ssDNA than that observed for Bs-CspB. Anisotropy measurements indicated that the reduction in molecular mobility of Cp-CspA upon Y-box binding is characterized by a cooperative process. DATABASE: Resonance assignment and structural data are available in the Biological Magnetic Resonance Data Bank and Protein Data Bank under accession number 26802 and 5O6F, respectively.

Molecular Dynamics Simulations Reveal Key Roles of the Interleukin-6 Alpha Receptor in the Assembly of the Human Interleukin-6 Receptor Complex.

Human interleukin-6 (hIL-6) is a pleiotropic cytokine with three distinct receptor epitopes, termed sites I, II, and III, which function to assemble a signaling complex. hIL-6 signals via a glycoprotein 130 (gp130) homodimer after initially forming a heterodimer with the nonsignaling α-receptor (IL-6Rα). The molecular description of the assembly of the hIL-6 signaling complex remains elusive because available structures provide descriptions of hIL-6 in its free and fully bound receptor forms, but not for intermediate steps that are crucial in the stepwise assembly of the signaling complex. In this report, molecular dynamics simulations provide atomic details describing the functional role of the initial hIL-6/IL-6Rα complex in facilitating subsequent interactions with gp130, which have not been previously shown. IL-6Rα binding to hIL-6 rigidifies the flexible N-terminus of the hIL-6 AB-loop through interactions with the D2 domain of IL-6Rα. This rigidification combined with repositioning of residues involved in gp130 receptor recognition promotes gp130 binding at site III. Binding of gp130 receptors at sites II and III is coupled with the release of the hIL-6 N-terminal AB-loop interaction and a pivoting of IL-6Rα around the hIL-6 helix bundle to the state of the hIL-6/IL-6Rα/gp130 complex.

Multiple WW domains of Nedd4-1 undergo conformational exchange that is quenched upon peptide binding.

The third WW domain (WW3*) of the ubiquitin ligase human neuronal precursor cell expressed developmentally downregulated gene 4-1 (hNedd4-1) was reported to bind its PY motif peptide by a coupled folding-binding equilibrium. However, it is unknown whether these thermodynamic properties are retained in the context of neighboring hNedd4-1 domains. In this report, NMR data show that the WW3* displays a fold-unfold equilibrium in the presence of neighboring WW domains, and that similar fold-unfold equilibria also likely exist for neighboring WW domains. These equilibria are quenched upon interaction with peptide. Thus, the binding mechanism of hNedd4-1 WW domains to proteins involves coupled folding and binding equilibria, and this mechanism may be a general feature that modulates peptide affinities of WW domains.

Using Neutron Reflectometry to Characterize Antimicrobial Protein Surface Coatings.

Understanding the interaction of adsorbed or covalently immobilized proteins with solid substrates at the molecular level guides the successful design of functionalized surfaces used in biomedical applications. In this report, neutron reflectometry (NR) was used to characterize the structure of surface-attached antimicrobial protein films, with antimicrobial activity assessed using an adaption of the Japanese Industrial Standard Test JIS Z 2801. NR allowed parameters influencing bioactivity to be measured at nanometer resolution and for conclusions about structural characteristics relating to bioactivity to be drawn. Hydramacin-1 (HM-1) and lysozyme were covalently attached to poly(methyl methacrylate) (PMMA) and 3-aminopropyltriethoxysilane (APTES) films in the presence and absence of a four-unit poly(ethylene glycol) PEG-based spacer and measured using NR, followed by antimicrobial assays. APTES-PEG-protein films were structurally unique, with a layer of 80% water directly beneath the protein layer, and were the only films that displayed antimicrobial activity against Escherichia coli and Bacillus subtilis. The hydration content of these films combined with the subtle difference in the PEG layer thickness of APTES versus PMMA films played a role in defining antimicrobial activity of the prepared surface coatings.

Data describing the solution structure of the WW3* domain from human Nedd4-1.

The third WW domain (WW3*) of human Nedd4-1 (Neuronal precursor cell expressed developmentally down-regulated gene 4-1) interacts with the poly-proline (PY) motifs of the human epithelial Na+ channel (hENaC) subunits at micromolar affinity. This data supplements the article (Panwalkar et al., 2015) [1]. We describe the NMR experiments used to solve the solution structure of the WW3* domain. We also present NOE network data for defining the rotameric state of side chains of peptide binding residues, and complement this data with χ 1 dihedral angles derived from (3) J couplings and molecular dynamics simulations data.

The Nedd4-1 WW Domain Recognizes the PY Motif Peptide through Coupled Folding and Binding Equilibria.

The four WW domains of human Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) interact with the PPxY (PY) motifs of the human epithelial Na(+) channel (hENaC) subunits, with the third WW domain (WW3*) showing the highest affinity. We have shown previously that the α-hENaC PY motif binding interface of WW3* undergoes conformational exchange on the millisecond time scale, indicating that conformational sampling plays a role in peptide recognition. To further understand this role, the structure and dynamics of hNedd4-1 WW3* were investigated. The nuclear Overhauser effect-derived structure of apo-WW3* resembles the domain in complex with the α-hENaC peptide, although particular side chain conformations change upon peptide binding, which was further investigated by molecular dynamics simulations. Model-free analysis of the (15)N nuclear magnetic resonance spin relaxation data showed that the apo and peptide-bound states of WW3* have similar backbone picosecond to nanosecond time scale dynamics. However, apo-WW3* exhibits pronounced chemical exchange on the millisecond time scale that is quenched upon peptide binding. (1)HN and (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments at various temperatures revealed that apo-WW3* exists in an equilibrium between the natively folded peptide binding-competent state and a random coil-like denatured state. The thermodynamics of the folding equilibrium was determined by fitting a thermal denaturation profile monitored by circular dichroism spectroscopy in combination with the CPMG data, leading to the conclusion that the unfolded state is populated to ∼ 20% at 37 °C. These results show that the binding of the hNedd4-1 WW3* domain to α-hENaC is coupled to the folding equilibrium.

Chemical synthesis of a pore-forming antimicrobial protein, caenopore-5, by using native chemical ligation at a glu-cys site.

The 2014 report from the World Health Organization (WHO) on antimicrobial resistance revealed an alarming rise in antibiotic resistance all around the world. Unlike classical antibiotics, with the exception of a few species, no acquired resistance towards antimicrobial peptides (AMPs) has been reported. Therefore, AMPs represent leads for the development of novel antibiotics. Caenopore-5 is constitutively expressed in the intestine of the nematode Caenorhabditis elegans and is a pore-forming AMP. The protein (82 amino acids) was successfully synthesised by using Boc solid-phase peptide synthesis and native chemical ligation. No γ-linked by-product was observed despite the use of a C-terminal Glu-thioester. The folding of the synthetic protein was confirmed by (1) H NMR spectroscopy and circular dichroism and compared with data recorded for recombinant caenopore-5. The permeabilisation activities of the protein and of shortened analogues were evaluated.

Functional implications of large backbone amplitude motions of the glycoprotein 130-binding epitope of interleukin-6.

Human interleukin (IL)-6 plays a pivotal role in the immune response, hematopoiesis, the acute-phase response, and inflammation. IL-6 has three distinct receptor epitopes, termed sites I, II, and III, that facilitate the formation of a signaling complex. IL-6 signals via a homodimer of glycoprotein 130 (gp130) after initially forming a heterodimer with the nonsignaling α-receptor [IL-6 α-receptor (IL-6R)] via site I. Here, we present the backbone dynamics of apo-IL-6 as determined by analysis of NMR relaxation data with the extended model-free formalism of Lipari and Szabo. To alleviate significant resonance overlap in the HSQC-type spectra, cell-free protein synthesis was used to selectively (15) N-label residues, thereby ensuring a complete set of residue-specific dynamics. The calculated order parameters [square of the generalized model-free order parameter (S(2))] showed significant conformational heterogeneity among clusters of residues in IL-6. In particular, the N-terminal region of the long AB-loop, which corresponds spatially to one of the gp130 receptor binding epitopes (i.e. site III), experiences substantial fluctuations along the conformation of the main chain (S(2) = 0.3-0.8) that are not observed at the other two epitopes or in other cytokines. Thus, we postulate that dynamic properties of the AB-loop are responsible for inhibiting the interaction of IL-6 with gp130 in the absence of the IL-6R, and that binding of IL-6R at site I shifts the dynamic equilibrium to favor interaction with gp130 at site III. In addition, molecular dynamics simulations corroborated the NMR-derived dynamics, and showed that the BC-loop adopts different substates that possibly play a role in facilitating receptor assembly.

Expression and purification of soluble HIV-2 viral protein R (Vpr) using a sandwich-fusion protein strategy.

Viral accessory proteins of the human immunodeficiency virus (HIV), including virus protein R (Vpr), are crucial for the efficient replication of the virus in the host organism. While functional data are available for HIV-1 Vpr, there is a paucity of data describing the function and structure of HIV-2 Vpr. In this report, the construction of a His6-MBP-intein1-Vpr-intein2-Cyt b5-His6 fusion protein is presented. Unlike previous research efforts where only microgram quantities of HIV-1 Vpr could be produced, this construct enabled soluble milligram yields via an Escherichia coli over-expression system. Straightforward protein purification of HIV-2 Vpr was achieved by standard chromatography routines and autocatalytic intein cleavage. Preliminary structural studies by circular dichroism (CD) and NMR spectroscopy revealed that the protein is stable in the presence of micellar concentrations of the detergent DPC and adopts an α-helix secondary structure.

Structural and functional characterization of the recombinant death domain from death-associated protein kinase.

Death-associated protein kinase (DAPk) is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD) that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic α-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD), NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the in vitro interaction between extracellular-regulated kinase-2 (ERK2) and various recombinant DAPk-DD constructs. Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1∶1 ratio with a K d in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent 'D-motif' located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural bioinformatics assignment of the three-dimensional structure.

Structure and dynamics of human Nedd4-1 WW3 in complex with the αENaC PY motif.

Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) is an E3 ubiquitin ligase that interacts with and negatively regulates the epithelial Na(+) channel (ENaC). The WW domains of Nedd4-1 bind to the ENaC subunits via recognition of PY motifs. Human Nedd4-1 (hNedd4-1) contains four WW domains with the third domain (WW3*) showing the strongest affinity to the PY motif. To understand the mechanism underlying this binding affinity, we have carried out NMR structural and dynamics analyses of the hNedd4-1 WW3* domain in complex with a peptide comprising the C-terminal tail of the human ENaC α-subunit. The structure reveals that the peptide interacts in a similar manner to other WW domain-ENaC peptide structures. Crucial interactions that likely provide binding affinity are the broad XP groove facilitating additional contacts between the WW3* domain and the peptide, compared to similar complexes, and the large surface area buried (83Å(2)) between R430 (WW3*) and L647' (αENaC). This corroborates the model-free analysis of the (15)N backbone relaxation data, which showed that R430 is the most rigid residue in the domain (S(2)=0.90±0.01). Carr-Purcell-Meiboom-Gill relaxation dispersion analysis identified two different conformational exchange processes on the µs-ms time-scale. One of these processes involves residues located at the peptide binding interface, suggesting conformational exchange may play a role in peptide recognition. Thus, both structural and dynamic features of the complex appear to define the high binding affinity. The results should aid interpretation of biochemical data and modeling interfaces between Nedd4-1 and other interacting proteins.

Structure and function of a unique pore-forming protein from a pathogenic acanthamoeba.

Human pathogens often produce soluble protein toxins that generate pores inside membranes, resulting in the death of target cells and tissue damage. In pathogenic amoebae, this has been exemplified with amoebapores of the enteric protozoan parasite Entamoeba histolytica. Here we characterize acanthaporin, to our knowledge the first pore-forming toxin to be described from acanthamoebae, which are free-living, bacteria-feeding, unicellular organisms that are opportunistic pathogens of increasing importance and cause severe and often fatal diseases. We isolated acanthaporin from extracts of virulent Acanthamoeba culbertsoni by tracking its pore-forming activity, molecularly cloned the gene of its precursor and recombinantly expressed the mature protein in bacteria. Acanthaporin was cytotoxic for human neuronal cells and exerted antimicrobial activity against a variety of bacterial strains by permeabilizing their membranes. The tertiary structures of acanthaporin's active monomeric form and inactive dimeric form, both solved by NMR spectroscopy, revealed a currently unknown protein fold and a pH-dependent trigger mechanism of activation.

Structure, dynamics and domain organization of the repeat protein Cin1 from the apple scab fungus.

Venturia inaequalis is a hemi-biotrophic fungus that causes scab disease of apple. A recently-identified gene from this fungus, cin1 (cellophane-induced 1), is up-regulated over 1000-fold in planta and considerably on cellophane membranes, and encodes a cysteine-rich secreted protein of 523 residues with eight imperfect tandem repeats of ~60 amino acids. The Cin1 sequence has no homology to known proteins and appears to be genus-specific; however, Cin1 repeats and other repeat domains may be structurally similar. An NMR-derived structure of the first two repeat domains of Cin1 (Cin1-D1D2) and a low-resolution model of the full-length protein (Cin1-FL) using SAXS data were determined. The structure of Cin1-D1D2 reveals that each domain comprises a core helix-loop-helix (HLH) motif as part of a three-helix bundle, and is stabilized by two intra-domain disulfide bonds. Cin1-D1D2 adopts a unique protein fold as DALI and PDBeFOLD analysis identified no structural homology. A (15)N backbone NMR dynamic analysis of Cin1-D1D2 showed that a short stretch of the inter-domain linker has large amplitude motions that give rise to reciprocal domain-domain mobility. This observation was supported by SAXS data modeling, where the scattering length density envelope remains thick at the domain-domain boundary, indicative of inter-domain dynamics. Cin1-FL SAXS data models a loosely-packed arrangement of domains, rather than the canonical parallel packing of adjacent HLH repeats observed in α-solenoid repeat proteins. Together, these data suggest that the repeat domains of Cin1 display a "beads-on-a-string" organization with inherent inter-domain flexibility that is likely to facilitate interactions with target ligands.

Caenopore-5: the three-dimensional structure of an antimicrobial protein from Caenorhabditis elegans.

The caenopore-5 protein encoded by the spp-5 gene is one of the 33 caenopores identified in Caenorhabditis elegans and is a pore-forming peptide which plays an important role in the elimination of Escherichia coli ingested by the worm. Thus, caenopore-5 appears to contribute to the nutrition of the worm while simultaneously protecting the organism against pathogens. Here, three-dimensional heteronuclear NMR spectroscopy was used to solve the solution structure of caenopore-5. The NMR data revealed that two conformers of caenopore-5 exist in solution which differ by the isomerization of the peptide bond of Pro-81. The overall structure of the two caenopore-5 conformers consists of five amphiphatic helices connected by three disulfide bonds. The five helices are arranged in a folded leaf which is the characteristic signature of the SAPLIP family. The structure presented here is the first of an effector protein of the defensive system elucidated for the well-known model organism C. elegans.

Cell-free synthesis and combinatorial selective 15N-labeling of the cytotoxic protein amoebapore A from Entamoeba histolytica.

Amoebapore A is a pore-forming protein produced by the pathogenic parasite Entamoeba histolytica, which causes human amoebic dysentery. The pore-forming activity of amoebapore A is regulated by pH-dependent dimerization, a prerequisite for membrane insertion and pore formation. Understanding of these important processes has been hampered by the cytotoxicity of amoebapore A, which prevents the production of this protein in cell-based expression systems. In this study, a protocol for the cell-free production of active recombinant amoebapore A is presented. Protein yields of approximately 500 microg/ml of cell-free reaction were achieved. Recombinant amoebapore A was purified using a three-step procedure. To facilitate the structural characterization of the dimeric and pore forms, we adapted the cell-free system to isotope label amoebapore A for NMR studies. The preliminary assignment of a 2D 1H-15N HSQC spectrum of a uniformly 13C/15N-labeled sample was achieved using a combinatorial selective 15N-labeling approach coupled with available 1H(N) chemical shift data, resulting in the unambiguous assignment of resonances from 55 of the 77 residues. To confirm these results and obtain the full sequence-specific assignments of the 2D 1H-15N HSQC spectrum, a 3D HNCA spectrum was recorded. These assignment data will be used to aid the characterization of amoebapore A dimer formation and membrane insertion.