The mechanism of the β-lactam antibacterials is the functionally irreversible acylation of the enzymes that catalyze the cross-linking steps in the biosynthesis of their peptidoglycan cell wall. The Gram-positive pathogen Staphylococcus aureus uses one primary resistance mechanism based on an enzyme, called penicillin-binding protein 2a (PBP2a), which is involved in this biosynthetic pathway being able to discriminate effectively against the β-lactam antibiotics as potential inhibitors, and in favor of the peptidoglycan substrate. The basis for this discrimination is an allosteric site, distal from the active site, that when properly occupied concomitantly opens the gatekeeper residues within the active site and realigns the conformation of key residues to permit catalysis. Throughout a combination of different techniques (X-ray crystallography and computational analysis by molecular dynamics and quantum mechanics), our results provide critical information about the regulation mechanism of PBP2a, a key protein in the primary resistance mechanism against antibiotics, giving us detailed information about the structural basis of communication between the allosteric and catalytic sites. Furthermore, this study reveals how β-lactam antibiotics mimicry the peptidoglycan substrates, as foundational to the mechanistic understanding of emerging PBP2a resistance mutations. This is part of a collaborative effort between the IQFR and the Univ. of Notre Dame (Indiana, USA).
Mahasenan, K.; Molina, R.; Bouley, R.; Batuecas, M.; Fisher, J.; Hermoso, J.A.; Chang, M. and Mobashery*, S. “Conformational dynamics in penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus, allosteric communication network and enablement of catalysis”. J. Am. Chem. Soc. (2017).
The oriented attachment of molecules in general and proteins in particular at the interface of nanoparticles is currently a challenge in (bio)nanotechnology. In this work carried out by research groups of the Institute of Physical Chemistry Rocasolano, Institute of Material Science of Madrid and Institute of Catalysis and Petroleochemistry, agarose-coated magnetic nanoparticles have been prepared and characterized. They have shown that these nanoparticles constitute an excellent experimental platform for the oriented attachment of recombinant proteins tagged with the β-trefoil lectin LSL150. Optimization of the preparation of the agarose-coated magnetic nanoparticles as followed by a survey of techniques such as dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric studies, required the decoupling of particle formation from agarose coating. LSL150 interacted with these agarose-coated nanoparticles exclusively through the recognition of the sugars of the polymer, forming highly stable complexes. The marked topological polarity of this small lectin makes it an excellent molecular adaptor for the oriented attachment of proteins at the nanoparticle interface since they always face the bulk solvent. This fact opens up new possibilities for the design of novel and more efficient (bio)sensors.
Iván Acebrón, Amalia G. Ruiz-Estrada, Yurena Luengo, María del Puerto Morales, José Manuel Guisán, and José Miguel Mancheño. “Oriented Attachment of Recombinant Proteins to Agarose-Coated Magnetic Nanoparticles by Means of a β‑Trefoil Lectin Domain”. Bioconjugate Chemistry (2016) 27, 2734−2743.
Apoptin is a small protein from the chicken anemia virus which selectively induces apoptosis (programmed cell suicide) in over 80 different cancer cell lines, yet does not harm healthy cells. Apoptin is a promising cancer lead and is progressing through clinical trials. Nevertheless, Apoptin's strong tendency to oligomerize limits its ability to enter cells and thwarts studies of its structure. Therefore, we have prepared and characterized a monomeric Apoptin variant that retains most of the wild type protein's selective anti-cancer activity. Using NMR spectroscopy, this variant was shown to be intrinsically disordered and dynamic on ps-ms time scales. The conformational ensemble is not significantly affected by specific phosphorylation, addition of Mg++, pH changes or red/ox conditions. These findings support a model for Apoptin's mechanism of action in which cancer specific kinases phosphorylate Apoptin, leading to its accumulation in the nucleus and activation of p53-independent apoptosis.
1. "Insights into the mechanism of Apoptin's exquisitely selective anti-tumor action from atomic level characterization of its conformation and dynamics." Ruiz-Martínez S, Pantoja-Uceda D, Castro J, Vilanova M, Ribó M, Bruix M, Benito A, Laurents DV. Arch Biochem Biophys. (2017) 614:53-64.
2. "A truncated Apoptin protein variant selectively kills cancer cells." Ruiz-Martínez S, Castro J, Vilanova M, Bruix M, Laurents DV, Ribó M, Benito A. Invest New Drugs (2017).
The protein complex formed by the Ca2+ sensor NCS-1 and the guanine exchange factor Ric8a co-regulates in a antagonistic manner synapse number and probability of neurotransmitter release, emerging as a potential therapeutic target for diseases affecting synapses such as Fragile X syndrome (FXS), the most common heritable autism disorder. By combining crystallographic and chemoinformatic methodologies, a new and small phenothiazine derivative has been found to inhibit this protein complex, whose contact surface is big and complex. The administration of the compound reduces the aberrant excess of synapse number to normal levels and improves associative learning in a Drosophila FXS model. Finally, the structure-function studies have demonstrated the mechanism of action of this new molecule. This work opens the path to the generation of new drugs to treat neuronal diseases affecting synapse function, such as Autism or Alzheimer.
This work has been carried out by researchers from three CSIC Institutes (Instituto de Química-Física “Rocasolano”, Instituto Cajal and Centro de Investigaciones Biológicas) and the BSRC “Alexander Fleming” in Greece.
Alicia Mansilla, Antonio Chaves-Sanjuan, Nuria E. Campillo, Ourania Semelidou, Loreto Martínez-González, Lourdes Infantes, Juana María González-Rubio, Carmen Gil, Santiago Conde, Efthimio M. C. Skoulaki, Alberto Ferrús, Ana Martínez, María José Sánchez-Barrena. “Interference of the complex between NCS-1 and Ric8a with phenothiazines regulates synaptic function and is an approach for fragile X syndrome”. Proc. Nat. Acad. Sci., PNAS (2017).
EFE press release
CSIC press release
A complex link exists between cell-wall recycling/repair and the manifestation of resistance to β-lactam antibiotics in many Enterobacteriaceae and Pseudomonas aeruginosa. This process is mediated by specific cell-wall-derived muropeptide products. These muropeptides are internalized into the cytoplasm and bind to the transcriptional regulator AmpR, which controls the cytoplasmic events that lead to expression of β-lactamase, an antibiotic-resistance determinant. By a combination of X-ray crystallography, mass spectrometry and molecular dynamics techniques we have characterized the effector-binding domain (EBD) of AmpR. Our results provide insights on the muropeptides triggering antibiotics resistance and revises the dogma in the field.
This is part of a collaborative effort between the IQFR and the Univ. of Notre Dame (Indiana, USA).
Dik, D.A.; Domínguez-Gil, T.; Lee, M.; Hesek, D.; Byun, B.; Fishovitz, J.; Boggess, B.; Hellman, L.M.; Fisher, J. F.; Hermoso, J.A.; Mobashery, S. “Muropeptide Binding and the X-Ray Structure of the Effector Domain of the Transcriptional Regulator AmpR of Pseudomonas aeruginosa”. J. Am. Chem. Soc. (2017).