RESUMO
Gram-negative bacteria expressing class A ß-lactamases pose a serious health threat due to their ability to inactivate all ß-lactam antibiotics. The acyl-enzyme intermediate is a central milestone in the hydrolysis reaction catalyzed by these enzymes. However, the protonation states of the catalytic residues in this complex have never been fully analyzed experimentally due to inherent difficulties. To help unravel the ambiguity surrounding class A ß-lactamase catalysis, we have used ultrahigh-resolution X-ray crystallography and the recently approved ß-lactamase inhibitor avibactam to trap the acyl-enzyme complex of class A ß-lactamase CTX-M-14 at varying pHs. A 0.83-Å-resolution CTX-M-14 complex structure at pH 7.9 revealed a neutral state for both Lys73 and Glu166. Furthermore, the avibactam hydroxylamine-O-sulfonate group conformation varied according to pH, and this conformational switch appeared to correspond to a change in the Lys73 protonation state at low pH. In conjunction with computational analyses, our structures suggest that Lys73 has a perturbed acid dissociation constant (pKa) compared with acyl-enzyme complexes with ß-lactams, hindering its function to deprotonate Glu166 and the initiation of the deacylation reaction. Further NMR analysis demonstrated Lys73 pKa to be â¼5.2 to 5.6. Together with previous ultrahigh-resolution crystal structures, these findings enable us to follow the proton transfer process of the entire acylation reaction and reveal the critical role of Lys73. They also shed light on the stability and reversibility of the avibactam carbamoyl acyl-enzyme complex, highlighting the effect of substrate functional groups in influencing the protonation states of catalytic residues and subsequently the progression of the reaction.
Assuntos
Compostos Azabicíclicos/química , Compostos Azabicíclicos/farmacologia , Prótons , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/química , beta-Lactamases/efeitos dos fármacos , Acilação , Compostos Azabicíclicos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/efeitos dos fármacos , Sítios de Ligação , Catálise , Cristalografia por Raios X , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Inibidores de beta-Lactamases/química , beta-Lactamases/metabolismoRESUMO
The prevalence of multidrug-resistant Pseudomonas aeruginosa has led to the reexamination of older "forgotten" drugs, such as temocillin, for their ability to combat resistant microbes. Temocillin is the 6-α-methoxy analogue of ticarcillin, a carboxypenicillin with well-characterized antipseudomonal properties. The α-methoxy modification confers resistance to serine ß-lactamases, yet temocillin is ineffective against P. aeruginosa growth. The origins of temocillin's inferior antibacterial properties against P. aeruginosa have remained relatively unexplored. Here, we analyze the reaction kinetics, protein stability, and binding conformations of temocillin and ticarcillin with penicillin-binding protein 3 (PBP3), an essential PBP in P. aeruginosa We show that the 6-α-methoxy group perturbs the stability of the PBP3 acyl-enzyme, which manifests in an elevated off-rate constant (koff) in biochemical assays comparing temocillin with ticarcillin. Complex crystal structures with PBP3 reveal similar binding modes of the two drugs but with important differences. Most notably, the 6-α-methoxy group disrupts a high-quality hydrogen bond with a conserved residue important for ligand binding while also being inserted into a crowded active site, possibly destabilizing the active site and enabling water molecule from bulk solvent to access and cleave the acyl-enzyme bond. This hypothesis is supported by the observation that the acyl-enzyme complex of temocillin has reduced thermal stability compared with ticarcillin. Furthermore, we explore temocillin's mechanism of ß-lactamase inhibition with a high-resolution complex structure of CTX-M-14 class A serine ß-lactamase. The results suggest that the α-methoxy group prevents hydrolysis by locking the compound into an unexpected conformation that impedes access of the catalytic water to the acyl-enzyme adduct.
Assuntos
Penicilinas/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Ticarcilina/farmacologia , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Pseudomonas aeruginosa/genética , beta-Lactamases/genética , beta-Lactamas/metabolismoRESUMO
Designing organic saccharide sensors for use in aqueous solution is a nontrivial endeavor. Incorporation of hydrogen bonding groups on a sensor's receptor unit to target saccharides is an obvious strategy but not one that is likely to ensure analyte-receptor interactions over analyte-solvent or receptor-solvent interactions. Phenylboronic acids are known to reversibly and covalently bind saccharides (diols in general) with highly selective affinity in aqueous solution. Therefore, recent work has sought to design such sensors and understand their mechanism for allowing fluorescence with bound saccharides. In past work, binding orientations of several saccharides were determined to dimethylaminomethylphenylboronic acid (DMPBA) receptors with an anthracene fluorophore; however, the binding orientation of d-fructose to such a sensor could not be determined. In this work, we investigate the potential binding modes by generating 20 possible bidentate and six possible tridentate modes between fructose and DMPBA, a simplified receptor model. Gas phase and implicit solvent geometry optimizations, with a myriad functional/basis set pairs, were carried out to identify the lowest energy bidentate and tridentate binding modes of d-fructose to DMPBA. An interesting hydrogen transfer was observed during selected bidentate gas phase optimizations; this transfer suggests a strong sharing of the hydrogen atom between the boronate hydroxyl and amine nitrogen.
Assuntos
Ácidos Borônicos/química , Frutose/análise , Frutose/química , Espectrometria de Fluorescência/instrumentação , Modelos Moleculares , Conformação MolecularRESUMO
Primaquine (PQ) is the only commercially available drug that clears dormant liver stages of malaria and blocks transmission to mosquito vectors. Although an old drug, much remains to be known about the mechanism(s) of action. Herein we develop a fluorescent tagged PQ to discover cellular localization in the human malaria parasite, Plasmodium falciparum. Successful synthesis and characterization of a primaquine-coumarin fluorescent probe (PQCP) demonstrated potency equivalent to the parent drug and the probe was not cytotoxic to HepG2 carcinoma cells. Cellular localization was found primarily in the cytosol of the asexual erythrocytic and gametocyte stages of parasite development.
Assuntos
Antimaláricos/química , Corantes Fluorescentes/química , Plasmodium falciparum/metabolismo , Primaquina/química , Aminoquinolinas/química , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Eritrócitos/parasitologia , Células Hep G2 , Humanos , Estágios do Ciclo de Vida , Malária Falciparum/tratamento farmacológico , Microscopia de Fluorescência , Plasmodium falciparum/química , Plasmodium falciparum/efeitos dos fármacos , Primaquina/metabolismo , Primaquina/farmacologia , Primaquina/uso terapêuticoRESUMO
In the Firmicutes phylum, GpsB is a membrane associated protein that coordinates peptidoglycan synthesis with cell growth and division. Although GpsB has been studied in several bacteria, the structure, function, and interactome of Staphylococcus aureus GpsB is largely uncharacterized. To address this knowledge gap, we solved the crystal structure of the N-terminal domain of S. aureus GpsB, which adopts an atypical, asymmetric dimer, and demonstrates major conformational flexibility that can be mapped to a hinge region formed by a three-residue insertion exclusive to Staphylococci. When this three-residue insertion is excised, its thermal stability increases, and the mutant no longer produces a previously reported lethal phenotype when overexpressed in Bacillus subtilis. In S. aureus, we show that these hinge mutants are less functional and speculate that the conformational flexibility imparted by the hinge region may serve as a dynamic switch to fine-tune the function of the GpsB complex and/or to promote interaction with its various partners. Furthermore, we provide the first biochemical, biophysical, and crystallographic evidence that the N-terminal domain of GpsB binds not only PBP4, but also FtsZ, through a conserved recognition motif located on their C-termini, thus coupling peptidoglycan synthesis to cell division. Taken together, the unique structure of S. aureus GpsB and its direct interaction with FtsZ/PBP4 provide deeper insight into the central role of GpsB in S. aureus cell division.
Assuntos
Proteínas de Bactérias , Proteínas do Citoesqueleto , Ligação Proteica , Conformação Proteica , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Staphylococcus aureus/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/química , Cristalografia por Raios X , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/química , Modelos MolecularesRESUMO
Low-barrier hydrogen bonds (LBHBs) are a special type of short hydrogen bond (HB) that is characterized by the equal sharing of a hydrogen atom. The existence and catalytic role of LBHBs in proteins has been intensely contested. Advancements in X-ray and neutron diffraction methods has revealed delocalized hydrogen atoms involved in potential LBHBs in a number of proteins, while also demonstrating that short HBs are not necessarily LBHBs. More importantly, a series of experiments on ketosteroid isomerase (KSI) have suggested that LBHBs are significantly stronger than standard HBs in the protein microenvironment in terms of enthalpy, but not free energy. The discrepancy between the enthalpy and free energy of LBHBs offers clues to the challenges, and potential solutions, of the LBHB debate, where the unique strength of LBHBs plays a special role in the kinetic processes of enzyme function and structure, together with other molecular forces in a pre-organized environment.
Assuntos
Biocatálise , Hidrogênio/química , Proteínas/metabolismo , Esteroide Isomerases/metabolismo , Animais , Bactérias/química , Bactérias/enzimologia , Humanos , Ligação de Hidrogênio , Plantas/química , Plantas/enzimologia , Conformação Proteica , Proteínas/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Esteroide Isomerases/química , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The Asp233-Asp246 pair is highly conserved in Class A ß-lactamases, which hydrolyze ß-lactam antibiotics. Here, we characterize its function using CTX-M-14 ß-lactamase. The D233N mutant displayed decreased activity that is substrate-dependent, with reductions in kcat /Km ranging from 20% for nitrocefin to 6-fold for cefotaxime. In comparison, the mutation reduced the binding of a known reversible inhibitor by 10-fold. The mutant structures showed movement of the 213-219 loop and the loss of the Thr216-Thr235 hydrogen bond, which was restored by inhibitor binding. Mutagenesis of Thr216 further highlighted its contribution to CTX-M activity. These results demonstrate the importance of the aspartate pair to CTX-M hydrolysis of substrates with bulky side chains, while suggesting increased protein flexibility as a means to evolve drug resistance.
Assuntos
Ácido Aspártico/genética , Sequência Conservada , Mutação/genética , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/química , beta-Lactamases/genética , Cristalografia por Raios X , Ligantes , Proteínas Mutantes/química , Especificidade por Substrato , Tetrazóis/química , Tetrazóis/farmacologia , Inibidores de beta-Lactamases/química , beta-Lactamases/metabolismoRESUMO
Compared to aryl-aryl π-stacking interactions, the analogous stacking of heteroarenes on amide π systems is less well understood and vastly underutilized in structure-based drug design. Recent theoretical studies have delineated the important geometric coordinates of the interaction, some of which have been confirmed with synthetic model systems based on Rebek imides. Unfortunately, a broadly useful and tractable protein-ligand model system of this interaction has remained elusive. Here we employed a known inhibitor scaffold to study π-stacking of diverse heteroarene substituents on the amide face of Gly238 in the cephalosporinases CTX-M-14 and CTX-M-27. Biochemical inhibition constants (K i) and biophysical binding constants (K d) were determined for nineteen new analogues against both enzymes, while multiple high-resolution co-crystal structures revealed remarkably consistent placement of the probe heteroarene on Gly238. The data presented support the predicted importance of opposing dipoles in amide-heteroarene interactions and should be useful for evaluating other theoretical predictions concerning these interactions.
RESUMO
CXCL12 is a human chemokine that recognizes the CXCR4 receptor and is involved in immune responses and metastatic cancer. Interactions between CXCL12 and CXCR4 are an important drug target but, like other elongated protein-protein interfaces, present challenges for small molecule ligand discovery due to the relatively shallow and featureless binding surfaces. Calculations using an NMR complex structure revealed a binding hot spot on CXCL12 that normally interacts with the I4/I6 residues from CXCR4. Virtual screening was performed against the NMR model, and subsequent testing has verified the specific binding of multiple docking hits to this site. Together with our previous results targeting two other binding pockets that recognize sulfotyrosine residues (sY12 and sY21) of CXCR4, including a new analog against the sY12 binding site reported herein, we demonstrate that protein-protein interfaces can often possess multiple sites for engineering specific small molecule ligands that provide lead compounds for subsequent optimization by fragment based approaches.