Regulation of mammalian cell behavior trough designed four-helical bundle protein oligomerization

Sposobnost načrtovanja proteinskih interakcij predstavlja pomembno orodje pri ustvarjanju sintetičnih omrežij za uravnavanje celičnega odziva in številnih drugih funkcij, kot sta lokalizacija in sestavljanje kompleksnih celičnih struktur. Dodatna orodja za uravnavanje tvorbe proteinskih kompleksov bi imela velik prispevek k znanosti, tako kot orodje za raziskave kot tudi za biotehnološke in biomedicinske aplikacije. Svežnji štirih vijačnic so proteinske strukture, primerne za de novo načrtovanje. Na podlagi osnovnega sestavljanja iz dveh domen, kjer vsaka domena obsega dve vijačnici, so svežnje štirih vijačnic predhodno uporabili za (hetero)dimerizacijo proteinov. V sklopu doktorskega dela smo preučevali potencial različnih segmentacij načrtovanega svežnja štirih vijačnic (4HB) kot modelne strukture za načrtovanje dimerizacijskih, trimerizacijskih in tetramerizacijskih modulov iz enega proteina. Z metodo segmentacije smo razširili repertoar ortogonalnih oligomerizacijskih domen iz ene same strukture 4HB, kar zmanjša potrebo po de novo načrtovanju podobnih struktur. Eksperimentalno smo preučili strategijo segmentacije na več svežnjih štirih vijačnic, s čimer smo želeli vpeljati široko uporabo strategije na podobne načrtovane strukture. Poleg možnosti priprave več oligomerizacijskih domen predstavlja segmentacija dodaten vpogled v prispevke posameznih ? vijačnic k tvorbi 4HB ter s tem prepoznavanje problematičnih področij za nadaljnjo optimizacijo načrtovanih struktur. Tovrstne informacije so bile pogosto spregledane v procesu ovrednotenja načrtovanih struktur, kjer je bilo glavno merilo uspešnosti načrtovanja stabilnost celotne strukture. Poleg svoje vrednosti za raziskovanje de novo načrtovanih struktur imajo novo ustvarjeni oligomerizacijski moduli potencial za različne biološke aplikacije. Tekom doktorskega dela smo sestavljanje oligomerizacijskih domen v načrtovano strukturo ovrednotili kot rekonstitucijo razcepljenega poročevalskega proteina kresničkine luciferaze. Pripravljene module smo uporabili za posredovanje informacij različnih bioloških procesov v sesalskih celicah preko izgradnje sintezno-bioloških sistemov za: sočasno in neodvisno uravnavanje izražanja genov preko TALE-VP16 sistemaod rapaloga odvisno inducibilno aktivacijo rekonstitucije razcepljene kresničkine luciferaze ter vpeljavo SbMVp-PPVp proteazne kaskadein sklopljeno zaznavanje CD19 ter CD20 ligandov na površini rakavih celic s prenosom signala v celice CAR-T na področju imunoterapije.The ability to design synthetic protein-protein interactions provides an important tool in creating synthetic networks for the regulation of the cellular response and numerous other functions, such as localization and construction of cellular structures. Additional tools for the regulation of protein assemblies would have strong impact on science, both as a tool for research as well as for versatile applications, from biotech to therapy. Four helical bundles are protein domains that can be designed and have been used to mediate protein (hetero)dimerization based on splitting them into two subdomains each comprising two helices. For this PhD work we decided to further investigate the potentials of diverse segmentation strategy of a de novo designed four helical bundle (4HB) as a platform for generation dimerization, trimerization and tetramerization modules from a single protein. Segmentation strategy can thus extend the repertoire of orthogonal oligomerization domains from a single 4HB structures and reduces the need for de novo designs. We provide a demonstration of generalizability of segmentation strategy on several four helical bundles. Additionally, segmentation provides further detail in four helical bundle structure assembly and contribution of each of the four single peptides to its formation. This information has been often overlooked in the protein design workflow, where only the stability of final structure is assessed. In addition to its value for the investigation of designed structures, newly generated oligomerization modules have potentials for diverse biological applications. We have evaluated the level of affinity of designed oligomerization modules and reconstitution into designed four helical bundle based on split firefly luciferase reconstitution and activation. Further, we demonstrated in mammalian cells, that they can act as mediators of information towards diverse biological processes, such as: simultaneous and independent regulation of gene expression via the TALE-VP16 systemrapalog dependent inducible activation of split firefly luciferase reconstitution and construction of a SbMVp-PPVp protease cascadeand detection of CD19 and CD20 ligands on the surface of cancer cells with signal transduction into CAR-T cells in the field of immunotherapy

Segmentation strategy of de novo designed four-helical bundles expands protein oligomerization modalities for cell regulation

New protein assemblies can be introduced through the fusion of selected proteins with di/oligomerization domains, which interact specifically with their partners but not with other cellular proteins. Here the authors demonstrate that a single four-helical bundle protein can be segmented into several different parts, defining up to four interacting molecules for enzyme reconstitution, gene expression, or CAR-T cell regulation

Metal ion–regulated assembly of designed modular protein cages

Coiled-coil (CC) dimers are versatile, customizable building modules for the design of diverse protein architectures unknown in nature. Incorporation of dynamic self-assembly, regulated by a selected chemical signal, represents an important challenge in the construction of functional polypeptide nanostructures. Here, we engineered metal binding sites to render an orthogonal set of CC heterodimers Zn(II)-responsive as a generally applicable principle. The designed peptides assemble into CC heterodimers only in the presence of Zn(II) ions, reversibly dissociate by metal ion sequestration, and additionally act as pH switches, with low pH triggering disassembly. The developed Zn(II)-responsive CC set is used to construct programmable folding of CC-based nanostructures, from protein triangles to a two-chain bipyramidal protein cage that closes and opens depending on the metal ion. This demonstrates that dynamic self-assembly can be designed into CC-based protein cages by incorporation of metal ion–responsive CC building modules that act as conformational switches and that could also be used in other contexts

Modulation of Coiled-Coil Dimer Stability through Surface Residues while Preserving Pairing Specificity

The coiled-coil dimer is a widespread protein structural motif and, due to its designability, represents an attractive building block for assembling modular nanostructures. The specificity of coiled-coil dimer pairing is mainly based on hydrophobic and electrostatic interactions between residues at positions a, d, e, and g of the heptad repeat. Binding affinity, on the other hand, can also be affected by surface residues that face away from the dimerization interface. Here we show how design of the local helical propensity of interacting peptides can be used to tune the stabilities of coiled-coil dimers over a wide range. By designing intramolecular charge pairs, regions of high local helical propensity can be engineered to form trigger sequences, and dimer stability is adjusted without changing the peptide length or any of the directly interacting residues. This general principle is demonstrated by a change in thermal stability by more than 30 °C as a result of only two mutations outside the binding interface. The same approach was successfully used to modulate the stabilities in an orthogonal set of coiled-coils without affecting their binding preferences. The stability effects of local helical propensity and peptide charge are well described by a simple linear model, which should help improve current coiled-coil stability prediction algorithms. Our findings enable tuning the stabilities of coiled-coil-based building modules match a diverse range of applications in synthetic biology and nanomaterials