How Allosteric Control of Staphylococcus aureus Penicillin-Binding Protein 2a Enables Methicillin-Resistance and Physiological Function

The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The highmolecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain - a remarkable 60 Å distant from the DD-transpeptidase active site - discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.Fil: Otero, Lisandro Horacio. Consejo Superior de Investigaciones Científicas. Instituto de Química Física; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Rojas Altuve, Alzoray. Consejo Superior de Investigaciones Científicas. Instituto de Química Física; EspañaFil: Llarrull, Leticia Irene. University of Notre Dame; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Carrasco López, Cesar. Consejo Superior de Investigaciones Científicas. Instituto de Química Física; EspañaFil: Kumarasiri, Malika. University of Notre Dame; Estados UnidosFil: Lastochkin, Elena. University of Notre Dame; Estados UnidosFil: Fishovitz, Jennifer. University of Notre Dame; Estados UnidosFil: Dawley, Matthew. University of Notre Dame; Estados UnidosFil: Hesek, Dusan. University of Notre Dame; Estados UnidosFil: Lee, Mijoon. University of Notre Dame; Estados UnidosFil: Johnson, Jarrod W.. University of Notre Dame; Estados UnidosFil: Fisher, Jed F.. University of Notre Dame; Estados UnidosFil: Chang, Mayland. University of Notre Dame; Estados UnidosFil: Mobashery, Shahriar. University of Notre Dame; Estados UnidosFil: Hermoso, Juan A.. Consejo Superior de Investigaciones Científicas. Instituto de Química Física; Españ

Utilization of Mechanistic Enzymology to Evaluate the Significance of ADP Binding to Human Lon Protease

Lon, also known as Protease La, is one of the simplest ATP-dependent proteases. It is a homooligomeric enzyme comprised of an ATPase domain and a proteolytic domain in each enzyme subunit. Despite sharing about 40% sequence identity, human and Escherichia coli Lon proteases utilize a highly conserved ATPase domain found in the AAA+ family to catalyze ATP hydrolysis, which is needed to activate protein degradation. In this study, we utilized mechanistic enzymology techniques to show that despite comparable kcat and Km parameters found in the ATPase activity, human and E. coli Lon exhibit significantly different susceptibility to ADP inhibition. Due to the low affinity of human Lon for ADP, the conformational changes in human Lon generated from the ATPase cycle are also different. The relatively low affinity of human Lon for ADP cannot be accounted for by reversibility in ATP hydrolysis, as a positional isotope exchange experiment demonstrated both E. coli Lon and human Lon catalyzed ATP hydrolysis irreversibly. A limited tryptic digestion study however indicated that human and E. coli Lon bind to ADP differently. Taken together, the findings reported in this research article suggest that human Lon is not regulated by a substrate-promoted ADP/ATP exchange mechanism as found in the bacterial enzyme homolog. The drastic difference in structural changes associated with ADP interaction with the two protease homologs offer potential for selective inhibitor design and development through targeting the ATPase sites. In addition to revealing unique mechanistic differences that distinguish human vs. bacterial Lon, this article underscores the benefit of mechanistic enzymology in deciphering the physiological mechanism of action of Lon proteases and perhaps other closely related ATP-dependent proteases in the future

Phosphorylation of BlaR1 in Manifestation of Antibiotic Resistance in Methicillin-Resistant Staphylococcus aureus and Its Abrogation by Small Molecules

Methicillin-resistant Staphylococcus aureus (MRSA), an important human pathogen, has evolved an inducible mechanism for resistance to β-lactam antibiotics. We report herein that the integral membrane protein BlaR1, the β- lactam sensor/signal transducer protein, is phosphorylated on exposure to β-lactam antibiotics. This event is critical to the onset of the induction of antibiotic resistance. Furthermore, we document that BlaR1 phosphorylation and the antibioticresistance phenotype are both reversed in the presence of synthetic protein kinase inhibitors of our design, restoring susceptibility of the organism to a penicillin, resurrecting it from obsolescence in treatment of these intransigent bacteria.Fil: Boudreau, Marc A.. University Of Notre Dame-Indiana; Estados UnidosFil: Fishovitz, Jennifer. University Of Notre Dame-Indiana; Estados UnidosFil: Llarrull, Leticia Irene. University Of Notre Dame-Indiana; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Xiao, Qiaobin. University Of Notre Dame-Indiana; Estados UnidosFil: Mobashery, Shahriar. University Of Notre Dame-Indiana; Estados Unido

Processive Degradation of Unstructured Protein by Escherichia coli Lon Occurs via the Slow, Sequential Delivery of Multiple Scissile Sites Followed by Rapid and Synchronized Peptide Bond Cleavage Events

Processive protein degradation is a common feature found in ATP-dependent proteases. This study utilized a physiological substrate of Escherichia coli Lon protease known as the lambda N protein (λN) to initiate the first kinetic analysis of the proteolytic mechanism of this enzyme. To this end, experiments were designed to determine the timing of three selected scissile sites in λN approaching the proteolytic site of ELon and their subsequent cleavages to gain insight into the mechanism by which ATP-dependent proteases attain processivity in protein degradation. The kinetic profile of peptide bond cleavage at different regions of λN was first detected by the iTRAQ/mass spectrometry technique. Fluorogenic λN constructs were then generated as reporter substrates for transient kinetic characterization of the ATP- versus AMPPNP-dependent peptide bond cleavage and the delivery of the scissile sites near the amino- versus carboxyl-terminal of the λN protein to the proteolytic site of ELon. Collectively, our results support a mechanism by which the cleavage of multiple peptide bonds awaits the “almost complete” delivery of all the scissile sites in λN to the proteolytic site in an ATP-dependent manner. Comparing the time courses of delivery to the active site of the selected scissile sites further implicates the existence of a preferred directionality in the final stage of substrate delivery, which begins at the carboxyl-terminal. The subsequent cleavage of the scissile sites in λN, however, appears to lack a specific directionality and occurs at a much faster rate than the substrate delivery step

The Tipper–Strominger Hypothesis and Triggering of Allostery in Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus (MRSA)

The transpeptidases involved in the synthesis of the bacterial cell wall (also known as penicillin-binding proteins, PBPs) have evolved to bind the acyl-d-Ala-d-Ala segment of the stem peptide of the nascent peptidoglycan for the physiologically important cross-linking of the cell wall. The Tipper–Strominger hypothesis stipulates that β-lactam antibiotics mimic the acyl-d-Ala-d-Ala moiety of the stem and, thus, are recognized by the PBPs with bactericidal consequences. We document that this mimicry exists also at the allosteric site of PBP2a of methicillin-resistant Staphylococcus aureus (MRSA). Interactions of different classes of β-lactam antibiotics, as mimics of the acyl-d-Ala-d-Ala moiety at the allosteric site, lead to a conformational change, across a distance of 60 Å to the active site. We directly visualize this change using an environmentally sensitive fluorescent probe affixed to the protein loops that frame the active site. This conformational mobility, documented in real time, allows antibiotic access to the active site of PBP2a. Furthermore, we document that this allosteric trigger enables synergy between two different β-lactam antibiotics, wherein occupancy at the allosteric site by one facilitates occupancy by a second at the transpeptidase catalytic site, thus lowering the minimal-inhibitory concentration. This synergy has important implications for the mitigation of facile emergence of resistance to these antibiotics by MRSA

AAC(3)-XI, a New Aminoglycoside 3- N

Corynebacterium striatum BM4687 was resistant to gentamicin and tobramycin but susceptible to kanamycin A and amikacin, a phenotype distinct among Gram-positive bacteria. Analysis of the entire genome of this strain did not detect any genes for known aminoglycoside resistance enzymes. Yet, annotation of the coding sequences identified 12 putative acetyltransferases or GCN5-related N-acetyltransferases. A total of 11 of these coding sequences were also present in the genomes of other Corynebacterium spp. The 12th coding sequence had 55 to 60% amino acid identity with acetyltransferases in Actinomycetales. The gene was cloned in Escherichia coli, where it conferred resistance to aminoglycosides by acetylation. The protein was purified to homogeneity, and its steady-state kinetic parameters were determined for dibekacin and kanamycin B. The product of the turnover of dibekacin was purified, and its structure was elucidated by high-field nuclear magnetic resonance (NMR), indicating transfer of the acetyl group to the amine at the C-3 position. Due to the unique profile of the reaction, it was designated aminoglycoside 3-N-acetyltransferase type XI

Disruption of Allosteric Response as an Unprecedented Mechanism of Resistance to Antibiotics

Ceftaroline, a recently approved β-lactam antibiotic for treatment of infections by methicillinresistant Staphylococcus aureus (MRSA), is able to inhibit penicillin-binding protein 2a (PBP2a) by triggering an allosteric conformational change that leads to the opening of the active site. The opened active site is now vulnerable to inhibition by a second molecule of ceftaroline, an event that impairs cell-wall biosynthesis and leads to bacterial death. The triggering of the allosteric effect takes place by binding of the first antibiotic molecule 60 Å away from the active site of PBP2a within the core of the allosteric site. We document, by kinetic studies and by determination of three X-ray structures of the mutant variants of PBP2a that result in resistance to ceftaroline, that the effect of these clinical mutants is the disruption of the allosteric trigger in this important protein in MRSA. This is an unprecedented mechanism for antibiotic resistance.Fil: Fishovitz, Jennifer. University of Notre Dame. Department of Chemistry and Biochemistry; Estados UnidosFil: Rojas Altuve, Alzoray. Consejo Superior de Investigaciones Cientificas. Instituto de Quimica Fisica; EspañaFil: Otero, Lisandro Horacio. Consejo Superior de Investigaciones Cientificas. Instituto de Quimica Fisica; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: Dawley, Matthew. University of Notre Dame. Department of Chemistry and Biochemistry; Estados UnidosFil: Carrasco López, Cesar. Consejo Superior de Investigaciones Cientificas. Instituto de Quimica Fisica; EspañaFil: Chang, Mayland. University of Notre Dame. Department of Chemistry and Biochemistry; Estados UnidosFil: Hermoso, Juan Antonio. Consejo Superior de Investigaciones Cientificas. Instituto de Quimica Fisica; EspañaFil: Mobashery, Shahriar. University of Notre Dame. Department of Chemistry and Biochemistry; Estados Unido

Active-Site-Directed Chemical Tools for Profiling Mitochondrial Lon Protease

Lon and ClpXP are the only soluble ATP-dependent proteases within the mammalian mitochondria matrix, which function in protein quality control by selectively degrading misfolded, misassembled, or damaged proteins. Chemical tools to study these proteases in biological samples have not been identified, thereby hindering a clear understanding of their respective functions in normal and disease states. In this study, we applied a proteolytic site-directed approach to identify a peptide reporter substrate and a peptide inhibitor that are selective for Lon but not ClpXP. These chemical tools permit quantitative measurements that distinguish Lon-mediated proteolysis from that of ClpXP in biochemical assays with purified proteases, as well as in intact mitochondria and mitochondrial lysates. This chemical biology approach provides needed tools to further our understanding of mitochondrial ATP-dependent proteolysis and contributes to the future development of diagnostic and pharmacological agents for treating diseases associated with defects in mitochondrial protein quality