Fehérjék konformációs dinamikája mint a biomolekuláris felismerés és jelátvitel meghatározó eleme = Protein conformational dynamics as a key determinant in biomolecular recognition and signal transmission

A térszerkezet alapján, a konformációs dinamika figyelembevételével kíséreltük meg az intramolekuláris és molekulák közötti jelátvitel megértését atomi felbontással. Kísérleti objektumok: a komplement rendszer, azon belül is a nemrég felfedezett lektin út fehérjekomplexei, a flagelláris exportrendszer valamint moduláris monomer, dimer és oligomer felépítésű enzimek álltak. Megállapítottuk, hogy FliI ATPáz, amely képes az exportálandó fehérjéje kitekerésére, a FliJ, FliH és FliS komponensekkel együtt képez olyan szupramolekuláris komplexet, amely képes az export szubsztrátumok felismerésére. Leírtuk a foszfoglicerát kináz enzim alloszterikus működési mechanizmusát, atomi felbontással. Feltártuk az izopropilmalát dehidrogenáz molekuláris csuklóinak működését és szerepét az alegységek kölcsönhatásaiban. Szelektív inhibitorokkal a tankönyvi tézissel ellentétes felismerésre jutottunk, miszerint a komplement rendszer lektin útjának meghatározó aktivátora a MASP-1 szerin proteáz. Így a komplement aktiválással összefüggő betegségek új gyógyszercélpont molekuláját azonosítottuk. Felfedeztük, hogy a MASP-1 képes a kininogén hasítása útján, bradikinint felszabadítva, komplement függő gyulladást keltésére. Felfedeztük, hogy a trombinhoz hasonlóan a MASP-1, PAR-4 receptoron keresztül endotél sejteket aktivál. Bizonyítékot találtunk arra, hogy a fehérjék konformációs dinamikája meghatározza a szerkezet evolúciójának lehetséges irányait, több milliárd éves időskálán is. | The CUB2 domain of C1r without calcium has disordered structure. This flexibility, necessary for autocativation of C1r inside the C1 complex, is regulated by calcium. Using MASP-selective inhibitors we proved that, in contrast to the previous textbook picture, MASP-1 is the exclusive activator of MASP-2. Blocking the proteolytic activity of MASP-1 prevents activation of the lectin pathway, therefore MASP-1 is a new target in treating complement related diseases. We solved the structure of the catalytic region of MASP-1. The structure explains the special enzymatic characteristics of this complement protease. We discovered a new, inflammation related function of the complement system: MASP-1 is able to directly activate endothelial cells through cleaving protease activated receptor-4. We discovered that MASP-1 is able to cleave kininogen and liberates bradykinin. In this way MASP-1 can contribute to the local inflammatory reaction triggered by complement activation. The allosteric mechanismnof human PGK has been explored at atomic details. In the dimeric enzyme IPMDH structural and site-directed mutagenesis studies revealed the operation of the two main molecular hinges and their relationship with the subunit interactions. We have shown that conformational motions are linked to protein evolution by producing structural variants that can be evolutionarily stabilized. This process is exemplified by segment-swapped proteins, a new group of proteins discovered by us

Neuronal-specific septin-3 binds Atg8/LC3B, accumulates and localizes to autophagosomes during induced autophagy

In synapses that show signs of local apoptosis and mitochondrial stress and undergo neuro-immunological synapse pruning, an increase in the levels of the presynaptic protein, neuronal-specific septin-3 can be observed. Septin-3 is a member of the septin GTPase family with the ability to form multimers and contribute to the cytoskeleton. However, the function of septin-3 remains elusive. Here, we provide evidence that septin-3 is capable of binding the most-studied autophagy protein Atg8 homolog microtubule-associated protein 1 light chain 3B (LC3B), besides another homolog, GABA receptor-associated protein-like 2 (GABARAPL2). Moreover, we demonstrate that colocalization of septin-3 and LC3B increases upon chemical autophagy induction in primary neuronal cells. Septin-3 is accumulated in primary neurons upon autophagy enhancement or blockade, similar to autophagy proteins. Using electron microscopy, we also show that septin-3 localizes to LC3B positive membranes and can be found at mitochondria. However, colocalization results of septin-3 and the early mitophagy marker PTEN-induced kinase 1 (PINK1) do not support that binding of septin-3 to mitochondria is mitophagy related. We conclude that septin-3 correlates with synaptic/neuronal autophagy, binds Atg8 and localizes to autophagic membranes that can be enhanced with chemical autophagy induction. Based on our results, elevated septin-3 levels might indicate enhanced or impeded autophagy in neurons

Ligand-induced conformational rearrangements regulate the switch between membrane-proximal and distal functions of Rho kinase 2

Rho-associated protein kinase 2 (ROCK2) is a membrane-anchored, long, flexible, multidomain, multifunctional protein. Its functions can be divided into two categories: membrane-proximal and membrane-distal. A recent study concluded that membrane-distal functions require the fully extended conformation, and this conclusion was supported by electron microscopy. The present solution small-angle X-ray scattering (SAXS) study revealed that ROCK2 population is a dynamic mixture of folded and partially extended conformers. Binding of RhoA to the coiled-coil domain shifts the equilibrium towards the partially extended state. Enzyme activity measurements suggest that the binding of natural protein substrates to the kinase domain breaks up the interaction between the N-terminal kinase and C-terminal regulatory domains, but smaller substrate analogues do not. The present study reveals the dynamic behaviour of this long, dimeric molecule in solution, and our structural model provides a mechanistic explanation for a set of membrane-proximal functions while allowing for the existence of an extended conformation in the case of membrane-distal functions