20240105_membrane digest – Membrane Protein Biochemistry Lab

Membrane’s digest

* Zampaloni, C., Mattei, P., Bleicher, K. et al.

A novel antibiotic class targeting the lipopolysaccharide transporter.

Nature (2024).

https://doi-org.insb.bib.cnrs.fr/10.1038/s41586-023-06873-0

No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years.

Here: identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against Carbapenem-resistant Acinetobacter baumannii (CRAB).

Mechanism of action = blocking of the transport of bacterial LPS from the IM to its destination on the OM, through inhibition of the LptB2FGC complex.

A clinical candidate effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms.

* Pahil, K.S., Gilman, M.S.A., Baidin, V. et al.

A new antibiotic traps lipopolysaccharide in its intermembrane transporter.

Nature (2024).

https://doi-org.insb.bib.cnrs.fr/10.1038/s41586-023-06799-7

A new class of antibiotics that targets the LPS transport machine in Acinetobacter was recently identified (see Zampaloni et al, above).

Here: using structural, biochemical and genetic approaches, authors show that these antibiotics trap a substrate-bound conformation of the LPS transporter that stalls this machine.

* Tao K, Narita SI, Okada U, Murakami S, Tokuda H.

Dissection of an ABC transporter LolCDE function analyzed by photo-crosslinking.

J Biochem. 2023 Dec 29:mvad118.

doi: 10.1093/jb/mvad118.

The Lol system (= 5 Lol proteins), is responsible for the trafficking of lipoproteins to the OM. LolCDE binds to lipoproteins and transfers them to the periplasmic chaperone LolA.

Cryo-EM structures of E. coli LolCDE have been reported but the mechanisms by which OM lipoproteins are transferred to LolA remain elusive.

Here: investigation of the interaction between LolCDE and lipoproteins using site-specific photo-crosslinking: photo-crosslinkable amino acid into different locations in the lipoprotein-binding cavity =>identification of domains that crosslink with PG-associated lipoprotein (Pal) in vivo.

Using one of the derivatives containing the photo-crosslinkable amino acid, authors developed an in vitro system to analyze the binding of lipoproteins to LolCDE.

* Pata J, Moreno A, Wiseman B, Magnard S, Lehlali I, Dujardin M, Banerjee A, Högbom M, Boumendjel A, Chaptal V, Prasad R, Falson P.

Purification and characterization of Cdr1, the drug-efflux pump conferring azole resistance in Candida species.

Biochimie. 2023 Dec 27:S0300-9084(23)00331-0.

doi: 10.1016/j.biochi.2023.12.007.

Cdr1 from Candida albicans and C. glabrata => resistance to antifungals. Here: purification and characterization of Cdr1, functionally expressed in baker yeast, tagged at their C-terminal end with either a His-tag for the glabrata version or a green fluorescent protein (GFP) preceded by a proteolytic cleavage site for the albicans version.

Assay several detergents and stabilizers => probing of level of extraction and the ATPase activity.

IMAC and SEC => homogenous samples.

* Maharana J, Sano FK, Sarma P, Yadav MK, Duan L, Stepniewski TM, Chaturvedi M, Ranjan A, Singh V, Saha S, Mahajan G, Chami M, Shihoya W, Selent J, Chung KY, Banerjee R, Nureki O, Shukla AK.

Molecular insights into atypical modes of β-arrestin interaction with seven transmembrane receptors.

Science. 2024 Jan 5;383(6678):101-108.

doi: 10.1126/science.adj3347.

Interactions between GPCRs and their downstream signaling partners are often weak, transient, or mediated by short sequences or structural motifs and are thus difficult to study in detail.

Here: cryoEM structures of β-arrestin in complex with full receptors or a phosphopeptide corresponding to the receptor C terminus.

=> visualization of the peptide-bound conformation; the protein’s C terminus adopts an α-helical conformation that the authors propose is important for dynamic recognition of different receptors and phosphorylation patterns.

Membranes

* Karan D. Sharma, Milka Doktorova, M. Neal Waxham, Frederick A. Heberle.

Cryo-EM images of phase separated lipid bilayer vesicles analyzed with a machine learning approach

bioRxiv 2023.12.31.573132;

doi: https://doi.org/10.1101/2023.12.31.573132

Study showing that cryo-EM can detect phase separation in liposomes based on differences in bilayer thickness.

However, the considerable noise within cryo-EM data = challenge for accurately determining the membrane phase state at high spatial resolution.

=> image processing pipeline that utilizes machine learning to predict the bilayer phase in projection images of lipid vesicles.

The approach can correctly identify the bilayer phase at 5 nm lateral resolution with > 90% accuracy => isolate the intensity profiles of coexisting Ld and Lo phases + accurate determination of domain size distributions, number of domains, and phase area fractions.

Methods

* Zhang Y, Gan Y, Zhao W, Zhang X, Zhao Y, Xie H, Yang J.

Membrane Protein Structures in Native Cellular Membranes Revealed by Solid-State NMR Spectroscopy.

JACS Au. 2023 Nov 21;3(12):3412-3423.

doi: 10.1021/jacsau.3c00564.

Residue site-specific structural analysis of MPs in native membranes by ssNMR = challenge (poor spectral sensitivity and serious interference from background protein signals).

Here: new protocol enabling to reveal the secondary structure of the MscL from Methanosarcina acetivorans (MaMscL) in Escherichia coli IM.

Use of the BL21(DE3) is crucial for effectively suppressing background protein labeling without compromising the sensitivity of the target protein.

* Jiko C, Morimoto Y, Tsukihara T, Gerle C.

Large-scale column-free purification of bovine F-ATP synthase.

J Biol Chem. 2023 Dec 28:105603.

doi: 10.1016/j.jbc.2023.105603.

Purification approach for the isolation of bovine F-ATP synthase from heart muscle mitochondria: combination of buffer conditions favoring IF1 binding and sucrose density gradient ultracentrifugation to yield stable complexes at high purity in the milligram range.

GDN : LMNG ratio in a final gradient => fractions either enriched in tetrameric or monomeric F-ATP synthase.

Microbials

* ’Explainable’ AI identifies a new class of antibiotics

An artificial-intelligence graph neural network was trained on experimental data and used to identify chemical substructures that underlie selective antibiotic activity in more than 12 million compounds. This led to the discovery of a class of antibiotics with in vitro and in vivo activity against Gram-positive bacteria, including Staphylococcus aureus.

Wong, F., Zheng, E.J., Valeri, J.A. et al.

Discovery of a structural class of antibiotics with explainable deep learning.

Nature (2023).

https://doi.org/10.1038/s41586-023-06887-8

* A new class of antibiotics is cause for cautious celebration — but the economics must be fixed.

Nature 625, 7 (2024)

doi: https://doi-org.insb.bib.cnrs.fr/10.1038/d41586-023-04086-z

Two studies published in Nature (see Zampaloni et al and Pahil et al Nature papers above) introduce a new class of drug candidates for combating infections caused by the resilient bacterium Acinetobacter baumannii, which poses a significant global health threat. The compounds, including one in clinical trials called zosurabalpin, target the bacterium’s ability to transport essential building blocks. However, challenges in antibiotic development, such as economic incentives and the risk of resistance, underscore the need for international efforts to advance promising drugs and address the growing threat of antimicrobial resistance.

Miscellaneous

* How CRISPR gene editing could help treat Alzheimer’s

Nature 625, 13-14 (2024)

doi: https://doi-org.insb.bib.cnrs.fr/10.1038/d41586-023-03931-5

Some researchers hoping that gene-editing technology can conquer forms of Alzheimer’s caused by genetic mutations.

* The world’s largest proteins? These mega-molecules turn bacteria into predators

Nature 625, 16-18 (2024)

doi: https://doi-org.insb.bib.cnrs.fr/10.1038/d41586-023-03937-z