Implementation of ribosome display technique for studying protein-membrane interactions

Od holesterola odvisni citolizini (ang. cholesterol-dependent cytolysins, CDC) imajo pomembno vlogo pri virulentnosti bakterij. Razumevanje njihovega delovanja je pomembno za razvoj učinkovin za zaviranje njihove aktivnosti ter za možnost njihove uporabe v terapevtske, biotehnološke in sinteznobiološke namene. S tehniko predstavitve na ribosomih smo pridobili vezavno proteinsko ogrodje na osnovi racionalno načrtovanega proteina z ankirinskimi ponovitvami (DARPin-a, ang. designed ankyrin repeat protein), ki se je vezalo na Y406A, ki je od pH odvisen mutantni CDC. Izbrani DARPin se je z veliko afiniteto (KD ~ 100 nM) vezal na Y406A na tak način, da je še vedno omogočal vezavo Y406A na membrano, vendar do nastanka pore ni prišlo, hkrati pa je bila vezava DARPin-a na Y406A mogoča, ko je bil slednji že vezan na membrano. Z vpeljavo tehnike predstavitve na ribosomih smo pridobili tudi različice domene CDC za vezavo na membrano (PFOD4), ki so se na membrano vezale z različnimi vezavnimi parametri. Divji tip PFOD4 se je vezal na membrano s 50 molarnimi odstotki holesterola z afiniteto (KD) v nanomolarnem območju. Testirali smo dve različici PFOD4, od katerih se je ena vezala na membranski holesterol manj specifično, in sicer na dve različno dostopni populaciji membranskega holesterola. Obe različici sta imeli KD v enakem območju kot divji tip PFOD4. Na osnovi selektivnega in specifičnega zaviranja aktivnosti CDC z DARPin-om smo postavili proteinska logična vrata na membrani in pokazali princip delovanja takega sistema v sintezni biologiji. Tehnika predstavitve na ribosomih se je izkazala kot primerna tudi za pridobitev različic membranskovezavnih domen CDC s spremenjenimi vezavnimi lastnostmi. V kombinaciji z uveljavljenimi tehnikami za merjenje interakcij predstavlja predstavitev na ribosomih dobrodošlo dodatno orodje za proučevanje interakcij proteinov z membranami, pri čemer so njene glavne prednosti predvsem prilagodljivost, omogočanje številnih kombinacij zamenjav aminokislinskih ostankov ter možnost zorenja afinitete.Cholesterol-dependent cytolysins (CDCs) are important virulence factors. An in-depth understanding of their membrane binding and pore formation mechanism is crucial for the development of their inhibitors and for their utilization in therapeutic, biotechnological, and synthetic biology applications. The ribosome display technique enabled us to gain the protein binding scaffold based on designed ankyrin protein (DARPin) with a high binding affinity (KD ~ 100 nM) towards the Y406A, which is a pH-dependent mutant of CDC. Selected DARPin bound to the Y406A and inhibited its pore forming activity, although the membrane-binding step of Y406A was not affected. The DARPin was able to bind to the Y406A also when the Y406A was already inserted into a membrane. Implementation of ribosome display was successful also for selection of CDC membrane-binding domain variants (PFOD4) with different binding parameters. The wild-type PFOD4 bound to the membrane with 50 molar percent of cholesterol with a nanomolar affinity (KD). Between the two tested PFOD4 variants, one bound to the membrane cholesterol less specifically, namely to two differently accessible populations of membrane cholesterol, whereas both variants had the KDs in a similar range as the wild-type PFOD4. Based on selective and specific inhibition of the CDC with the DARPin, we constructed the protein logic gate on a membrane and showed a proof of concept for such a system in synthetic biology. Ribosome display proved appropriate also for enrichment of membrane-binding domains with altered binding characteristics. Together with established techniques for measuring molecular interactions, ribosome display is an appreciated additional tool for studying protein-membrane interactions, with its main advantages being flexibility, allowance of numerous combinations of amino acid substitutions, and the possibility of affinity maturation

Inhibition of Pore-Forming Proteins

Perforation of cellular membranes by pore-forming proteins can affect cell physiology, tissue integrity, or immune response. Since many pore-forming proteins are toxins or highly potent virulence factors, they represent an attractive target for the development of molecules that neutralize their actions with high efficacy. There has been an assortment of inhibitors developed to specifically obstruct the activity of pore-forming proteins, in addition to vaccination and antibiotics that serve as a plausible treatment for the majority of diseases caused by bacterial infections. Here we review a wide range of potential inhibitors that can specifically and effectively block the activity of pore-forming proteins, from small molecules to more specific macromolecular systems, such as synthetic nanoparticles, antibodies, antibody mimetics, polyvalent inhibitors, and dominant negative mutants. We discuss their mechanism of inhibition, as well as advantages and disadvantages

Design of protein logic gate system operating on lipid membranes

Lipid membranes are becoming increasingly popular in synthetic biology due to their biophysical properties and crucial role in communication between different compartments. Several alluring protein–membrane sensors have already been developed, whereas protein logic gates designs on membrane-embedded proteins are very limited. Here we demonstrate the construction of a two-level protein–membrane logic gate with an OR-AND logic. The system consists of an engineered pH-dependent pore-forming protein listeriolysin O and its DARPin-based inhibitor, conjugated to a lipid vesicle membrane. The gate responds to low pH and removal of the inhibitor from the membrane either by switching to a reducing environment, protease cleavage, or any other signal depending on the conjugation chemistry used for inhibitor attachment to the membrane. This unique protein logic gate vesicle system advances generic sensing and actuator platforms used in synthetic biology and could be utilized in drug delivery

Pore-forming moss protein bryoporin is structurally and mechanistically related to actinoporins from evolutionarily distant cnidarians

Pore-forming proteins perforate lipid membranes and consequently affect their integrity and cell fitness. Therefore, it is not surprising that many of these proteins from bacteria, fungi, or certain animals act as toxins. While pore-forming proteins have also been found in plants, there is little information about their molecular structure and mode of action. Bryoporin is a protein from the moss Physcomitrium patens, and its corresponding gene was found to be upregulated by various abiotic stresses, especially dehydration, as well as upon fungal infection. Based on the amino acid sequence, it was suggested that bryoporin was related to the actinoporin family of pore-forming proteins, originally discovered in sea anemones. Here, we provide the first detailed structural and functional analysis of this plant cytolysin. The crystal structure of monomeric bryoporin is highly similar to those of actinoporins. Our cryo-EM analysis of its pores showed an actinoporin-like octameric structure, thereby revealing a close kinship of proteins from evolutionarily distant organisms. This was further confirmed by our observation of bryoporin’s preferential binding to and formation of pores in membranes containing animal sphingolipids, such as sphingomyelin and ceramide phosphoethanolamine; however, its binding affinity was weaker than that of actinoporin equinatoxin II. We determined bryoporin did not bind to major sphingolipids found in fungi or plants, and its membrane-binding and pore-forming activity was enhanced by various sterols. Our results suggest that bryoporin could represent a part of the moss defense arsenal, acting as a pore-forming toxin against membranes of potential animal pathogens, parasites, or predators

Sequestration of membrane cholesterol by cholesterol-binding proteins inhibits SARS-CoV-2 entry into Vero E6 cells

Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4$^{E458L}$ to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and beta-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small beta-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The beta-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the beta-annulus peptide for vaccine design

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and β-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small β-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The β-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the β-annulus peptide for vaccine design