Investigation of Coiled-Coil Interactions between Proteins of the Spindle Pole Body by Nora Zizlsperger B.S. Biochemistry Simmons College, 2000 SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY FEBRUARY 2010 © 2010 Massachusetts Institute of Technology All rights reserved. Signature of Author………………………………………………………………………………… Nora Zizlsperger Department of Biology November 19, 2009 Certified by………………………………………………………………………………………… Amy E. Keating Associate Professor of Biology Thesis Supervisor Accepted by………………………………………………………………………………………... Steven P. Bell Chairman, Department Committee on Graduate Students 1 2 Investigation of Coiled-Coil Interactions between Proteins of the Spindle Pole Body by Nora Zizlsperger Submitted to the Department of Biology on November 19, 2009 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology ABSTRACT The spindle pole body (SPB) is a large multi-protein complex that organizes microtubules in yeast. Through its function of nucleating and anchoring microtubules, the SPB is essential for cell viability. High-resolution structures of the SPB have not been achieved using x-ray crystallography, due to its low copy number, large size, heterogeneous composition, and association with the nuclear membrane. However, structural information may be deciphered through a variety of other techniques. Cryo-electron microscopy images have provided a low- resolution model of the SPB. Experiments testing which proteins interact provide additional structural data, although in most cases, it is not known precisely how these interactions occur. Interestingly, a large proportion of SPB proteins are predicted to contain one or more coiled coils. The coiled coil is a common protein-protein interaction domain, consisting of two or more supercoiled α-helices. The high frequency of coiled coils predicted in SPB proteins suggests that this structure may be important for establishing the overall architecture of the complex. This thesis describes work towards determining whether coiled coils form interactions within or between SPB proteins. All coiled-coil regions predicted in SPB proteins were produced and tested for interactions as individual peptides, taking advantage of the often-observed ability of coiled coils to fold and interact cooperatively, isolated from the rest of the protein. Many self- associating coiled coils and several hetero-associating coiled coils were identified, and structural features of several complexes were determined. The results suggest that several SPB coiled coils form supports between layers of the SPB and other coiled coils mediate homo- and hetero- associations within layers or sub-complexes. The coiled-coil interaction data were incorporated with other structural information described in the literature to generate a model of the spindle pole body structure. Thesis Supervisor: Amy E. Keating Title: Associate Professor of Biology 3 4 Table of Contents List of Figures ................................................................................................................................ 7 List of Tables ................................................................................................................................. 8 CHAPTER 1 Introduction ............................................................................................. 9 Structure determination of large protein complexes through integration of diverse data .......... 9 Methods for obtaining structural information about complexes ............................................... 10 High-resolution structural studies ......................................................................................... 10 Computational structure prediction and annotation .............................................................. 10 Electron microscopy ............................................................................................................. 11 Biochemical identification of components and interactions ................................................. 11 Models of complex structures ................................................................................................... 12 Progress towards structural models of particular large protein complexes .............................. 13 The proteasome ..................................................................................................................... 14 The kinetochore .................................................................................................................... 14 The nuclear pore complex ..................................................................................................... 15 The spindle pole body ............................................................................................................... 16 Coiled coils – predicted structural features in the SPB ............................................................ 18 Summary of thesis .................................................................................................................... 21 CHAPTER 2 Analysis of coiled-coil interactions between core proteins of the spindle pole body ................................................................................... 23 ABSTRACT .............................................................................................................................. 23 INTRODUCTION .................................................................................................................... 24 MATERIAL AND METHODS ................................................................................................ 25 Coiled-coil prediction ........................................................................................................... 25 Domain annotation ................................................................................................................ 29 Protein production ................................................................................................................. 29 Circular dichroism spectroscopy ........................................................................................... 30 Fluorescence resonance energy transfer (FRET) assay ........................................................ 30 Analytical ultracentrifugation ............................................................................................... 31 Crosslinking .......................................................................................................................... 31 Crystallization ....................................................................................................................... 31 Structure determination ......................................................................................................... 31 RESUTS .................................................................................................................................... 32 Many coiled coils, but few other domains, are predicted in core SPB protein sequences. ... 32 Several core SPB coiled coils self-associate ......................................................................... 33 Several core SPB coiled coils hetero-associate..................................................................... 42 Characterization of core SPB coiled-coil interactions .......................................................... 44 Crystal structure of Spc42_1 ................................................................................................. 47 DISCUSSION ........................................................................................................................... 50 5 CHAPTER 3 Specific coiled-coil interactions contribute to a global model of the structure of the spindle pole body ........................................................ 53 ABSTRACT .............................................................................................................................. 53 INTRODUCTION .................................................................................................................... 53 MATERIALS AND METHODS .............................................................................................. 55 Coiled-coil prediction and production. ................................................................................. 55 Circular dichroism spectroscopy, analytical ultracentrifugation, and crosslinking experiments ........................................................................................................................... 56 Fluorescence resonance energy transfer (FRET) assay ........................................................ 56 Fluorescence anisotropy (FA) assay ..................................................................................... 56 Domain annotation. ............................................................................................................... 57 RESULTS ................................................................................................................................. 57 Many coiled coils are predicted in membrane-associated and half-bridge SPB proteins. .... 57 Many coiled coils from SPB membrane-associated proteins self-associate. ........................ 57 Characterization of homo-oligomerizing coiled coils in membrane-associated proteins ..... 62 Coiled coils from Bbp1 and Mps2 hetero-associate.