TECHNISCHE UNIVERSITÄT MÜNCHEN Department Chemie Lehrstuhl für Biotechnologie Co-chaperone interactions in the Hsp90 chaperone cycle Jing Li Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof.Dr. L. Hintermann Prüfer der Dissertation : 1. Univ.-Prof.Dr.J.Buchner 2. Univ.-Prof.Dr.S.Weinkauf 3. Univ.-Prof.Dr.M.Sattler Die Dissertation wurde am 20.10.2011 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 09.12.2011 angenommen To My Family Index Dissertation Jing Li 1. Summary .......................................................................................................................... 1 2. Introduction ...................................................................................................................... 5 2.1 Protein folding ............................................................................................................. 5 2.2 Protein folding in the cell ............................................................................................. 6 2.3 Catalyzed protein folding ............................................................................................ 7 2.4 Molecular chaperones ................................................................................................. 9 2.4.1 Hsp60/Chaperonin family ....................................................................................10 2.4.2 Hsp100 family .....................................................................................................11 2.4.3 Hsp70 family .......................................................................................................11 2.4.4 Small heat shock proteins ...................................................................................12 2.4.5 Hsp90 family .......................................................................................................13 2.5 Hsp90 chaperone machinery .....................................................................................13 2.5.1 Structure and conformational dynamics of Hsp90 ................................................13 2.5.2 Hsp90 co-chaperones .........................................................................................17 2.5.3 The chaperone cycle of Hsp90 ............................................................................27 2.5.4 Regulation of Hsp90 cycle by posttranslational modifications ..............................29 2.5.5 Hsp90 client protein reorganization .....................................................................30 2.5.6 Hsp90 and protein degradation ...........................................................................33 2.5.7 Hsp90, inhibitors and human diseases ................................................................33 3. Objective .........................................................................................................................36 4. Results and discussion ....................................................................................................38 4.1 Asymmetic Hsp90/co-chaperone complexes are important for the progression of the reaction cycle ...................................................................................................................38 4.1.1 Co-chaperone interaction in the yeast Hsp90 cycle .............................................38 4.1.1.1 TPR co-chaperone interactions with Hsp90 ..................................................38 4.1.1.2 Characterization of Sti1 inhibition on Hsp90 ATPase activity ........................39 4.1.1.3 Interaction between Sti1, Hsp90 and Cpr6 ....................................................40 4.1.1.4 Setting up a FRET system to study Hsp90–co-chaperone interaction ...........41 4.1.1.5 Sti1 and Cpr6 form a ternary complex in vivo ................................................43 4.1.1.6 Analysis of asymmetric Hsp90 complex by analytical ultracentrifugation .......44 4.1.1.7 Asymmetric complexes are disrupted by AMP-PNP and p23 ........................48 4.1.2 Asymmetric complexes are conserved in the Hsp90 cycle ..................................50 4.1.2.1 Co-chaperone interaction in human Hsp90 cycle ..........................................50 I Index Dissertation Jing Li 4.1.2.2 Asymmetric co-chaperone complex is covserved in human hsp90 cycle ...... 52 4.1.3 Discussion: the Hsp90 co-chaperone cycle ......................................................... 56 4.2 Synergistic binding of Aha1 and Cpr6 to Hsp90 promotes the progression of chaperone cycle .............................................................................................................. 59 4.2.1 Aha1/Hch1 and Cpr6 prefer the closed conformation of Hsp90 ........................... 59 4.2.2 Aha1 and Cpr6 form ternary complex with Hsp90 ............................................... 62 4.2.3 Aha1 provides additional driving forces for the progression of the Hsp90 cycle ... 66 4.2.4 Aha1 and Cpr6 stimulate the ATPase activity of Hsp90....................................... 67 4.2.5 p23 releases Aha1 from Hsp90 ........................................................................... 68 4.2.6 Discussion: Aha1 in the co-chaperone cycle of Hsp90 ........................................ 70 4.3 Comparative studies of AIP/Xap2 and AIPL1 ............................................................. 73 4.3.1 AIP and AIPL1 show high sequence and structural similarity but distinct differences in the C-terminal region.............................................................................. 73 4.3.2 AIP and AIPL1 are inactive PPIase with altered function ..................................... 77 4.3.3 AIPL1 but not AIP shows molecular chaperone activity ....................................... 78 4.3.4 The Proline-rich domain is critical for the chaperone activity of AIPL1 ................. 79 4.3.5 The Proline-rich domain is a negative regulator of Hsp90 interaction .................. 81 4.3.6 Discussion: Chaperone function of AIP and AIPL1 .............................................. 83 5. Material and methods ...................................................................................................... 85 5.1 Material ..................................................................................................................... 85 5.1.1 Chemicals ........................................................................................................... 85 5.1.2 Fluorophors ......................................................................................................... 86 5.1.3 Size and molecular mass standard kits ............................................................... 87 5.1.4 Protein and antibodies......................................................................................... 87 5.1.5 Chromatographic material ................................................................................... 88 5.1.6 Miscellaneous material ........................................................................................ 88 5.1.7 Equipment ........................................................................................................... 89 5.1.8 Computer software .............................................................................................. 91 5.2 Organisms and cultivation ......................................................................................... 92 5.2.1 Strains ................................................................................................................. 92 5.2.2 Media and antibiotics .......................................................................................... 93 5.2.3 Growth and storage of E. coli .............................................................................. 93 5.2.4 Growth and storage of yeast cells ....................................................................... 94 II Index Dissertation Jing Li 5.3 Methods in molecular biology .....................................................................................94 5.3.1 Plasmids and constructs ......................................................................................94 5.3.2 Molecular biological solutions ..............................................................................96 5.3.3 Preparation of plasmid DNA from E. coli .............................................................96 5.3.4 Separation of DNA by agarose gel electrophoresis .............................................97 5.3.5 DNA isolation from agarose gels .........................................................................97 5.3.6 Purification of PCR products and plasmids ..........................................................97 5.3.7 DNA sequencing analysis ....................................................................................97 5.3.8 Transformation of E. coli ......................................................................................97 5.3.9 PCR amplification ................................................................................................98 5.3.10 DNA digestion by restriction endonucleases ......................................................99 5.3.11 Dephosphorylation of DNA ends .......................................................................99 5.3.12 Ligation of DNA fragments .............................................................................