Molekularna evolucija proteinske domene za vezavo na površino celic

Lenarčič, Tea (author), Anderluh, Gregor (mentor)

Številni taksonomsko nesorodni organizmi v svoje okolje izločajo enodomenske proteine z zgradbo beta-sendviča, ki specifično prepoznajo različne komponente celičnih membran. Nekateri med njimi so toksični tako, da v membranah tvorijo pore, drugi pa svojo funkcijo opravijo že s samo vezavo na površino celic. V okviru doktorskega dela smo se podrobneje ukvarjali s proteini NLP in egerolizini. Proteine NLP izločajo različni fitopatogeni mikroorganizmi in s specifičnim prepoznavanjem tarčnega lipida GIPC v membranah rastlinskih celic povzročajo nekrozo, hkrati pa stimulirajo rastlinski imunski sistem. Z določitvijo tridimenzionalnih zgradb kompleksov toksičnega proteina NLPPya z glukozaminom oziroma manozaminom smo razložili interakcijo med proteini NLP in sladkornim delom tarčnega lipida GIPC v membranah tarčnih celic. Za študije razlik med zgradbami toksičnih in netoksičnih predstavnikov proteinov NLP smo določili tridimenzionalno zgradbo proteina HaNLP3. S strukturno-sekvenčno analizo smo določili pomembne aminokislinske zamenjave v zankah, ki zapirajo osrednji žleb in tako pripomorejo k omejeni plastičnosti vezavnega mesta, to pa se posledično odraža v netoksičnosti. Prav tako smo določili tridimenzionalni zgradbi egerolizinov OlyA6 in RahU, s čimer smo obogatili nabor zgradb proteinov, ki specifično prepoznavajo komponente v membrani. Na podlagi primerjav tridimenzionalnih zgradb proteinskih družin NLP, aktinoporinov, aktinoporinom podobnega proteina, egerolizinov, termostabilnih direktnih hemolizinov, glivnih lektinov in proteinov TLP smo opazili razporejanje proteinov v dve skupini: proteini, ki se vežejo na komponente membran, in proteini, ki se ne vežejo na membrane. S podrobnejšo analizo topoloških diagramov vseh sedmih družin smo izluščili dva strukturna motiva, ki sta drugačna kot pri podobnih beta-sendvičih, prisotnih v naravi. S tem se te družine uvrščajo v nov razred beta-sendvičev, ki se je tekom evolucije prilagodil življenjskim potrebam. S poznavanjem značilnosti tovrstnih beta-sendvičev lahko na isto osnovo združimo več vezavnih mest, kar bi v bodoče lahko uporabili kot dostavne mehanizme ali za označevanje lipidnih skupkov v membranah pri celični biologiji.

• Language: Slovenian
• Keywords: Proteini NLP, rastlinski patogen, evolucija, primerjava tridimenzionalnih zgradb, beta-sendvič
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2018
• PID: 20.500.12556/RUL-100586
• COBISS.SI-ID: 3903508
• Publication date in RUL: 31.03.2018

Secondary language

• Language: English
• Title: Molecular evolution of cell surface-binding domain

Abstract

Many taxonomic unrelated organisms produce small beta-structured proteins that specifically bind to various cell membrane components. Some exhibit toxic activity by forming pores in the membranes, while other elicit molecular response just by binding to the cell surface. In this work two protein families were studied thoroughly. NLP proteins are a group of proteins, produced by various phytopathogenic microorganisms and cause necrosis upon binding to target glycolipids, GIPCs, in dicot plant membranes. These proteins also stimulate plant immune response. By determining crystal structures of the NLPPya in complex with glucosamine and mannosamine, respectively, we gained structural insight into the interaction between NLP proteins and glycosyl part of GIPCs. To study differences between toxic and non-toxic NLP proteins we also determined crystal structure of non-toxic HaNLP3 protein. Close examination of HaNLP3 structure in comparison to toxic NLPPya pinpointed a set of unique structural features in the loops that contribute to the loss of binding site plasticity, and therefore, HaNLP3 toxic inactivity. We also determined structures of aegerolysins OlyA6 and RahU, which resulted in enriching the set of protein structures that specifically recognize cell membrane components. Based on crystal structure comparison of NLP proteins, actinoporins, actinoporine-like protein, aegerolysins, thermostable direct hemolysins, fungal lectins and TLP proteins we observed general distribution to two main groups: proteins that bind to the cell membrane components and proteins that do not. Detailed topology analysis uncovered two structural motifs, present only in these protein families in contrast to other similar beta-sandwiches in nature. As a result, these proteins can be classified as a new group of beta-sandwiches which have adapted to their needs through evolution. Understanding the structural properties of such beta-sandwiches together with incorporating different binding sites to the same molecular basis can be used as delivery mechanisms or cell surface labeling in cell biology.

• Keywords: NLP proteins, plant pathogen, evolution, structural comparison, beta-sandwich