Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase.

There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.

Structure snapshots reveal the mechanism of a bacterial membrane lipoprotein N-acyltransferase.

Bacterial lipoproteins (BLPs) decorate the surface of membranes in the cell envelope. They function in membrane assembly and stability, as enzymes, and in transport. The final enzyme in the BLP synthesis pathway is the apolipoprotein N-acyltransferase, Lnt, which is proposed to act by a ping-pong mechanism. Here, we use x-ray crystallography and cryo-electron microscopy to chart the structural changes undergone during the progress of the enzyme through the reaction. We identify a single active site that has evolved to bind, individually and sequentially, substrates that satisfy structural and chemical criteria to position reactive parts next to the catalytic triad for reaction. This study validates the ping-pong mechanism, explains the molecular bases for Lnt's substrate promiscuity, and should facilitate the design of antibiotics with minimal off-target effects.

Structural basis of the membrane intramolecular transacylase reaction responsible for lyso-form lipoprotein synthesis.

Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis.

In meso crystallogenesis. Compatibility of the lipid cubic phase with the synthetic digitonin analogue, glyco-diosgenin.

Digitonin has long been used as a mild detergent for extracting proteins from membranes for structure and function studies. As supplied commercially, digitonin is inhomogeneous and requires lengthy pre-treatment for reliable downstream use. Glyco-diosgenin (GDN) is a recently introduced synthetic surfactant with features that mimic digitonin. It is available in homogeneously pure form. GDN is proving to be a useful detergent, particularly in the area of single-particle cryo-electron microscopic studies of membrane integral proteins. With a view to using it as a detergent for crystallization trials by the in meso or lipid cubic phase method, it was important to establish the carrying capacity of the cubic mesophase for GDN. This was quantified in the current study using small-angle X-ray scattering for mesophase identification and phase microstructure characterization as a function of temperature and GDN concentration. The data show that the lipid cubic phase formed by hydrated monoolein tolerates GDN to concentrations orders of magnitude in excess of those used for membrane protein studies. Thus, having GDN in a typical membrane protein preparation should not deter use of the in meso method for crystallogenesis.

Membrane (and Soluble) Protein Stability and Binding Measurements in the Lipid Cubic Phase Using Label-Free Differential Scanning Fluorimetry.

Label-free differential scanning fluorimetry (DSF) is a relatively new method for evaluating the stability of proteins. It can be used as a screening tool for downstream applications such as crystallization. The method is attractive in that it requires miniscule quantities of proteins, it can be performed using intrinsic tryptophan and tyrosine fluorescence, and, with the right equipment, it is easy to perform. To date, the method has been used with proteins in liquid solutions and dispersions. It was of interest to determine if DSF could be used with membrane proteins in the lipid cubic phase (LCP), which increasingly is being used for crystallization in support of structure-function studies. The cubic phase is viscous. Furthermore, in coexistence with excess aqueous solution, as happens during crystallization trials, it can become turbid and scatter light. The concern was that these features may render the mesophase unsuitable for DSF analysis. However, using lysozyme and four integral membrane proteins we demonstrate that the method works with all tested proteins in solution and in the LCP. Of note is the observation that some of the test membrane proteins are more stable while others are less so in the mesophase. The method also works in ligand binding measurements. Thus, DSF should prove useful as an analytical tool for identifying host and additive lipids, detergents, precipitants and chemical probes that support the generation of quality crystals by the cubic phase method. Microscale thermophoresis was used to supplement the DSF study and was also shown to work with proteins in the mesophase. Measurements with lysozyme highlight the utility of the cubic mesophase as a model system in which to perform confinement studies.

Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis.

As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase.

The cubicon method for concentrating membrane proteins in the cubic mesophase.

The lipid cubic phase (in meso) method is an important approach for generating crystals and high-resolution X-ray structures of integral membrane proteins. However, as a consequence of instability, it can be impossible-using traditional methods-to concentrate certain membrane proteins and complexes to values suitable for in meso crystallization and structure determination. The cubicon method described here exploits the amphiphilic nature of membrane proteins and their natural tendency to partition preferentially into lipid bilayers from aqueous solution. Using several rounds of reconstitution, the protein concentration in the bilayer of the cubic mesophase can be ramped up stepwise from less than a milligram per milliliter to tens of milligrams per milliliter for crystallogenesis. The general applicability of the method is demonstrated with five integral membrane proteins: the ß2-adrenergic G protein-coupled receptor (ß2AR), the peptide transporter (PepTSt), diacylglycerol kinase (DgkA), the alginate transporter (AlgE) and the cystic fibrosis transmembrane conductance regulator (CFTR). In the cases of ß2AR, PepTSt, DgkA and AlgE, an effective 20- to 45-fold concentration was realized, resulting in a protein-laden mesophase that allowed the formation of crystals using the in meso method and structure determination to resolutions ranging from 2.4 Å to 3.2 Å. In addition to opening up in meso crystallization to a broader range of integral membrane protein targets, the cubicon method should find application in situations that require membrane protein reconstitution in a lipid bilayer at high concentrations. These applications include functional and biophysical characterization studies for ligand screening, drug delivery, antibody production and protein complex formation. A typical cubicon experiment can be completed in 3-5 h.

Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis.

Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein's invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects.

Structure-guided development of dual ß2 adrenergic/dopamine D2 receptor agonists.

Aiming to discover dual-acting ß2 adrenergic/dopamine D2 receptor ligands, a structure-guided approach for the evolution of GPCR agonists that address multiple targets was elaborated. Starting from GPCR crystal structures, we describe the design, synthesis and biological investigation of a defined set of compounds leading to the identification of the benzoxazinone (R)-3, which shows agonist properties at the adrenergic ß2 receptor and substantial G protein-promoted activation at the D2 receptor. This directed approach yielded molecular probes with tuned dual activity. The congener desOH-3 devoid of the benzylic hydroxyl function was shown to be a ß2 adrenergic antagonist/D2 receptor agonist with Ki values in the low nanomolar range. The compounds may serve as a promising starting point for the investigation and treatment of neurological disorders.

Covalent molecular probes for class A G protein-coupled receptors: advances and applications.

Covalent modification of G protein-coupled receptors (GPCRs) by employing specific molecular probes has for decades provided a successful strategy to facilitate the elucidation of the structure and function of this pharmacologically important class of membrane proteins. The ligands typically comprise a pharmacophore that generates affinity for a given GPCR and contain a reactive functionality that may form a covalent bond with a suitably positioned amino acid residue. Covalent ligands have been successfully applied to circumvent poor affinity of compounds when stable labeling of receptor populations was required, and they have been used in the isolation, purification, and pharmacological characterization of specific subtypes of GPCRs. Recently, structural studies have demonstrated that covalent molecular probes are effective at stabilizing GPCRs to obtain X-ray crystal structures, thus providing valuable insights for the development of novel therapeutics. Herein, we review covalently binding molecular probes for class A GPCRs with a focus on ligands comprising cross-linking groups that do not require photoactivation and further highlight their significant and diverse applications.

Molecular determinants of biased agonism at the dopamine D2 receptor.

The development of biased (functionally selective) ligands provides a formidable challenge in medicinal chemistry. In an effort to learn to design functionally selective molecular tools for the highly therapeutically relevant dopamine D2 receptor, we synthesized a collection of agonists based on structurally distinct head groups derived from canonical or atypical dopaminergic pharmacophores. The test compounds feature a long lipophilic appendage that was shown to mediate biased signaling. By employing functional assays and molecular dynamics simulations, we could show that atypical dopamine surrogates of type 1 and 2 promote biased signaling, while ligands built from classical dopaminergic head groups (type 3 and 4) typically elicit more balanced signaling profiles. Besides this, we found a strong influence of the stereochemistry of type 4 aminotetraline-derived agonists on functional selectivity at D2 receptors. Whereas the (S)-enantiomer behaved as a full agonist, the biased ligand (R)-4 induced poor G protein coupling but substantial ß-arrestin recruitment.

Covalent agonists for studying G protein-coupled receptor activation.

Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating Gs-, Gi-, and Gq-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the ß2-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis.