Structural and biochemical characterization of the Rod-Zwilch-Zw10 complex
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Date
2015
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Abstract
In höher entwickelten Eukaryoten ist der aus drei Proteinen bestehende Rod-
Zwilch-Zw10 (RZZ) Komplex essentieller Bestandteil des “spindle assembly
checkpoint” (SAC). Der RZZ-Komplex wird für die Lokalisation des Mad1/Mad2
SAC Komplexes sowie des Microtubuli Motorproteins Dynein benötigt. Dadurch
trägt der RZZ-Komplex zur Ausbildung von Kinetochor-Microtubuli-Bindungen bei.
Außerdem ist der RZZ-Komplex in den Prozess involviert bei dem SAC Proteine in
Abhängigkeit von Dynein vom Kinetochor weg zu den Spindelpolen transportiert
werden, sobald der SAC zufrieden gestellt ist („kinetochore shedding“). Wie genau
der RZZ-Komplex diese unterschiedlichen Funktionen erfüllt und Signale von der
Kinetochor-Microtubuli Schnittstelle integriert ist bislang nicht bekannt.
In meiner Doktorarbeit beschreibe ich die erfolgreiche Aufreinigung des
rekombinant hergestellten, 813 kDa großen RZZ-Komplexes sowie dessen
vorläufige kristallographische Analyse. In einem multidisziplinären Ansatz
bestehend aus strukturbiologischen und biochemischen Methoden habe ich die
strukturelle Organisation und Funktion des RZZ-Komplexes untersucht. Eine
Kombination aus Kryo Elektronenmikroskopie und quervernetzender
Massenspektroskopie Analyse führte zu Erkenntnissen über die strukturelle
Organisation des RZZ-Komplexes. Das herausgearbeitete Modell zeigt, dass der
RZZ-Komplex ein Hexamer mit einer 2:2:2 Stöchiometrie bildet. Darin formen zwei
Rod Moleküle ein längliches antiparalleles Dimer. An beiden Enden der Struktur
betten der C-Terminus und der N-Terminale beta-propeller von zwei
unterschiedlichen Rod Molekülen ein Zwilch Molekül zwischen sich ein. Zw10
verbindet die zentralen Bereiche von beiden Rod Molekülen miteinander.
Zusammen bilden Rod, Zwilch und Zw10 eine extensive Dimer-Berührungsfläche
aus. Außerdem beschreibe ich in meiner Arbeit umfangreiche Interaktionsuntersuchungen
zwischen dem RZZ-Komplex und anderen Kinetochor Proteinen,
welche möglicherweise die RZZ Funktionen beeinflussen. Ich zeige in meiner
Arbeit erstmals, dass eine direkte Binding zwischen dem RZZ-Komplex und
Spindly stattfindet und dass diese von der Farnesylierung der C-terminalen
CAAX-Box von Spindly abhängt.
Diese Untersuchungen ebnen den Weg zu einer detaillierten strukturellen und
funktionalen Untersuchung des RZZ-Komplexes und favorisieren ein Modell, in
welchem der Prozess des „kinetochore shedding“ Farnesylierung von SpindlyZusammenfassung
13
erfordert. Unser Modell schlägt vor, dass nach SAC Aktivierung Spindly
farnesyliert wird und daraufhin an RZZ bindet. Das wiederum ermöglicht die
Bindung von Dynein/Dynactin. Durch die Binding des zum Minus-Ende gerichteten
Motorproteins Dynein werden SAC Proteine vom Kinetochore weg und zu den
Spindlepolen hin transportiert.
The 3-subunit Rod-Zwilch-Zw10 (RZZ) complex is a crucial component of the spindle assembly checkpoint (SAC) in higher eukaryotes. It is required for kinetochore localization of the Mad1/Mad2 checkpoint complex and of the microtubule motor Dynein, thus contributing to kinetochore-microtubule attachment as well as to the Dynein-dependent stripping of SAC components upon checkpoint satisfaction. How the RZZ fulfills these different roles and integrates signals from the kinetochore-microtubule interface remains unclear. In my doctoral thesis, I report the successful recombinant production and a preliminary crystallographic analysis of the 813 kDa RZZ complex from coexpression of its three subunits Rod, Zwilch, and Zw10. Additionally the organization and function of the RZZ complex was studied by using a multidisciplinary approach that combines structural biology and biochemistry. Cryo electron microscopy combined with cross-linking mass spectrometry analysis provided insight into the structural organization of the RZZ complex. The resulting molecular model shows that the RZZ complex is a 2:2:2 hexamer in which the two Rod molecules form an elongated antiparallel dimer. At either end of the structure the Rod heads interact with Zwilch, using both the N-terminal beta-propeller domain, and its C-terminal region. Zw10 bridges the middle parts of both Rod molecules. Together, Rod, Zwilch and Zw10 create a very extensive dimerization interface that explains the stoichiometry of the complex. Furthermore, I describe an extensive series of interaction studies of the RZZ complex with other kinetochore components that have been suggested to be involved in the RZZ function. I demonstrate for the first time a direct interaction between the RZZ complex and Spindly that strictly depends on farnesylation of the C-terminal CAAX-box of Spindly. Our studies pave the way to a detailed structural and functional characterization of the RZZ complex and support a model by which Spindly farnesylation promotes its interaction with kinetochores and mediates its biological function. We propose that upon SAC activation Spindly becomes farnesylated by a Farnesyltransferase and binds to the RZZ complex. This in turn enables further recruitment of Dynein/Dynactin to the kinetochore. Upon binding of the minus-end directed motor protein Dynein, checkpoint proteins are transported away from kinetochores towards the spindle poles.
The 3-subunit Rod-Zwilch-Zw10 (RZZ) complex is a crucial component of the spindle assembly checkpoint (SAC) in higher eukaryotes. It is required for kinetochore localization of the Mad1/Mad2 checkpoint complex and of the microtubule motor Dynein, thus contributing to kinetochore-microtubule attachment as well as to the Dynein-dependent stripping of SAC components upon checkpoint satisfaction. How the RZZ fulfills these different roles and integrates signals from the kinetochore-microtubule interface remains unclear. In my doctoral thesis, I report the successful recombinant production and a preliminary crystallographic analysis of the 813 kDa RZZ complex from coexpression of its three subunits Rod, Zwilch, and Zw10. Additionally the organization and function of the RZZ complex was studied by using a multidisciplinary approach that combines structural biology and biochemistry. Cryo electron microscopy combined with cross-linking mass spectrometry analysis provided insight into the structural organization of the RZZ complex. The resulting molecular model shows that the RZZ complex is a 2:2:2 hexamer in which the two Rod molecules form an elongated antiparallel dimer. At either end of the structure the Rod heads interact with Zwilch, using both the N-terminal beta-propeller domain, and its C-terminal region. Zw10 bridges the middle parts of both Rod molecules. Together, Rod, Zwilch and Zw10 create a very extensive dimerization interface that explains the stoichiometry of the complex. Furthermore, I describe an extensive series of interaction studies of the RZZ complex with other kinetochore components that have been suggested to be involved in the RZZ function. I demonstrate for the first time a direct interaction between the RZZ complex and Spindly that strictly depends on farnesylation of the C-terminal CAAX-box of Spindly. Our studies pave the way to a detailed structural and functional characterization of the RZZ complex and support a model by which Spindly farnesylation promotes its interaction with kinetochores and mediates its biological function. We propose that upon SAC activation Spindly becomes farnesylated by a Farnesyltransferase and binds to the RZZ complex. This in turn enables further recruitment of Dynein/Dynactin to the kinetochore. Upon binding of the minus-end directed motor protein Dynein, checkpoint proteins are transported away from kinetochores towards the spindle poles.