Febs Letters

doi: 10.1002/1873-3468.13418pmid: N/A

Turk, Martin; Baumeister, Wolfgang

doi: 10.1002/1873-3468.13948pmid: 33020915

Structural biologists have traditionally approached cellular complexity in a reductionist manner in which the cellular molecular components are fractionated and purified before being studied individually. This ‘divide and conquer’ approach has been highly successful. However, awareness has grown in recent years that biological functions can rarely be attributed to individual macromolecules. Most cellular functions arise from their concerted action, and there is thus a need for methods enabling structural studies performed in situ, ideally in unperturbed cellular environments. Cryo‐electron tomography (Cryo‐ET) combines the power of 3D molecular‐level imaging with the best structural preservation that is physically possible to achieve. Thus, it has a unique potential to reveal the supramolecular architecture or ‘molecular sociology’ of cells and to discover the unexpected. Here, we review state‐of‐the‐art Cryo‐ET workflows, provide examples of biological applications, and discuss what is needed to realize the full potential of Cryo‐ET.

Riccio, Paolo; Rossano, Rocco

doi: 10.1002/1873-3468.13946pmid: 33011965

The recent explosive increase in the number of works on gut microbiota has been accompanied by the spread of rather vague or improper definitions, chosen more for common use than for experimental evidence. Among them are those defining the human gut microbiota as an organ of our body or as a commensal. But, is the human gut microbiota an organ or a commensal? Here, we address this issue to spearhead a reflection on the real roles of the human gut microbiota in our life. Actually, the misuse of the vocabulary used to describe the properties and functions of the gut microbiota may generate confusion and cause misunderstandings both in the scientific community and among the general public.

Chen, Yixi; Kunath, Tilo; Simpson, Joanna; Homer, Natalie; Sylantyev, Sergiy

doi: 10.1002/1873-3468.13910pmid: 33073864

Human embryonic stem cell (hESC)‐derived midbrain dopamine (DA) neurons stand out as a cell source for transplantation with their sustainability and consistency superior to the formerly used fetal tissues. However, multiple studies of DA neurons in culture failed to register action potential (AP) generation upon synaptic input. To test whether this is due to deficiency of NMDA receptor (NMDAR) coagonists released from astroglia, we studied the functional properties of neural receptors in hESC‐derived DA neuronal cultures. We find that, apart from an insufficient amount of coagonists, lack of interneuronal crosstalk is caused by hypofunction of synaptic NMDARs due to their direct inhibition by synaptically released DA. This inhibitory tone is independent of DA receptors and affects the NMDAR coagonist binding site.

Majorina, Maria A.; Balobanov, Vitaly A.; Uversky, Vladimir N; Melnik, Bogdan S.

doi: 10.1002/1873-3468.13905pmid: 32770670

Apomyoglobin is a widely used model for studying the molecular mechanisms of globular protein folding. This work aimed to analyze the effects of rigidity and length of loops linking protein secondary structure elements on the stability of the molten globule intermediate state. For this purpose, we studied folding/unfolding of mutant apomyoglobin forms with substitutions of loop‐located proline residues to glycine and with loop extension by three or six glycine residues. The kinetic and equilibrium experiments performed gave an opportunity to calculate free energies of different apomyoglobin states. Our analysis revealed that the mutations introduced into the apomyoglobin loops have a noticeable effect on the stability of the intermediate state compared to the unfolded state.

Ansari, Mohammed Yousuf; Batra, Sakshi D.; Ojha, Hina; Dhiman, Kanika; Ganguly, Ashish; Tyagi, Jaya S.; Mande, Shekhar C.

doi: 10.1002/1873-3468.13906pmid: 32808291

Among the two GroEL paralogs in Mycobacterium tuberculosis, GroEL1 and GroEL2, GroEL1 has a characteristic histidine‐rich C terminus. Since histidine richness is likely to be involved in metal binding, we attempted to decipher the role of GroEL1 in chelating metals and the consequence on M. tuberculosis physiology. Isothermal titration calorimetry showed that GroEL1 binds copper and other metals. Mycobacterial viability assay, redox balance, and DNA protection assay concluded that GroEL1 protects from copper stress in vitro. Solution X‐ray scattering and constrained modeling of GroEL1 −/+ copper ions showed reorientation of the apical domain as seen in functional assembly. We conclude that the duplication of chaperonin genes in M. tuberculosis might have led to their evolutionary divergence and consequent functional divergence of chaperonins.

Dobenecker, Marc‐Werner; Marcello, Jonas; Becker, Annette; Rudensky, Eugene; Bhanu, Natarajan V.; Carrol, Thomas; Garcia, Benjamin A.; Prinjha, Rabinder; Yurchenko, Vyacheslav; Tarakhovsky, Alexander

doi: 10.1002/1873-3468.13903pmid:

Oeemig, Jesper S.; Beyer, Hannes M.; Aranko, A. Sesilja; Mutanen, Justus; Iwaï, Hideo

doi: 10.1002/1873-3468.13909pmid: 32805768

Inteins catalyze self‐excision from host precursor proteins while concomitantly ligating the flanking substrates (exteins) with a peptide bond. Noncatalytic extein residues near the splice junctions, such as the residues at the −1 and +2 positions, often strongly influence the protein‐splicing efficiency. The substrate specificities of inteins have not been studied for many inteins. We developed a convenient mutagenesis platform termed “QuickDrop”‐cassette mutagenesis for investigating the influences of 20 amino acid types at the −1 and +2 positions of different inteins. We elucidated 17 different profiles of the 20 amino acid dependencies across different inteins. The substrate specificities will accelerate our understanding of the structure–function relationship at the splicing junctions for broader applications of inteins in biotechnology and molecular biosciences.

Omeis, Fatima; Santos Seica, Ana Filipa; Ermolova, Natalia; Kaback, H. Ronald; Hellwig, Petra

doi: 10.1002/1873-3468.13907pmid: 32780424

The monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic side of lactose permease (LacY) of Escherichia coli and inhibits H+/lactose symport and lactose efflux under nonenergized conditions. At the same time, ligand binding and translocation reactions that do not involve net H+ translocation remain unaffected by 4B1. In this study, surface‐enhanced infrared absorption spectroscopy applied to the immobilized LacY was used to study the pH‐dependent changes in LacY and to access in situ the effect of the 4B1 antibody on the pKa of Glu325, the primary functional H+‐binding site in LacY. A small shift of the pK value from 10.5 to 9.5 was identified that can be corroborated with the inactivation of LacY upon 4B1 binding.

Pierzynowska, Karolina; Gaffke, Lidia; Węgrzyn, Grzegorz

doi: 10.1002/1873-3468.13908pmid: 32880920

We used transcriptomic (RNA‐seq) analyses to determine whether patients suffering from all types and subtypes of mucopolysaccharidosis (MPS), a severe inherited metabolic disease, may be more susceptible to coronavirus disease 2019 (COVID‐19). The expression levels of genes encoding proteins potentially involved in SARS‐CoV‐2 development were estimated in MPS cell lines. Four genes (GTF2F2, RAB18, TMEM97, PDE4DIP) coding for proteins potentially facilitating virus development were down‐regulated, while two genes (FBN1, MFGE8), the products of which potentially interfere with virus propagation, were up‐regulated in most MPS types. Although narrowing of respiratory tract and occurrence of thick mucus, characteristic of MPS, are risk factors for COVID‐19, transcriptomic analyses suggest that MPS cells might be less, rather than more, susceptible to SARS‐CoV‐2 infection.