Structural basis of broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Broad-spectrum ß-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden1,2. In clinical strains, resistance is largely controlled by BlaR13, a receptor that senses ß-lactams through the acylation of its sensor domain, inducing transmembrane signalling and activation of the cytoplasmic-facing metalloprotease domain4. The metalloprotease domain has a role in BlaI derepression, inducing blaZ (ß-lactamase PC1) and mecA (ß-lactam-resistant cell-wall transpeptidase PBP2a) expression3-7. Here, overcoming hurdles in isolation, we show that BlaR1 cleaves BlaI directly, as necessary for inactivation, with no requirement for additional components as suggested previously8. Cryo-electron microscopy structures of BlaR1-the wild type and an autocleavage-deficient F284A mutant, with or without ß-lactam-reveal a domain-swapped dimer that we suggest is critical to the stabilization of the signalling loops within. BlaR1 undergoes spontaneous autocleavage in cis between Ser283 and Phe284 and we describe the catalytic mechanism and specificity underlying the self and BlaI cleavage. The structures suggest that allosteric signalling emanates from ß-lactam-induced exclusion of the prominent extracellular loop bound competitively in the sensor-domain active site, driving subsequent dynamic motions, including a shift in the sensor towards the membrane and accompanying changes in the zinc metalloprotease domain. We propose that this enhances the expulsion of autocleaved products from the active site, shifting the equilibrium to a state that is permissive of efficient BlaI cleavage. Collectively, this study provides a structure of a two-component signalling receptor that mediates action-in this case, antibiotic resistance-through the direct cleavage of a repressor.

Structure and Mechanism of Staphylococcus aureus TarS, the Wall Teichoic Acid ß-glycosyltransferase Involved in Methicillin Resistance.

In recent years, there has been a growing interest in teichoic acids as targets for antibiotic drug design against major clinical pathogens such as Staphylococcus aureus, reflecting the disquieting increase in antibiotic resistance and the historical success of bacterial cell wall components as drug targets. It is now becoming clear that ß-O-GlcNAcylation of S. aureus wall teichoic acids plays a major role in both pathogenicity and antibiotic resistance. Here we present the first structure of S. aureus TarS, the enzyme responsible for polyribitol phosphate ß-O-GlcNAcylation. Using a divide and conquer strategy, we obtained crystal structures of various TarS constructs, mapping high resolution overlapping N-terminal and C-terminal structures onto a lower resolution full-length structure that resulted in a high resolution view of the entire enzyme. Using the N-terminal structure that encapsulates the catalytic domain, we furthermore captured several snapshots of TarS, including the native structure, the UDP-GlcNAc donor complex, and the UDP product complex. These structures along with structure-guided mutants allowed us to elucidate various catalytic features and identify key active site residues and catalytic loop rearrangements that provide a valuable platform for anti-MRSA drug design. We furthermore observed for the first time the presence of a trimerization domain composed of stacked carbohydrate binding modules, commonly observed in starch active enzymes, but adapted here for a poly sugar-phosphate glycosyltransferase.

Structure and mechanism of Staphylococcus aureus TarM, the wall teichoic acid α-glycosyltransferase.

Unique to Gram-positive bacteria, wall teichoic acids are anionic glycopolymers cross-stitched to a thick layer of peptidoglycan. The polyol phosphate subunits of these glycopolymers are decorated with GlcNAc sugars that are involved in phage binding, genetic exchange, host antibody response, resistance, and virulence. The search for the enzymes responsible for GlcNAcylation in Staphylococcus aureus has recently identified TarM and TarS with respective α- and ß-(1-4) glycosyltransferase activities. The stereochemistry of the GlcNAc attachment is important in balancing biological processes, such that the interplay of TarM and TarS is likely important for bacterial pathogenicity and survival. Here we present the crystal structure of TarM in an unusual ternary-like complex consisting of a polymeric acceptor substrate analog, UDP from a hydrolyzed donor, and an α-glyceryl-GlcNAc product formed in situ. These structures support an internal nucleophilic substitution-like mechanism, lend new mechanistic insight into the glycosylation of glycopolymers, and reveal a trimerization domain with a likely role in acceptor substrate scaffolding.

A method for integrative structure determination of protein-protein complexes.

MOTIVATION: Structural characterization of protein interactions is necessary for understanding and modulating biological processes. On one hand, X-ray crystallography or NMR spectroscopy provide atomic resolution structures but the data collection process is typically long and the success rate is low. On the other hand, computational methods for modeling assembly structures from individual components frequently suffer from high false-positive rate, rarely resulting in a unique solution. RESULTS: Here, we present a combined approach that computationally integrates data from a variety of fast and accessible experimental techniques for rapid and accurate structure determination of protein-protein complexes. The integrative method uses atomistic models of two interacting proteins and one or more datasets from five accessible experimental techniques: a small-angle X-ray scattering (SAXS) profile, 2D class average images from negative-stain electron microscopy micrographs (EM), a 3D density map from single-particle negative-stain EM, residue type content of the protein-protein interface from NMR spectroscopy and chemical cross-linking detected by mass spectrometry. The method is tested on a docking benchmark consisting of 176 known complex structures and simulated experimental data. The near-native model is the top scoring one for up to 61% of benchmark cases depending on the included experimental datasets; in comparison to 10% for standard computational docking. We also collected SAXS, 2D class average images and 3D density map from negative-stain EM to model the PCSK9 antigen-J16 Fab antibody complex, followed by validation of the model by a subsequently available X-ray crystallographic structure.

Structural investigation of the C-terminal catalytic fragment of presenilin 1.

The gamma-secretase complex has a decisive role in the development of Alzheimer's disease, in that it cleaves a precursor to create the amyloid beta peptide whose aggregates form the senile plaques encountered in the brains of patients. Gamma-secretase is a member of the intramembrane-cleaving proteases which process their transmembrane substrates within the bilayer. Many of the mutations encountered in early onset familial Alzheimer's disease are linked to presenilin 1, the catalytic component of gamma-secretase, whose active form requires its endoproteolytic cleavage into N-terminal and C-terminal fragments. Although there is general agreement regarding the topology of the N-terminal fragment, studies of the C-terminal fragment have yielded ambiguous and contradictory results that may be difficult to reconcile in the absence of structural information. Here we present the first structure of the C-terminal fragment of human presenilin 1, as obtained from NMR studies in SDS micelles. The structure reveals a topology where the membrane is likely traversed three times in accordance with the more generally accepted nine transmembrane domain model of presenilin 1, but contains unique structural features adapted to accommodate the unusual intramembrane catalysis. These include a putative half-membrane-spanning helix N-terminally harboring the catalytic aspartate, a severely kinked helical structure toward the C terminus as well as a soluble helix in the assumed-to-be unstructured N-terminal loop.

Transmembrane segment enhanced labeling as a tool for the backbone assignment of alpha-helical membrane proteins.

Recent advances in cell-free expression protocols have opened a new avenue toward high-resolution structural investigations of membrane proteins by x-ray crystallography and NMR spectroscopy. One of the biggest challenges for liquid-state NMR-based structural investigations of membrane proteins is the significant peak overlap in the spectra caused by large line widths and limited chemical shift dispersion of alpha-helical proteins. Contributing to the limited chemical shift dispersion is the fact that approximately 60% of the amino acids in transmembrane regions consist of only six different amino acid types. This principle disadvantage, however, can be exploited to aid in the assignment of the backbone resonances of membrane proteins; by (15)N/(13)C-double-labeling of these six amino acid types, sequential connectivities can be obtained for large stretches of the transmembrane segments where number and length of stretches consisting exclusively of these six amino acid types are enhanced compared with the remainder of the protein. We show by experiment as well as by statistical analysis that this labeling scheme provides a large number of sequential connectivities in transmembrane regions and thus constitutes a tool for the efficient assignment of membrane protein backbone resonances.

The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour-1 kg-1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

Cell-free expression and stable isotope labelling strategies for membrane proteins.

Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems provides a very interesting alternative to cell-based systems, since it circumvents many problems such as toxicity or necessity for the transportation of the synthesized protein to the membrane, and constitutes the only system that allows for direct production of membrane proteins in membrane-mimetic environments which may be suitable for liquid state NMR measurements. The unique advantages of the cell-free expression system, including strong expression yields as well as the direct incorporation of almost any combination of amino acids with very little metabolic scrambling, has allowed for the development of a wide-array of isotope labelling techniques which facilitate structural investigations of proteins whose spectral congestion and broad line-widths may have earlier rendered them beyond the scope of NMR. Here we explore various labelling strategies in conjunction with cell-free developments, with a particular focus on alpha-helical transmembrane proteins which benefit most from such methods.

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection.

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection. ONE SENTENCE SUMMARY: LY-CoV555, an anti-spike antibody derived from a convalescent COVID-19 patient, potently neutralizes SARS-CoV-2 and protects the upper and lower airways of non-human primates against SARS-CoV-2 infection.

Reduced expression of hypoxia-inducible factor-1alpha in perinecrotic regions of solid tumors.

Hypoxia that develops in solid tumors stabilizes the hypoxia-inducible factor-1alpha (HIF-1alpha) subunit of the HIF-1 transcription factor, leading to up-regulation of dozens of hypoxia-regulated genes that increase glycolysis and oxygen delivery. HIF-1alpha and its downstream target gene CA9 have both been used as surrogate hypoxia markers, and, in general, high expression predicts for a poor response to treatment. Combinations of hypoxia markers offer the opportunity to measure changes in tumor oxygenation that may be relevant to tumor response to treatment. We compared the degree of colocalization of two endogenous markers for hypoxia, HIF-1alpha and carbonic anhydrase IX (CAIX), with a chemical marker for hypoxia, pimonidazole. Unexpectedly, expression of HIF-1alpha was reduced in the most hypoxic regions that border necrosis in xenograft tumors composed of SiHa cervical carcinoma, WiDr colon carcinoma, or M006 astrocytoma cells. Similar results were obtained for samples from three cervical cancer biopsies. However, CAIX was present in these perinecrotic cells that were also capable of metabolizing and binding a chemical marker for hypoxia, pimonidazole. In vitro experiments using tumor cells and tumor cubes incubated under anoxic conditions indicated that nutrient deprivation seems to be largely responsible for the lack of HIF-1alpha expression in perinecrotic regions. The half-life of CAIX was sufficiently long that, once formed, it remained for days in the absence of continued HIF-1alpha expression. These results have implications for the use of HIF-1alpha as an indicator of tumor hypoxia and aggressiveness as well as development of hypoxia-directed antitumor therapies based on the expression of HIF-1alpha.

One ring to rule them all: Current trends in combating bacterial resistance to the ß-lactams.

From humble beginnings of a contaminated petri dish, ß-lactam antibiotics have distinguished themselves among some of the most powerful drugs in human history. The devastating effects of antibiotic resistance have nevertheless led to an "arms race" with disquieting prospects. The emergence of multidrug resistant bacteria threatens an ever-dwindling antibiotic arsenal, calling for new discovery, rediscovery, and innovation in ß-lactam research. Here the current state of ß-lactam antibiotics from a structural perspective was reviewed.

Fortifying the wall: synthesis, regulation and degradation of bacterial peptidoglycan.

Growth and maintenance of the protective peptidoglycan cell wall are vital to bacterial growth and morphogenesis. Thus, the relative rate and spatiotemporal control of the synthesis and degradation of this net-like polymer defines bacterial cell shape. In recent years, our understanding of the processes that govern this delicate metabolic balance has improved and should lend insight into how to therapeutically target the system in the future.

Strategies for the cell-free expression of membrane proteins.

Cell-free expression offers an interesting alternative method to produce membrane proteins in high amounts. Elimination of toxicity problems, reduced proteolytic degradation and a nearly unrestricted option to supply potentially beneficial compounds like cofactors, ligands or chaperones into the reaction are general advantages of cell-free expression systems. Furthermore, the membrane proteins may be translated directly into appropriate hydrophobic and membrane-mimetic surrogates, which might offer significant benefits for the functional folding of the synthesized proteins. Cell-free expression is a rapidly developing and highly versatile technique and several systems of both, prokaryotic and eukaryotic origins, have been established. We provide protocols for the cell-free expression of membrane proteins in different modes including their expression as precipitate as well as their direct synthesis into detergent micelles or lipid bilayers.

Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems.

Cell-free expression is emerging as a prime method for the rapid production of preparative quantities of high-quality membrane protein samples. The technology facilitates easy access to large numbers of proteins that have been extremely difficult to obtain. Most frequently used are cell-free systems based on extracts of Escherichia coli cells, and the reaction procedures are reliable and efficient. This protocol describes the preparation of all essential reaction components such as the E. coli cell extract, T7 RNA polymerase, DNA templates as well as the individual stock solutions. The setups of expression reactions in analytical and preparative scales, including a variety of reaction designs, are illustrated. We provide detailed reaction schemes that allow the preparation of milligram amounts of functionally folded membrane proteins of prokaryotic and eukaryotic origin in less than 24 h.