Chemoproteomic identification of CO2-dependent lysine carboxylation in proteins.

Carbon dioxide is an omnipresent gas that drives adaptive responses within organisms from all domains of life. The molecular mechanisms by which proteins serve as sensors of CO2 are, accordingly, of great interest. Because CO2 is electrophilic, one way it can modulate protein biochemistry is by carboxylation of the amine group of lysine residues. However, the resulting CO2-carboxylated lysines spontaneously decompose, giving off CO2, which makes studying this modification difficult. Here we describe a method to stably mimic CO2-carboxylated lysine residues in proteins. We leverage this method to develop a quantitative approach to identify CO2-carboxylated lysines of proteins and explore the lysine 'carboxylome' of the CO2-responsive cyanobacterium Synechocystis sp. We uncover one CO2-carboxylated lysine within the effector binding pocket of the metabolic signaling protein PII. CO2-carboxylatation of this lysine markedly lowers the affinity of PII for its regulatory effector ligand ATP, illuminating a negative molecular control mechanism mediated by CO2.

Computationally designed peptide macrocycle inhibitors of New Delhi metallo-ß-lactamase 1.

The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-ß-lactamase 1 (NDM-1), a bacterial enzyme that degrades ß-lactam antibiotics. We present structure-guided computational methods for designing peptide macrocycles built from mixtures of l- and d-amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a peptide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC50 values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets.

Potent and Selective Cell-Active Iminosugar Inhibitors of Human α-N-Acetylgalactosaminidase (α-NAGAL).

Glycoside hydrolases (GHs) are a class of enzymes with emerging roles in a range of disease. Selective GH inhibitors are sought to better understand their functions and assess the therapeutic potential of modulating their activities. Iminosugars are a promising class of GH inhibitors but typically lack the selectivity required to accurately perturb biological systems. Here, we describe a concise synthesis of iminosugar inhibitors of N-acetyl-α-galactosaminidase (α-NAGAL), the GH responsible for cleaving terminal α-N-acetylgalactosamine residues from glycoproteins and other glycoconjugates. Starting from non-carbohydrate precursors, this modular synthesis supported the identification of a potent (490 nM) and α-NAGAL selective (∼200-fold) guanidino-containing derivative DGJNGuan. To illustrate the cellular activity of this new inhibitor, we developed a quantitative fluorescence image-based method to measure levels of the Tn-antigen, a cellular glycoprotein substrate of α-NAGAL. Using this assay, we show that DGJNGuan exhibits excellent inhibition of α-NAGAL within cells using patient derived fibroblasts (EC50 =150 nM). Moreover, in vitro and in cell assays to assess levels of lysosomal ß-hexosaminidase substrate ganglioside GM2 show that DGJNGuan is selective whereas DGJNAc exhibits off-target inhibition both in vitro and within cells. DGJNGuan is a readily produced and selective tool compound that should prove useful for investigating the physiological roles of α-NAGAL.

Thermal Proteome Profiling Reveals the O-GlcNAc-Dependent Meltome.

Posttranslational modifications alter the biophysical properties of proteins and thereby influence cellular physiology. One emerging manner by which such modifications regulate protein functions is through their ability to perturb protein stability. Despite the increasing interest in this phenomenon, there are few methods that enable global interrogation of the biophysical effects of posttranslational modifications on the proteome. Here, we describe an unbiased proteome-wide approach to explore the influence of protein modifications on the thermodynamic stability of thousands of proteins in parallel. We apply this profiling strategy to study the effects of O-linked N-acetylglucosamine (O-GlcNAc), an abundant modification found on hundreds of proteins in mammals that has been shown in select cases to stabilize proteins. Using this thermal proteomic profiling strategy, we identify a set of 72 proteins displaying O-GlcNAc-dependent thermostability and validate this approach using orthogonal methods targeting specific proteins. These collective observations reveal that the majority of proteins influenced by O-GlcNAc are, surprisingly, destabilized by O-GlcNAc and cluster into distinct macromolecular complexes. These results establish O-GlcNAc as a bidirectional regulator of protein stability and provide a blueprint for exploring the impact of any protein modification on the meltome of, in principle, any organism.

The structure of a family 110 glycoside hydrolase provides insight into the hydrolysis of α-1,3-galactosidic linkages in λ-carrageenan and blood group antigens.

α-Linked galactose is a common carbohydrate motif in nature that is processed by a variety of glycoside hydrolases from different families. Terminal Galα1-3Gal motifs are found as a defining feature of different blood group and tissue antigens, as well as the building block of the marine algal galactan λ-carrageenan. The blood group B antigen and linear α-Gal epitope can be processed by glycoside hydrolases in family GH110, whereas the presence of genes encoding GH110 enzymes in polysaccharide utilization loci from marine bacteria suggests a role in processing λ-carrageenan. However, the structure-function relationships underpinning the α-1,3-galactosidase activity within family GH110 remain unknown. Here we focus on a GH110 enzyme (PdGH110B) from the carrageenolytic marine bacterium Pseudoalteromonas distincta U2A. We showed that the enzyme was active on Galα1-3Gal but not the blood group B antigen. X-ray crystal structures in complex with galactose and unhydrolyzed Galα1-3Gal revealed the parallel ß-helix fold of the enzyme and the structural basis of its inverting catalytic mechanism. Moreover, an examination of the active site reveals likely adaptations that allow accommodation of fucose in blood group B active GH110 enzymes or, in the case of PdGH110, accommodation of the sulfate groups found on λ-carrageenan. Overall, this work provides insight into the first member of a predominantly marine clade of GH110 enzymes while also illuminating the structural basis of α-1,3-galactoside processing by the family as a whole.

Structural analysis of avibactam-mediated activation of the bla and mec divergons in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections cause significant mortality and morbidity globally. MRSA resistance to ß-lactam antibiotics is mediated by two divergons that control levels of a ß-lactamase, PC1, and a penicillin-binding protein poorly acylated by ß-lactam antibiotics, PBP2a. Expression of genes encoding these proteins is controlled by two integral membrane proteins, BlaR1 and MecR1, which both have an extracellular ß-lactam-binding sensor domain. Here, we solved the X-ray crystallographic structures of the BlaR1 and MecR1 sensor domains in complex with avibactam, a diazabicyclooctane ß-lactamase inhibitor at 1.6-2.0 Å resolution. Additionally, we show that S. aureus SF8300, a clinically relevant strain from the USA300 clone of MRSA, responds to avibactam by up-regulating the expression of the blaZ and pbp2a antibiotic-resistance genes, encoding PC1 and PBP2a, respectively. The BlaR1-avibactam structure of the carbamoyl-enzyme intermediate revealed that avibactam is bound to the active-site serine in two orientations ∼180° to each other. Although a physiological role of the observed alternative pose remains to be validated, our structural results hint at the presence of a secondary sulfate-binding pocket that could be exploited in the design of future inhibitors of BlaR1/MecR1 sensor domains or the structurally similar class D ß-lactamases. The MecR1-avibactam structure adopted a singular avibactam orientation similar to one of the two states observed in the BlaR1-avibactam structure. Given avibactam up-regulates expression of blaZ and pbp2a antibiotic resistance genes, we suggest further consideration and research is needed to explore what effects administering ß-lactam-avibactam combinations have on treating MRSA infections.

Rational design of cell active C2-modified DGJ analogues for the inhibition of human α-galactosidase A (GALA).

We report the rational design and synthesis of C2-modified DGJ analogues to improve the selective inhibition of human GALA over other glycosidases. We prepare these analogues using a concise route from non-carbohydrate materials and demonstrate the most selective inhibitor 7c (∼100-fold) can act in Fabry patient cells to drive reductions in levels of the disease-relevant glycolipid Gb3.

Precision Mapping of O-Linked N-Acetylglucosamine Sites in Proteins Using Ultraviolet Photodissociation Mass Spectrometry.

Despite its central importance as a regulator of cellular physiology, identification and precise mapping of O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification (PTM) sites in proteins by mass spectrometry (MS) remains a considerable technical challenge. This is due in part to cleavage of the glycosidic bond occurring prior to the peptide backbone during collisionally activated dissociation (CAD), which leads to generation of characteristic oxocarbenium ions and impairs glycosite localization. Herein, we leverage CAD-induced oxocarbenium ion generation to trigger ultraviolet photodissociation (UVPD), an alternate high-energy deposition method that offers extensive fragmentation of peptides while leaving the glycosite intact. Upon activation using UV laser pulses, efficient photodissociation of glycopeptides is achieved with production of multiple sequence ions that enable robust and precise localization of O-GlcNAc sites. Application of this method to tryptic peptides originating from O-GlcNAcylated proteins TAB1 and Polyhomeotic confirmed previously reported O-GlcNAc sites in TAB1 (S395 and S396) and uncovered new sites within both proteins. We expect this strategy will complement existing MS/MS methods and be broadly useful for mapping O-GlcNAcylated residues of both proteins and proteomes.

Aspergillomarasmine A overcomes metallo-ß-lactamase antibiotic resistance.

The emergence and spread of carbapenem-resistant Gram-negative pathogens is a global public health problem. The acquisition of metallo-ß-lactamases (MBLs) such as NDM-1 is a principle contributor to the emergence of carbapenem-resistant Gram-negative pathogens that threatens the use of penicillin, cephalosporin and carbapenem antibiotics to treat infections. To date, a clinical inhibitor of MBLs that could reverse resistance and re-sensitize resistant Gram-negative pathogens to carbapenems has not been found. Here we have identified a fungal natural product, aspergillomarasmine A (AMA), that is a rapid and potent inhibitor of the NDM-1 enzyme and another clinically relevant MBL, VIM-2. AMA also fully restored the activity of meropenem against Enterobacteriaceae, Acinetobacter spp. and Pseudomonas spp. possessing either VIM or NDM-type alleles. In mice infected with NDM-1-expressing Klebsiella pneumoniae, AMA efficiently restored meropenem activity, demonstrating that a combination of AMA and a carbapenem antibiotic has therapeutic potential to address the clinical challenge of MBL-positive carbapenem-resistant Gram-negative pathogens.

Structural and functional insight into human O-GlcNAcase.

O-GlcNAc hydrolase (OGA) removes O-linked N-acetylglucosamine (O-GlcNAc) from a myriad of nucleocytoplasmic proteins. Through co-expression and assembly of OGA fragments, we determined the three-dimensional structure of human OGA, revealing an unusual helix-exchanged dimer that lays a structural foundation for an improved understanding of substrate recognition and regulation of OGA. Structures of OGA in complex with a series of inhibitors define a precise blueprint for the design of inhibitors that have clinical value.

Both Chemical and Non-Chemical Steps Limit the Catalytic Efficiency of Family 4 Glycoside Hydrolases.

The glycoside hydrolase family 4 (GH4) α-galactosidase from Citrobacter freundii (MelA) catalyzes the hydrolysis of fluoro-substituted phenyl α-d-galactopyranosides by utilizing two cofactors, NAD+ and a metal cation, under reducing conditions. In order to refine the mechanistic understanding of this GH4 enzyme, leaving group effects were measured with various metal cations. The derived ßlg value on V/ K for strontium activation is indistinguishable from zero (0.05 ± 0.12). Deuterium kinetic isotope effects (KIEs) were measured for the activated substrates 2-fluorophenyl and 4-fluorophenyl α-d-galactopyranosides in the presence of Sr2+, Y3+, and Mn2+, where the isotopic substitution was on the carbohydrate at C-2 and/or C-3. To determine the contributing factors to the virtual transition state (TS) on which the KIEs report, kinetic isotope effects on isotope effects were measured on these KIEs using doubly deuterated substrates. The measured D V/ K KIEs for MelA-catalyzed hydrolysis of 2-fluorophenyl α-d-galactopyranoside are closer to unity than the measured effects on 4-fluorophenyl α-d-galactopyranoside, irrespective of the site of isotopic substitution and of the metal cation activator. These observations are consistent with hydride transfer at C-3 to the on-board NAD+, deprotonation at C-2, and a non-chemical step contributing to the virtual TS for V/ K.

Structural Insights into Inhibition of Escherichia coli Penicillin-binding Protein 1B.

In Escherichia coli, the peptidoglycan cell wall is synthesized by bifunctional penicillin-binding proteins such as PBP1b that have both transpeptidase and transglycosylase activities. The PBP1b transpeptidase domain is a major target of ß-lactams, and therefore it is important to attain a detailed understanding of its inhibition. The peptidoglycan glycosyltransferase domain of PBP1b is also considered an excellent antibiotic target yet is not exploited by any clinically approved antibacterials. Herein, we adapt a pyrophosphate sensor assay to monitor PBP1b-catalyzed glycosyltransfer and present an improved crystallographic model for inhibition of the PBP1b glycosyltransferase domain by the potent substrate analog moenomycin. We elucidate the structure of a previously disordered region in the glycosyltransferase active site and discuss its implications with regards to peptidoglycan polymerization. Furthermore, we solve the crystal structures of E. coli PBP1b bound to multiple different ß-lactams in the transpeptidase active site and complement these data with gel-based competition assays to provide a detailed structural understanding of its inhibition. Taken together, these biochemical and structural data allow us to propose new insights into inhibition of both enzymatic domains in PBP1b.

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.

Multivalency To Inhibit and Discriminate Hexosaminidases.

A set of multivalent polyhydroxylated acetamidoazepanes based on ethylene glycol, glucoside, or cyclodextrin scaffolds was prepared. The compounds were assessed against plant, mammalian, and therapeutically relevant hexosaminidases. Multimerization was shown to improve the inhibitory potency with synergy, and to fine tune the selectivity profile between related hexosaminidases.

Molecular insights into the coding region determinant-binding protein-RNA interaction through site-directed mutagenesis in the heterogeneous nuclear ribonucleoprotein-K-homology domains.

The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function.

Structural insights into the lipoprotein outer membrane regulator of penicillin-binding protein 1B.

In bacteria, the synthesis of the protective peptidoglycan sacculus is a dynamic process that is tightly regulated at multiple levels. Recently, the lipoprotein co-factor LpoB has been found essential for the in vivo function of the major peptidoglycan synthase PBP1b in Enterobacteriaceae. Here, we reveal the crystal structures of Salmonella enterica and Escherichia coli LpoB. The LpoB protein can be modeled as a ball and tether, consisting of a disordered N-terminal region followed by a compact globular C-terminal domain. Taken together, our structural data allow us to propose new insights into LpoB-mediated regulation of peptidoglycan synthesis.

Assay for drug discovery: Synthesis and testing of nitrocefin analogues for use as ß-lactamase substrates.

We report on the synthesis of three nitrocefin analogues and their evaluation as substrates for the detection of ß-lactamase activity. These compounds are hydrolyzed by all four Ambler classes of ß-lactamases. Kinetic parameters were determined with eight different ß-lactamases, including VIM-2, NDM-1, KPC-2, and SPM-1. The compounds do not inhibit the growth of clinically important antibiotic-resistant gram-negative bacteria in vitro. These chromogenic compounds have a distinct absorbance spectrum and turn purple when hydrolyzed by ß-lactamases. One of these compounds, UW154, is easier to synthesize from commercial starting materials than nitrocefin and should be significantly less expensive to produce.

New Delhi metallo-ß-lactamase: structural insights into ß-lactam recognition and inhibition.

The ß-lactam antibiotics have long been a cornerstone for the treatment of bacterial disease. Recently, a readily transferable antibiotic resistance factor called the New Delhi metallo-ß-lactamase-1 (NDM-1) has been found to confer enteric bacteria resistance to nearly all ß-lactams, including the heralded carbapenems, posing a serious threat to human health. The crystal structure of NDM-1 bound to meropenem shows for the first time the molecular details of how carbapenem antibiotics are recognized by dizinc-containing metallo-ß-lactamases. Additionally, product complex structures of hydrolyzed benzylpenicillin-, methicillin-, and oxacillin-bound NDM-1 have been solved to 1.8, 1.2, and 1.2 Å, respectively, and represent the highest-resolution structural data for any metallo-ß-lactamase reported to date. Finally, we present the crystal structure of NDM-1 bound to the potent competitive inhibitor l-captopril, which reveals a unique binding mechanism. An analysis of the NDM-1 active site in these structures reveals key features important for the informed design of novel inhibitors of NDM-1 and other metallo-ß-lactamases.

Molecular mechanisms regulating O-linked N-acetylglucosamine (O-GlcNAc)-processing enzymes.

The post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) dynamically programmes cellular physiology to maintain homoeostasis and tailor biochemical pathways to meet context-dependent cellular needs. Despite diverse roles of played by O-GlcNAc, only two enzymes act antagonistically to govern its cycling; O-GlcNAc transferase installs the monosaccharide on target proteins, and O-GlcNAc hydrolase removes it. The recent literature has exposed a network of mechanisms regulating these two enzymes to choreograph global, and target-specific, O-GlcNAc cycling in response to cellular stress and nutrient availability. Herein, we amalgamate these emerging mechanisms from a structural and molecular perspective to explore how the cell exerts fine control to regulate O-GlcNAcylation of diverse proteins in a selective fashion.

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.