Konformacijska dinamika K-Ras4B v interakciji z membrano

magistrsko delo

Neli Sedej (Author), Jurij Reščič (Mentor), Franc Požgan (Thesis defence commission member), Jurij Lah (Thesis defence commission member), Franci Merzel (Co-mentor)

Abstract

K-Ras4B je protein udeležen v celični signalizaciji. Prehajanje med dvema stanjema, GDP-vezanim aktivnim in GDP-vezanim neaktivnim, mu omogoča, da deluje kot molekulsko stikalo in regulira intenziteto in trajanje celičnih signalov, ki vodijo v celične odzive kot sta rast in diferenciacija celic ter inhibicija apoptoze. K-Ras4B je eden najpogosteje mutiranih proteinov v rakavih celicah. Kljub desetletja trajajočim poskusom, je bil do sedaj uspeh na področju razvoja učinkovin, ki bi preko inhibicije K-Ras4B delovale kot zdravilo proti raku, omejen. Za nadaljni razvoj inhibitorjev K-Ras4B je potrebno globoko razumevanje bioloških in kemijskih mehanizmov delovanja tega proteina. V aktivni obliki je K-Ras4B preko neurejene C-terminalne regije zasidran v celično membrano. Katalitična domena K-Ras4B z membrano interagira le prehodno. Pokazano pa je bilo, da katalitična domena z membrano pretežno interagira v določenih orientacijah. V nekaterih izmed teh orientacij je zaradi položaja membrane ovirana interakcija s proteini, ki so pomembni za signalno funkcijo K-Ras4B, kar bi lahko vplivalo na njegovo aktivnost. Več dosedanjih raziskav je bilo usmerjeno v določanje prisotnih orientacij in prehodov med njimi, vpliv različnih orientacij na interno dinamiko katalitične domene pa še ni bil raziskan. V magistrskem delu smo izvedli simulacije molekulske dinamike in pripadajočimi analizami strukturne dinamike K-Ras4B divjega tipa ter K-Ras4B z onkogeno mutacijo G12D v GTP- in GDP- vezanem stanju v vodi in v dveh orientacijah glede na membrano, ki sta bili eksperimentalno pokazani kot dve najpogosteje zasedeni orientaciji. Potrdili smo eksperimentalno opaženo dejstvo, da je pri G12D-K-Ras4B v primerjavi z divjim tipom stabilnost orientacije z α-vijačnicami, orientiranimi paralelno glede na membrano (H1), večja od stabilnosti orientacije z α-vijačnicami, orientiranimi pravokotno (P1). Opazili smo konformacijske prehode, ki bi lahko bili ugodni za izmenjavo GDP z GTP v K-Ras4B-GDP v H1. S kvaziharmonsko analizo smo pokazali, da vezava proteina na membrano poveča njegovo vibracijsko entropijo, kar bi lahko olajšalo konformacijske spremembe, pomembne za funkcijo proteina. Opazili smo nizkofrekvenčne normalne načine nihanja, ki so specifični za protein, vezan na membrano in se razlikujejo med različnimi orientacijami. V teh načinih so prisotni odmiki atomov v regijah, pomembnih za interakcijo z vezavnimi partnerji K-Ras4B in bi lahko kazali na preferenco za specifične funkcijsko pomembne konformacijske spremembe v proteinu na membrani. Naši rezultati dajejo vpogled v vpliv orientacije proteina glede na membrano na dinamiko K-Ras4B, kar bi lahko prispevalo k razvoju inhibitorjev K-Ras, ki bi delovali preko modulacije orientacijske preference v korist neaktivnih orientacij.

Keywords

proteini;K-Ras4B;interakcije protein-membrana;simulacije molekulske dinamike;kvaziharmonska analiza;konformacijska analiza;magistrska dela;

Data

Language:	Slovenian
Year of publishing:	2020
Typology:	2.09 - Master's Thesis
Organization:	UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher:	[N. Sedej]
UDC:	577.322(043.2)
COBISS:	33552643
Views:	476
Downloads:	148
Average score:	0 (0 votes)
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Other data

Secondary language:	English
Secondary title:	Conformational dynamics of membrane-bound K-Ras4B
Secondary abstract:	K-Ras4B is a protein involved in cell signaling. By switching between active GTP-bound and inactive GDP-bound conformation, it functions as a molecular switch, regulating the intensity and the timescale of cellular signals leading to various cellular responses such as cell growth, differentiation and inhibition of apoptosis. K-Ras4B is one of the most frequently mutated proteins in human cancers. Despite decades of efforts, the success in treating cancer by inhibiting K-Ras4B has been limited. For further development of K-Ras4B inhibitors a deep understanding of the biological and chemical mechanisms of its actions is required. In its active form, K-Ras4B is anchored to the plasma membrane through a disordered C-terminal region. The catalytic domain interacts with the membrane transiently however, it has been shown, that the catalytic domain interacts with the membrane preferentially in a limited number of distinct orientations relative to the membrane. In some of the orientations, the interaction with proteins, important for signalling function of K-Ras4B, may be hindered by the membrane, which suggests, that the orientation could influence the activity of the protein. There has been a significant effort in characterizing the most populated orientations and their transitions, however, the effects of different orientations on the internal dynamics of the catalytic domain have not yet been investigated. In this study we performed molecular dynamics simulations with the corresponding analysis of structure and dynamics of K-Ras4B in wild type form and with an oncogenic mutation G12D in GTP- and GDP-bound states in water and in two orientations, which have been experimentally shown as the two dominant orientations. We have corroborated the experimentaly observed fact that in G12D-K-Ras4B the stability of the orientation with parallel helices (H2) relative to the membrane compared to the orientation with perpendicular helices (P1) is increased in G12D-K-Ras4B relative to the wild type. We observed conformational transitions that could be favorable GDP-GTP exchange in K-Ras4B-GDP in H1. Using quasiharmonic analysis we have shown, that the vibrational entropy of membrane-bound protein is increased, which could facilitate the conformational changes, important for protein function. We have observed low-frequency normal modes, which are specific for membrane-bound protein and differ between different orientations. These modes feature displacements of atoms in regions, important for interaction with K-Ras4B binding proteins and might indicate preferences for specific functional conformational changes on the membrane. Our results give insight into the effect of the membrane orientation on dynamics of K-Ras4B, which could contribute to the development of K-Ras4B inhibitors that would act by modulating the orientational preferences towards inactive orientations.
Secondary keywords:	protein-membrane interaction;molecular dynamics simulations;quasiharmonic analysis;
Type (COBISS):	Master's thesis/paper
Study programme:	1000375
Embargo end date (OpenAIRE):	1970-01-01
Thesis comment:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Kemija
Pages:	XVI, 71 str.
ID:	12036922