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Instruct-ERIC Webinar Series: Structure Meets Function - Webinar #23

Meeting
Date: 13-Dec-2022

Contact: John Dolan

Instruct

The latest webinar in the Instruct-ERIC Structure Meets Function series is a special edition. This month, guest speakers from Instruct's partner institutions in Latin America will give talks on the work they completed as part of the International Call. Register for the webinar here.

The international call was conducted in the context of the EU-LAC ResInfra project. This project aims to harbour collaborative efforts between Europe and Latin America and the Caribbean. This has been through access calls, staff exchanges, bi-regional events and more! Find out more about Instruct-ERIC's international collaboration here.

This month, both speakers are representing IBR-CONICET in Argentina: Leticia Llarrull and Alejandro Vila!

The webinar took place 15:00 CET (11:00 ART, 14:00 GMT), on 13 December

Take a look at the previous webinars in the series here.

 

 

Moderator: Ana Zeri, LNLS

 

Speaker 1: Leticia Llarrull, IBR-CONICET

Talk Title: Advances in structural and functional characterization of the sensor/transducer MecR1 protein of Staphylococcus aureus

Abstract: Methicillin resistant Staphylococcus aureus (MRSA) is a pathogen that poses a worldwide threat. Resistance to β-lactams in MRSA is inducible, and MecR1 regulates expression of PBP2a, the main resistance determinant. We are interested in unveiling how the presence of β-lactams activate this metalloprotease, resulting in manifestation of resistance, and on understanding the functional differences with its homologue, BlaR1 from S. aureus, which regulates expression of a serin-β-lactamase. To date, no high-resolution model of full-length MecR1 or BlaR1 is available. 

MecR1- and BlaR1-mediated induction of the mec and bla operons was evaluated using S. aureus RN4220 reporter strains, in which GFP expression is under control of either sensor/transducer protein. Fluorescence assays evidenced significant differences in expression of GFP under control of both systems, and suggested almost no induction mediated by MecR1. The differences in affinity for β-lactams of the sensor domains of BlaR1 and MecR1 (the latter displaying lower affinity) could lead to differences in activation at the low antibiotic concentrations that can be used in assays with the GFP reporter strains, which are β-lactam sensitive. Hence, we evaluated MecR1-regulated expression of PBP2a in S. aureus RN4220 (RN4220/mecA strain), using the complete mec operon from S. aureus N315. Strain RN4220/mecA remained sensible to β-lactams. However, qRT-PCR demonstrated β-lactam induced transcription of all the genes of the mec operon, including mecA. Although the strain was not resistant to β-lactams, PBP2a was detected in membranes by mass spectrometry and could be labelled with the fluorescent penicillin Bocillin-FL. Our assays demonstrated that transcription of the genes of the mec operon was regulated by MecR1 in response to β-lactams in RN4220, and this reporter strain gives us the possibility to study the effect of MecR1 mutations on its function. In addition, we showed that PBP2a did not confer resistance in RN4220, suggesting the absence of one or more factors that regulate its activity.
To structurally characterize MecR1, we have purified full-length MecR1. SEC-MALS and AUC experiments showed two oligomerization states in detergent micelles: a monomer (major species) and a dimer. Both species bound Bocillin-FL. The monomer fraction increased upon incubation of MecR1 in membranes with β-lactams, suggesting that the monomer is the physiologically relevant species. Cryogenic electron microscopy (cryo-EM) images of Mistic.MecR1 monomer sample were obtained. The 2D classes revealed some features and shapes. However, the original images had a low signal-to-noise ratio and, in 3D, the ab initio model was difficult to refine, producing a low-resolution map (in the range of 10 to 15 Å). We concluded that Mistic-MecR1 is not a homogeneous sample as it is in equilibrium between monomer and dimer, being the monomer the most abundant species after binding of β-lactams in the membrane. We obtained good preliminary cryo-EM data, that justifies optimization of sample and grids.

 

Speaker 2: Alejandro Vila, IBR-CONICET

Talk Title: In-cell kinetic stability is an essential trait in protein evolution: the case of Metallo-beta-lactamases

Abstract: Protein stability is essential for biological function. In contrast to the vast knowledge on the thermodynamics of protein stability in vitro, little is known about the factors governing the in-cell stability, that defines the lifetime of the native state of proteins within the cell. Here we show that the kinetic stability of the metallo-β-lactamase NDM-1 in the bacterial periplasm is optimized to face metal restriction at the host-pathogen interface. NDM-1 is one of the main responsible of providing resistance to carbapenems in pathogenic bacteria. Despite its high stability in vitro, the non-metalated (apo) NDM-1 is unstable in the bacterial periplasm. NMR reveals that metal dissociations endows the apo-enzyme with flexibility at the C-terminal helices, that enable recognition by specific periplasmic proteases, or to aggregation. Zn(II) binding renders the protein refractory to degradation by quenching this flexibility. Apo-NDM-1 is anchored to the outer membrane, a localization that renders it less accessible to the proteases and less prone to aggregate. More recent clinical variants of NDM accumulate mutations that quench this flexibility therefore enhancing their stability towards proteolysis. This work provides direct evidence of how the kinetic stability of a protein optimized within the bacterial cell, and links metallo-β-lactamase-mediated resistance with the cellular metabolism in the periplasm. In cell NMR also reveals information on the degradation pathway, providing insights into the physiology of the bacterial periplasm.

 

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