Structures of protein targeting complexes

Structures of protein targeting complexesMario Halic 11.112588#2911.231.2.14394#2201.2.25354#47761.2.378587#2139298#273563#18010111.2.412588#274550#4831.2.5131.2.6141516286#534587#294171.32172.1182.2192.2.1202.2.22.2.32.2.4212.2.52.32.3.1222.3.22.42.523242.5.125262.5.22.5.3272.5.4282.629302.7312.7.1322.83323.13334586#3563.2554#59435383#290574#32536586#423373839574#1873.2.1538#437403.2.241568#6094243550#5523.2.344540#6323.2.3.145537#323598#574463.33.3.147499#3993.3.2569#62148575#27149564#2523.450585#53851551#71652453544.155327#453586#280564.25758447#7234.2.14.3594.460Abbreviations Eidestattliche Erklärung:References:enTable of contentsHelpInstitut für Biochemie der Medizinischen Fakultät Charité
der Humboldt-Universität zu BerlinDissertationStructures of protein targeting complexesZur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)
im Fach Biologieeingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlinvon
Mario Halic geb. 06.04.1976 in Cakovec, Kroatien

Präsident/Präsidentin der Humboldt-Universität zu Berlin
Prof. Dr. Jürgen Mlynek

Dekan/Dekanin der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Thomas Buckhout Prof. Dr. Peter-M. Kloetzel Prof. Dr. Klaus Peter Hofmann Prof. Christian Spahn eingereicht: 31.01.05Datum der Promotion: 12.10.05 Abstract

Cotranslational translocation of proteins across or into membranes is a vital process in all kingdoms of life. It requires targeting of the translating ribosome to the membrane by the signal recognition particle (SRP), an evolutionary conserved ribonucleoprotein particle. SRP recognizes signal sequences of nascent protein chains emerging from the ribosome. Subsequent binding of SRP leads to pausing of peptide elongation and docking to the membrane-bound SRP receptor. Here, the 12 Å cryo-electron microscopy structure of a targeting complex is presented consisting of mammalian SRP bound to an active 80S ribosome carrying a signal sequence. A molecular model of SRP in this functional conformation was generated. The model reveals how the S-domain of SRP contacts the large ribosomal subunit at the nascent chain exit site to bind the signal sequence, and that the Alu-domain reaches into the elongation factor binding site of the ribosome explaining its elongation arrest activity. A molecular model of the first steps of protein targeting is presented. Moreover, also the docking step has been visualized by solving a cryo-EM structure of the ribosome-SRP complex bound to the SRP receptor. This structure provides first hints regarding the mechanism of ribosome transfer to the translocon. As a side result the position of the functionally significant ribosomal protein L30e has been identified in the high resolution maps of the wheat germ ribosome.

SRP SR Ribosome signal sequence protein targeting cryo-electron microscopy Zusammenfassung

Sowohl die kotranslationale Translokation von sekretorischen Proteinen durch die Membran als auch die Insertion von Membranproteinen sind essentielle Prozesse in allen lebenden Zellen. Sie erfordern die Sortierung des translatierenden Ribosoms zur Membran mittels des Signalerkenungspartikels (SRP), eines im Verlauf der Evolution konservierten Ribonukleoprotein-Partikels. SRP erkennt die Signalsequenz einer wachsenden Proteinkette, sobald diese aus dem Ribosom hervortritt. Die Bindung von SRP führt zum Anhalten der Peptidelongation (Elongationsarrest) und zum Andocken an den membrangebundenen SRP-Rezeptor (SR). In dieser Arbeit wird die 12 Å Kryo-Elektronenmikroskopie-Struktur eines Sortierungs-Komplexes dargestellt, der aus dem Säugetier-SRP gebunden an ein aktives Ribosom mit Signalsequenz besteht. Ein erstes molekulares Modell von SRP in dieser Konformation wurde erzeugt. Es zeigt wie die S-Domäne von SRP die große ribosomale Untereinheit nahe dem Peptidtunnel-Ausgang kontaktiert, um dort die Signalsequenz zu binden. Außerdem wird deutlich wie die Alu-Domäne von SRP in die Bindungsstelle für Elongationsfaktoren hineinreicht, wodurch die Elongationsarrest-Aktivität der Alu-Domäne erklärt wird. Auf dieser Basis konnte ein erstes Struktur-basiertes Modell der ersten Schritte der kotranslationalen Proteinsortierung entworfen werden. Darüberhinaus wurde auch der Schritt des Andockens an die Membran visualisiert, indem die Struktur des Ribosom-SRP-SR-Komplexes durch Kryo-EM gelöst wurde. Erste Schlüsse hinsichtlich des Mechanismus, der das Ribosom vom SRP zum Translokon transferiert, können hier gezogen werden. Als Nebenergebnis konnte durch die erreichte hohe Auflösung die Position des wichtigen ribosomalen Proteins L30e in der Kryo-EM-Struktur des Weizenkeim-Ribosoms idenifiziert werden.

SRP SR Kryo-Elektronenmikroskopie Signalsequenz Table of contents

1 Introduction
- 1.1 Protein Targeting
- 1.2 Signal recognition particle (SRP)
  - 1.2.1 SRP RNA
  - 1.2.2 Evolutionary conservation
  - 1.2.3 SRP assembly
  - 1.2.4 SRP54 and signal sequence recognition
  - 1.2.5 Elongation arrest
  - 1.2.6 GTPase cycle and SRP receptor
- 1.3 Goals
2 Materials and Methods
- 2.1 Purification of ribosome nascent chain complexes (RNCs)
- 2.2 Generation of DNA fragments by polymerase chain reaction
  - 2.2.1 Agarose gel electrophoresis
  - 2.2.2 Generation of RNA by DNA transcription
  - 2.2.3 Translation and RNC purification
  - 2.2.4 Protein precipitation and SDS PAGE
  - 2.2.5 Western Blot analysis
- 2.3 Reconstitution of SRP-RNC complex
  - 2.3.1 Reconstitution and sucrose gradient
  - 2.3.2 Grid preparation
- 2.4 Electron microscopy
- 2.5 Image processing
  - 2.5.1 Particle picking
  - 2.5.2 Alignment
  - 2.5.3 3D-reconstruction
  - 2.5.4 Refinement
- 2.6 Building the SRP model
- 2.7 High resolution structure of SRP-RNC complex
  - 2.7.1 L30 localization and the model
- 2.8 Structure of SR-SRP-RNC complex
3 Results
- 3.1 Ribosome nascent chain complex purification and reconstitution of SRP-RNC complex
- 3.2 Structure of the signal recognition particle interacting with the elongation arrested ribosome
  - 3.2.1 Environment and function of the Alu-domain
  - 3.2.2 Environment and function of the S-domain
  - 3.2.3 Functional states of SRP54
    - 3.2.3.1 A flexible domain linkage between SRP54M and SRP54NG
- 3.3 Structure of the signal recognition particle receptor interacting with the SRP-RNC complex
  - 3.3.1 Reconstitution of SR-SRP-RNC complex
  - 3.3.2 Cryo-EM map of SR-SRP-RNC complex
- 3.4 High resolution structure of the ribosome and localization of L30e
4 Discussion
- 4.1 Model of elongation arrest
- 4.2 Model of the first steps of the SRP cycle
  - 4.2.1 Regulation of GTP affinity
- 4.3 Structure of SR-SRP-RNC complex
- 4.4 The next steps
Abbreviations
Eidestattliche Erklärung:
References:

Tables

Table 1: Contacts between mammalian SRP and the 80S ribosome

Images

Figure 1: Signal sequence recognition and cotranslational targeting by SRP.
Figure 2: Secondary structure of the SRP RNA with RNA domains indicated
Figure 3: Evolutionary conservation of SRP.
Figure 4: Mammalian Alu domain assembly.
Figure 5: Structure of SRP S-domain assembly steps.
Figure 6: Model of SRP S-domain assembly.
Figure 7: A) Crystal structure of SRP54 in complex with helix 8 in a ribbons representation of the N- (green), G- (blue), Mn- (purple) and Mc-domains (red) and helix 8 (orange). The novel N-terminal part of the M-domain contains the linker helix (αML) and the closed finger loop is highlighted in purple. B) M-domain in a top view compared with A. The finger loop on top is folded into the hydrophobic groove, which is lined by helices αM1, αM1b, αM2 and αM5 [53].
Figure 8: A) Crystal structure of the SRP54 (Ffh) M-domain from T. aquaticus.
Figure 9: Structure of the heterodimeric FFH/FtsY NG domain complex.
Figure 10: Structure of SRX-SRβ(GTP) complex.
Figure 11: Purification of RNCs and reconstitution of the RNC-SRP complex.
Figure 12: Cryo-EM map of mammalian SRP bound to 80S RNC at 12.0 Å.
Figure 13: Resolution curve: Fourier shell correlation (FSC) curve for the cryo-EM map of the RNC-SRP complex.
Figure 14: Cryo-EM map of the SRP-RNC complex.
Figure 15: Molecular model of SRP.
Figure 16: Molecular model of SRP and the ribosome.
Figure 17: SRP Alu-domain interaction with 80S ribosome.
Figure 18: SRP S-domain interaction with 80S ribosome.
Figure 19: SRP S-domain interaction with the 80S ribosome.
Figure 20: Structures of the SRP core in the free and ribosome-bound state.
Figure 21: Dynamics of the SRP core.
Figure 22: Dynamics of the SRP core with secondary structure.
Figure 23: Purified DP90 RNCs were reconstituted with an excess of SR and SRP, subjected to sucrose density gradient centrifugation in the presence of 500 mM KOAc, fractionated and analyzed by SDS-PAGE and Sypro-Orange staining. SR-SRP-RNC complexes migrate in the 80S fraction indicating SR and SRP binding to RNCs. S, supernatant; p, pellet.
Figure 24: Cryo-EM maps of mammalian SRP bound to 80S RNC at 12 Å and mammalian SR and SRP bound to 80S RNC at 9.1 Å. Ribosomal and SRP/SR densities are shown at different contour levels and amplitude correction using B-factors was applied only to the ribosome.
Figure 25: Cryo-EM map of mammalian SR-SRP-RNC complex at 9.1 Å superimposed with SRP density from SRP-RNC complex.
Figure 26: Cryo-EM map of mammalian SR-SRP-RNC complex at 9.1 Å.
Figure 27: Localization of L30e in a 9.5 Å cryo-EM map of the 80S ribosome.
Figure 28: Molecular model of L30e and its ribosomal environment.
Figure 29: SRP Alu-domain interaction with 80S ribosome.
Figure 30: Signal sequence-dependent SRP-ribosome interaction.
Figure 31: Model for the first steps of the SRP cycle.