Model - Aims of the work 17 - Characterization of the ribosome-associated complex RAC from S. c

2. Aims of the work 17

3.6. Model

The insights gained by HX measurements, biochemical and in vivo assays as well as by EPR analysis lead to a model suggesting a novel mechanism for converting Zuo and Ssz chaperones into the functional dimer RAC (Fig 3.15).

The SBD of Ssz is shortened compared to canonical Hsp70 homologs, which use this domain for the transient ATP controlled substrate binding. So far no substrate binding of Ssz was shown, instead the SBD of Ssz is engaged in a stable interac-tion with the J-protein Zuo. Although ATP binding significantly stabilizes Ssz in its monomeric form, binding appears to be rather transient. In contrast, ATP binding in the heterodimer seems to strongly influence the stability of the NBD, leading to a pronounced stabilization even after 2 h compared to uncomplexed Ssz where ATP induced stabilization diminished after 2 min. However, ATP binding seems not to be essential to drive complex formation.

The flexible, unstructured N-terminal domain of Zuo seems to mimic an unfolded peptide structure and it is tempting to speculate that it is thereby recognized by the shortened peptide binding domain of Ssz. The additional contacts of Zuo with the ATPase domain of Ssz might represent a function comparable to that of ATP hydrolysis in canonical Hsp70s. This mechanism would suppress unspecific binding of peptides in the SBD of Ssz and ensure RAC formation only. Complex formation does not restrict Zuo’s mobility, instead, the part directly adjacent to the binding region stays rather flexible, acting as a linker to provide a high flexibility to the rest of Zuo, especially the J-domain, in relation to Ssz.

Zuo’s EPVG motif did not proof to be a specific motif, that is recognized by the SBD of Ssz. However, the motif might be a part of a peptide like structure which is recognized by the C-terminus of Ssz. In cooperation with the numerous prolines it could be responsible for the unstructured folding of the N-terminus of Zuo. Thus functionality is maintained as long as formation of secondary structure elements within the domain are repressed. Additionally, binding of Ssz to the N-terminus of Zuo transmits a signal to the adjacent J-domain, either by the linker itself or due to an additional contact side undetected so far. In this study it was not clarified whether (I) the J-domain undergoes a structural conformational change to reposition the HPD motif or whether (II) the J-domain itself is repositioned by a change at the base of the domain. Moreover, the HPD motif itself seems not to be changed in its flexibility upon

Figure 3.15.: Model for the RAC complex assembly and architecture ATP (green) binding strongly stabilizes the ATPase domain of Ssz (red). However, ATP binding is only transient, and complex formation (indicated by a "+") with Zuo (blue) is thought to be inde-pendent of the ATP status of Ssz. RAC assembly involves the SBD of Ssz and the N-terminus of Zuo, and leads to a pronounced stabilization of the engaged moieties in both proteins and to the formation of a kinetically very stable heterodimer. Domains with significant decrease in conformational dynamics upon ligand/partner binding are indicated in dark red (Ssz) or in dark blue (Zuo); regions with increased flexibility are indicated with light colors. Moreover, association of Zuo with Ssz results in increased dynamics of the Zuo J-domain region carrying the HPD motif, which is likely to contribute to the activation of RAC as co-chaperone. ATP binding to RAC has no effect on the conformational dynamics of Zuo but additionally stabilizes the ATPase domain of Ssz in the complex.

3.6. Model complex formation. However, the data indicate that the change within the J-domain

is necessary to ultimately activate RAC as a co-chaperone (Fig 3.15).

Our findings suggest a rather flexible complex, as this study reveals a linker region in Zuo right behind the complex forming site. This is in agreement with a recent study, which analyzed RAC by small angle X-ray scattering (SAXS), which revealed an elongated complex of RAC (Gartmann, 2009). The main length of the complex is provided by Zuo, which shows flexibility and can adopt different conformations. In contrast, Ssz shows a lower degree of flexibility. Although the limited resolution did neither provide information about the ribosomal binding site nor the J-domain, mea-surements with a truncated Zuo version (aa 1- 110) and structural fittings confirmed that indeed, the SBD of Ssz is involved in complex formation .

The results of this study provides the essential groundwork to understand the archi-tecture of this atypical complex. However, many characteristics and properties of this complex that are essential to its function are still unresolved. In particular, mechanism and kinetics of ribosome binding are still unresolved.

This work provided first insights into the formation of the ribosome-associated complex (RAC) from S. cerevisiae. Certainly, more information on structure and dynamics is needed to fully understand the molecular mechanism of complex formation and activation of RAC’s co-chaperone function on the ATPase activity of Ssb.

A basic open question is the role of Ssz within the stable complex. So far no distinct function could be assigned to the Hsp70 chaperone in RAC. This work provides the hint that one function of Ssz might be the activation of Zuo’s J-domain upon complex formation, to provide full functionality. However, how the J-domain of Zuo is finally converted to efficiently stimulate ATPase activity remains unclear. To understand the activation mechanism of the J-domain, EPR measurements using different spin labels, such as (iodoacetamido)proxyl (IPSL), could repeated. Further two spin active labels within one Zuo molecule could be used to determine changes in the orientation of the J-domain of Zuo.

Another open question regards the precise role of Ssz’s ATPase domain. Although this work did show an influence of ATP on the NBD of Ssz, it still remains unclear whether this is of any importance for Ssz’s function. HX measurements using ATP analogues could give further insight into the interrelations.

The function of the SBD of Ssz is not fully understood. This work revealed its involvement in complex formation which is in line with first SAXS analyses (Gartmann, 2009). However, the exact binding mechanism as well as the exact mechanism which restricts binding to its complex partner Zuo only, remains unclear.

Furthermore, this work could assign a function to the N-terminal region of Zuo.

The N-terminal region is directly involved in complex formation while the segment aa 53-79 provides high flexibility to the rest of the protein. It still remains unclear how complex formation finally transmits the signal from the very N-terminus to the J-domain, as well as why complex formation is necessary to gain full function as a co-chaperone.

Further HX measurements using mutant variants may give further insight into the interaction site of RAC. However, one major problem in investigating large protein-protein complexes by HX-MS is the complexity of data derived from peptic digestion in the HPLC setup. The development of new separation strategies, the establishment of sophisticated HPLC gradients or the usage of new mass spectrometers with higher mass accuracy and resolution can help to solve this problem. Finally, as HX-MS data analysis, due to its manual data selection, is very time-consuming and therefore rate-limiting for such studies, automation and acceleration is an important issue.

Clearly, determination of the crystal structure would significantly further clarify the characterization of Ssz’s and Zuo’s interaction. However, all attempts to crystallize RAC were unsuccessful. Therefore this work is the first to give an idea about the formation of an Hsp40 and an Hsp70 chaperone into a functional complex

Another question remaining to be answered is the binding of RAC to the ribosome.

Zuo mutants used in this study were not suited to solve this task, however, further mutant versions might be successful in reporting on ribosome binding using BADAN fluorescence label. Another approach to determine binding kinetics in solution could be isothermal titration calorimetry (ITC) in micro scale. The use of unlabeled, au-thentic protein would be a great advantage, however, large amounts of pure and highly concentrated protein are necessary for ITC measurements.

5.1. Materials

5.1.1. Dyes, Kits, Markers and Proteins

1kb DNA Ladder Gene Ruler Fermentas

apo-Myoglobin Sigma

Complete protease inhibitor cocktail tablets Roche Coomassie brilliant blue R-250 Roth

DNAse Sigma

dNTPs BioLabs

Oligonucleotides Thermo Fisher Scientific

Phusion HF DNA Polymerase Biolabs

Plasmid Preparation Kit Qiagen

Protein Marker unstained SM0661 Fermentas Protein Marker stained SM0671 Fermentas

Ulp1 (SUMO protease) Andreasson et al. (2008b)

QIAGEN Plasmid Midi Kit Qiagen

QIAquick PCR Purification Kit Qiagen

QIAquick Gel Extraction Kit Qiagen

Restriction Endonucleases Fermentas

Taq Polymerase BioLabs

T4- DNA Ligase Fermentas

5.1.2. Antibodies

Anti-Zuo, polyclonal antiserum, rabbit (1:10.000) lab collection Anti-Ssz, polyclonal antiserum, rabbit (1:10.000) lab collection Anti-rabbit, Alexa Fluorescence Dye 682 (1:8.000) Dyomics

5.1. Materials 5.1.3. Media

-HIS Minimal Medium 6,7 g Difco Yeast Nitrogen Base 0,77 g CMS-HIS Drop Out Mixture

LB medium 10 g Tryptone

5 g Yeast Extract 5 g NaCl

YPD medium 10 g Yeast Extract 20 g Peptone 2 % Glucose

(for plates, medium was supplemented with 15 g/l agar and antibiotics) 5.1.4. E.coli Strains

Table 5.1.: E.coli strains used in this study

Strain name Relevant Genotype Source DH5alpha SupE44 ∆lacU169 deoR (f80lacZAM15)

hsdR17 recA1 endA1 gyrA96 thi-1 relA1

lab collection

Table 5.2.: Yeast strains used in this study

Name Relevant Genotype Source

Y67 wt, his3∆1 leu2∆0 met15∆0 ura3∆0, Matα lab collection Y216 BY4741; his3∆1/ leu2∆0/ met15∆0/ ura3∆1,

ssz1::KANMX

Koplin et al. (2010) Y217 BY4741; his3∆1/ leu2∆0/ met15∆0/ ura3∆1,

zuo1::KANMX

Koplin et al. (2010)

5.1.6. Plasmids

Table 5.3.: Plasmids used and constructed in this study

Name Description Source

pRosetta rare tRNAs (cm^R) lab collection

pSUMO pET24 based with N-terminal His6-Smt3-Ulp1 site(SUMO)-tag,T7 pro-motor, kan^R

Andreasson et al.

2008b

pmCUP313 amp^R, HIS3, CUP1 lab collection

pSUMOZuo His₆-SUMO-Zuo, kan^R Koplin et al. (2010) pSUMOSsz His₆-SUMO-Ssz, kan^R Koplin et al. (2010) pSUMOSsz,Zuo His₆-SUMO-Ssz-Zuo, kan^R Koplin et al. (2010) pSUMOZuo∆N62 His₆-SUMO-Zuo ∆N62, kan^R this study

pSUMOZuoH128C/

C167S

His₆-SUMO-Zuo H128C C167S, kan^R

this study pmCUPZuoC167S pmCUP313-Zuo C167S, amp^R this study pmCUPZuo∆N62 pmCUP313-Zuo∆N62, amp^R this study

pmCUPZuo pmCUP313-Zuo, amp^R this study

pmCUPZuoE30R pmCUP313-Zuo E30R, amp^R this study pmCUPZuoP31A pmCUP313-Zuo P31A, amp^R this study pmCUPZuoG33W pmCUP313-Zuo G33W, amp^R this study pmCUPZuoP31A/

His₆-SUMO-Ssz C81S C86S -Zuo C167S A343C, kan^R

this study

5.1. Materials 5.1.7. Primer

Table 5.4.: Primer used in this study, relevant bases are indicated by lower cases

Primer Sequence(5’ → 3’)

pSUMOZuo-dN62 5’BsmBI CCAGTGcgtctcAGGTGGTATGACCGTTGatgAATCCAATGTC-GACCC

pSUMOZuo-dN62 3’XhoI CCAGTGctcgagtcaCACGAAGTAGGACAACAAGCTG

pmCUPZuo-dN62 5’BamHI cgggatccATGGGCTCTCATCACCATCATCACCATGGCTCTatgTTTT-CTTTACCTACC

pmCUPZuo 3’NotI CGCGCgcggccgctcaCACGAAGTAGGACAACAAGC

pmCUPZuo 5’BamHI cgggatccatgTTTTCTTTACCTACCC

Zuo H128C forw CAAGTTGTCAAGTACtgTCCAGACAAGC

Zuo H128C rev GCTTGTCTGGAcaGTACTTGACAACTTG

Zuo C167S forw GCTCAGTACGACTCATcTGATTTTGTTGC

Zuo C167C rev GCAACAAAATCAgATGAGTCGTACTGAGC

Zuo E30R forw CGTCCGGTCcgACCGGTTGGTAAGTTC

Zuo E30R rev GAACTTACCAACCGGTcgGACCGGACG

Zuo P31A forw CGTCCGGTCGAAgCGGTTGGTAAG

Zuo P31A rev CCAACCGcTTCGACCGGACG

Zuo G33W forw CGAACCGGTTtGgAAGTTCTTTTTGC

Zuo G33 rev GCAAAAAGAACTTcCaAACCGGTTCG

Zuo K341C forw GCAAAAGCTGACAAAtgtAAGGCTAAGGAAGC

Zuo K341C rev GCTTCCTTAGCCTTacaTTTGTCAGCTTTTGC

Zuo A343C forw GCTGACAAAAAGAAGtgTAAGGAAGCTGC

Zuo A343C rev GCAGCTTCCTTAcaCTTCTTTTTGTCAGC

Ssz C81S forw GCCATTTGACAAGTcTGATGTCAGC

Ssz C81S rev GCTGACATCAgACTTGTCAAATGGC

Ssz C86S forw GATGTCAGCAAGTcCGCTAACGG

Ssz C86S rev CCGTTAGCGGACTTGCTGACATc

5.1.8. Antibiotics

Ampicillin 100µg/ml (stock 100 mg/ml)

Chloramphenicol 25 µg/ml (stock 25 mg/ml)

Kanamycin 50 µg/ml (stock 50 mg/(ml)

All concentrations are final concentrations, stock solutions are filter-sterilized

5.1.9. Technical Equipment

Balance(s) Sartorius

Centrifuge (SS34) Sorvall

FastPrep MP

FPLC- ÄKTA Purifier GE Healthcare - Amersham

FPLC Workstation BioRad

French Press SIM AMINCO

L7-55 Ultracentrifuge Beckman

Mini UZ M150SE Sorvall

Multifuge 4 Heraeus

PCR BioRad

pH-Meter 766 Knick

QSTAR Pulsar i Hybrid MS/MS System Applied Biosystems/MDS SCIEX

Scanner Canon

Semi Dry Western Blot BioRad

Spectrofluorimeter Jasco

Spectrophotometer Ultraspec 3100 pro Amersham

Starion FLA 9000 FujiFilm

Vivaspin column 500, 10 kD MWCO Satorius stedim

5.1. Materials

2x Laemmli 100 mM Tris-HCl pH 6.8

4 mM EDTA

5x Laemmli 250 mM Tris-HCl pH 6.8

10 mM EDTA

HDX-KII buffer 25 mM HEPES-KOH pH 7.4

50 mM KCl 5 mM MgCl₂

HDX-Quench-buffer 0.4 M K-Phosphate pH 2.2 HEPES - Low Salt (HEPES-LS) 40 mM HEPES pH 7.4

100 mM K-Ac 5 mM MgCl₂ 5% (v/v) Glycerol

2 mMβ-Mercaptoethanol HEPES - High Salt (HEPES-HS) 40 mM HEPES pH 7.4

1 M K-Ac 5 mM MgCl₂ 5% (v/v) Glycerol

2 mMβ-Mercaptoethanol HEPES - non reducing Low Salt 40 mM HEPES pH 7.4

100 mM K-Ac 5 mM MgCl₂ 5% (v/v) Glycerol HEPES - Elutionbuffer 40 mM HEPES pH 7.4

100 mM K-Ac 5 mM MgCl₂ 300 mM Imidazole 5% (v/v) Glycerol

2 mMβ-Mercaptoethanol Power Stainer 0.5 % (w/v) Coomassie R250

50 % (v/v) methanol 10 % (v/v) acetic acid Power Destainer 45 % (v/v) methanol

10 % (v/v) acetic acid

5.1. Materials 10 mM MOPS pH 7.5

15 % (v/v) Glycerol SDS -stacking gel buffer 4x 0.5 M Tris-HCl pH 6.8

0.4 % (v/v) SDS (20%) SDS -running gel buffer 4x 50 mM Tris-HCl pH 8.8

0.4 % (v/v) SDS (20%) SDS sample buffer 5x 1.5 M Tris-HCl pH 6.8

2 mM EDTA

1% (v/v) SDS (20%)

1% (v/v) β-Mercaptoethanol 10% (v/v) Glycerol

Bromophenolblue SDS -running buffer 25 mM Tris

0.2 M Glycine

silver-stain 0.2 % (w/v) g AgNO₃

0.075 % (v/v) formaldehyde (37%) silver-developing 6% NaCO₃ *H₂O

0.015 % (v/v) formaldehyde

TBS 10x 100 mM Tris-HCl pH 8.0 1.5 M NaCl

TBS-TT 10x 100 mM Tris-HCl pH 8.0

1.5 M NaCl

5 % (v/v) Tween-20 Western Blotting Buffer 2.5 M Tris

2 M Glycine 0.5 % (w/v) SDS pH 8.0

5.2. Microbiological and Molecular Biological Methods

5.2.1. Cultivation and Conservation of E.coli Strains

Strains were streaked out from permanent cultures to form single colonies on LB agar plates supplemented with the appropriate antibiotics. Plates were incubated at 37^◦C for 12-16 h, if not stated otherwise, and stored at 4^◦C. Single colonies were used to inoculate liquid LB medium or new agar plates. Growth in liquid cultures was achieved using glass tubes in a roller drum or Erlenmeyer flasks in a shaking incubator.

Each liquid culture was inoculated with a single cell colony and grown at 37^◦C unless stated otherwise. Growth was followed spectroscopically by determining the optical cell density at 600 nm (OD₆₀₀). Permanent cell cultures were prepared from liquid cultures grown to exponential phase by mixing 800µl cell culture with 200µl DMSO.

The mixture was immediately frozen using liquid nitrogen and subsequently stored at -80^◦C.

5.2.2. Cultivation and Conservation of Yeast Strains

S.cerevisiae strains were streaked out from permanent cultures to form single colonies on YPD plates or the appropriate selection plate when containing a plasmid. Plates were incubated at 30^◦C for 2-3 days, if not stated otherwise, and stored at RT. Single colonies were used to inoculate liquid medium or new agar plates. Growth in liquid cultures was achieved using glass tubes in a roller drum or Erlenmeyer flasks in a shaking incubator. Each liquid culture was inoculated with a single cell colony and grown at 30^◦C unless stated otherwise. Growth was followed spectroscopically by determining the optical cell density at 600 nm (OD₆₀₀). Permanent cell cultures were prepared from liquid cultures grown to exponential phase by mixing 800µl cell culture with 200µl DMSO. The mixture was immediately frozen using liquid nitrogen and subsequently stored at -80^◦C.

5.2.3. Plasmid DNA Preparation

To purify plasmid DNA the QIAprep Spin Miniprep Kit (Qiagen) was used according to the protocol provided by the manufacturer.

5.2.4. PCR Mutagenesis

Using PCR technique various DNA-fragments can be amplified. A standard PCR reaction contained 1x reaction buffer, 1 µl of DNA template (50 - 100 ng), 100 µM of dNTP mix, 0.5µM of each primer and 0.5 µl of Phusion Polymerase in 50 µl final volume. The cycling parameters for a standard PCR are shown in Table 5.6.

Table 5.6.: Cycling Parameters for PCR Segment Temperature Time

Table 5.7.:Cycling Parameters for single site directed mutagenesis

The Single Site-Directed Mutagenesis can be used to introduce single site mutations into a DNA template using polymerase chain reaction (PCR). This technique was used to generate the various cystein mutations in Zuotin. A standard ssm-PCR reaction contained 1x reaction buffer, 1 µl of DNA template, 100 µM of dNTP mix, 0.5 µM of each primer and 0.5 µl of Phusion Polymerase in 50 µl final volume. The cycling parameters for the mutagenesis are shown in Table 5.7. The reaction products were treated with the restriction endonucleaseDpnI at 37^◦C overnight, to digest the parental DNA template. The DpnI endonuclease with the target sequence: 5’-Gm6ATC-3’ is specific for methylated and hemimethylated DNA (Nelson et al., 1992). Subsequently the multiply mutated single stranded DNA was transformed into DH5α competent cells.

5.2.6. Restriction Digest

Restriction digests were performed using the corresponding restriction endonuclease. 5 - 20µl of DNA sample (plasmid or PCR product) were incubated with 3µl 10 x reaction buffer and 0.2 - 1 U of the corresponding enzyme (w/o BSA) in a total volume of 30µl.

5.2. Microbiological and Molecular Biological Methods After incubation at the enzyme-specific temperature for 5 - 16 h the digest was used

for further work.

5.2.7. Ligation

Ligases link DNA ends by catalyzing the phosphodiester-bonding. DNA fragments with matching digested ends were incubated in no more than 10 µl total volume with 40 U T4-DNA-ligase and the recommended ligation buffer. The optimal ratio of vector to insert DNA was 1:3 for “sticky end” ligations. Different conditions for incubation were used: over night at 16^◦C, 2 h at room temperature or 30 min at 30^◦C.

5.2.8. Preparation of Chemically Competent E. coli Cells

A 50 ml culture was inoculated with 0.5 ml of an overnight culture and grown in medium (LB broth with 20 mM MgSO₄, 10mM KCl) to mid-logarithmic phase. The cells were kept on ice for 10 min, pelleted at 1,500g for 10 min at 4^◦C, resuspended gently in 150 ml cold TFB1 and incubated for 15 min on ice. The cells were sedimented again at 1,500g for 10 min at 4^◦C and resuspended in 20 ml cold TFB2. The cells were aliquoted in 250 µl portions, frozen in liquid nitrogen and stored at -80^◦C.

5.2.9. Transformation of Chemically Competent E. coli Cells

For transformation, 1-5 µl DNA were incubated with 50µl competent cells for 30 min on ice, then the cells were heat-shocked for 90 s at 42^◦C and subsequently kept on ice for 2 min. 1 ml of LB medium was added and the mixture was shaken for 45 min at 37^◦C to allow bacterial recovery and expression of the antibiotic resistance gene.

The transformed cells were plated onto LB agar plates containing the appropriate antibiotics.

5.2.10. Transformation of Yeast Cells

3 µl of 10 mg/ml boiled carrier DNA and 1 µg plasmid were mixed with 100 µl transformation mix (400 mM lithium acetate, 40 % PEG-3350, 130 mM β-Me). For transformation one large yeast colony was resuspended in the mix and vortexed. The mixture was then incubated rotating for 30 min at 37^◦C. The transformed cells were

pelleted, resuspended in H₂O and subsequently plated onto agar plates containing the selective media.

5.2.11. Spot Test

Yeast cells were grown to exponential phase and adjusted to OD₆₀₀ = 0.2. Serial dilutions were spotted on agar plates containing selective media and appropriate aux-otrophy marker.

5.2.12. DNA - Gel-Electrophoresis / Agarose Gel

Agarose gel electrophoresis is used to separate DNA strands by size and can be utilize to estimate the size of the DNA. Negatively charged DNA molecules migrate through the agarose matrix using an electric field, with shorter molecules moving faster than longer ones. Usually 1% agarose gels prepared in 1xTAE electrophoresis buffer were used in a casting tray. One-sixth volume of a 6 x concentrated loading dye is added to each sample, mixed and loaded into the wells. The samples are separated at 50 -100 V (depending on the size of the gel) until the required separation is achieved. To visualize and photograph the DNA fragments, a long wave UV light box is used.

5.2.13. DNA Sequencing

All mutations were verified by DNA sequencing performed at GATC - Biotech, Kon-stanz.

5.3. Protein Biochemical Techniques

5.3.1. Protein Expression in E.coli

To ascertain that the Zuotin mutant proteins are over-expressed and soluble, the ex-pression and solubility of the mutant Zuo proteinsin vivowere first analyzed. A single colony of the E.coli strain expressing the desired Zuotin variant was used to inoculate 5 ml LB kan/cm media to grow over night. A 1:100 dilution of the overnight culture was then grown at 37^◦ to an OD₆₀₀ of 0.6 - 0.7 and a 1 ml sample of the culture was taken (S1). Then the cultures were shifted to 30^◦C and the protein expression was induced by addition of 0.1 mM IPTG. After 3 additional h of growth a second

5.3. Protein Biochemical Techniques 1 ml sample was taken (S2). The rest of the culture was collected (S3). To analyze

the expression level, samples S1 and S2 were analyzed by SDS-PAGE. To analyze the solubility of the overexpressed protein, the cells of sample S3 were harvested (16,000 x g, 3 min) and resuspended in HEPES-LS buffer. The cells were lysed using FastPrep (1ml cells + 1g acid washed beads). Finally, the sample was centrifuged (14,000 x g, 20 min, 4^◦C) and the supernatant (soluble fraction) as well as the pellet (insoluble

Im Dokument Characterization of the ribosome-associated complex RAC from S. cerevisiae (Seite 63-0)