Filiz CIVRIL
SEMM PhD Student
Via Adamello, 16
20139, Milano
2008 JULY PROGRESS REPORT
ABSTRACT
Kinetochores (KTs) are large protein scaffolds that connect centromeric DNA to spindlemicrotubules (MTs), thus contributing to the mechanism of sister chromatid separation during
anaphase. KTs ensure the fidelity of sister chromatid separation by supporting the spindle
assembly checkpoint, a device that delays anaphase until all sister chromatids are bipolarly
attached.
Many aspects of the KT architecture remain elusive. To contribute to the knowledge of KT
organization, we tried to characterize the structural and functional properties of the RZZ
complex, which is known to contain proteins named ROD, ZW10 and Zwilch, and their core KT
interaction partner Zwint-1 .
PROGRESS:
In vitro attempts to reconstitute RZZ-Zwint-1 network
We have used bacterial expression systems to obtain recombinant fragments of the human RZZ
complex subunits and Zwint-1 to reconstitute their network of interactions in vitro. Since the
Rod protein was challenging for bacterial system because of its size, initially we focused on the
expression and purification of Zw10, Zwilch and Zwint-1. The purified soluble proteins (Figure
1) were used for GST-pulldown assays and gel filtration analysis to detect any binary
interactions between Zw10, Zwilch and Zwint-1. However, with these in vitro methods we failed
to find any significant interaction.
Figure 1. Soluble human Zwint-1, Zw10, Zwilch proteins are
expressed recombinantly in bacterial system as GST-tagged and
affinity purified with Glutathione beads and cleaved from the Tag
with the help of Prescission® protease.
We also attempted to co-express the Zwint-1, Zwilch and Zw10 as pairs from a single bicistronic vector (pGEX6p-2rbs by Anna De Antoni) in which one of the proteins was tagged and
any interaction would result in the co-purification of the second protein. However, this attempt
was again unsuccessful and failed to map any binary interaction. These negative observations
suggested that the presence of the other member of the complex, ROD might be required for
binding.
ROD is the largest protein among the components of the RZZ complex, with a molecular weight
of approximately 250 kDa. It might be very difficult to express the full-length ROD protein
recombinantly in a bacterial system. The approach we have taken was to express the protein as
separate segments to obtain soluble segments of ROD that we could use in in vitro assays for
interaction mapping. Particularly, we have created expression vectors to express four consecutive
segments of the protein based on secondary structure predictions. Among the four consecutive
segments only the C-terminal fragment was soluble. This segment was used to analyze if ZW10,
Zwilch or Zwint-1 were able to bind in vitro but unfortunately the results were negative. We
tried to clone the four consecutive segments into the bi-cistronic vector with ZW10 and to check
if the dual expression could convert the insolubility of Rod fragments because of a possible
interaction with ZW10. However, the results were again negative. Next, we in vitro translated the
Rod segments and pulled them down using the recombinant GST-tagged soluble proteins. GSTZwilch readily pulled down the N-terminus of Rod (Figure 2).
Figure 2. GST-pull down of in vitro translated Rod
segments either with GST-Zwilch or GST-Zw10.
In order to further characterize the interaction among Zwilch and the N-terminus of Rod we have
analyzed Rod sequence in silico. The sequence and structure analyses of Rod has shown that the
protein has a potential β-propeller motif at the N-terminus followed by ARM (armadillo) repeats.
Providing the presence of a protein-protein binding motif at the N-terminus and the interaction of
Zwilch and N-terminus of Rod we have created a bi-cictronic construct with probable β-propeller
motif of Rod and Zwilch. Our co-purifications confirmed the interaction (Figure 3) however the
Rod fragment was smaller than the expected size suggesting degradation of the polypeptide. The
coverage of the sequence by mass-spectroscopy analysis directed us to create new constructs and
at the moment we are working on those constructs to identify the smallest Rod domain which
interacts with Zwilch.
Figure 3. Co-purification of GST-Rod β-propeller and Zwilch.
Soluble protein fraction was loaded after the cleavage of GSTtag.
A recently developed cross-linking method with labeled chemicals allows using massspectroscopy to identify interacting domains in complexes in vitro (Maiolica, Cittaro et al.). The
technique was developed for recombinant proteins but we wanted to try it on the endogenous
complex. After purification of the endogenous complex by immunoprecipitation, the labeled
cross-linkers are added to the sample in vitro and the cross-linked peptides are analyzed either on
the gel or in solution after trypsin digestion. Because Rod, Zw10 and Zwilch form a stable
complex and can be pulled down together (Williams, Li et al. 2003) we targeted Zw10 for
immunoprecipitating the whole complex. We created stable cell lines expressing FLAG-Zw10
and we used Sigma ANTI-FLAG® M1 Agarose Affinity Gel for immunoprecipitation. As shown
in Figure 4, we succeeded to pull down the whole endogenous complex from mitotic cells.
However, although we were able to cross-link this material to a very high molecular complex,
we failed to detect any cross-linked peptides that might be revealing inter-subunit interactions,
most likely because of the insufficient sensitivity of the signals from cross-linked peptides during
mass spectrometry analysis.
Figure 4 . A. Colloidal Blue Staining of Sigma ANTI-FLAG® immuno-precipitation from
Nocodazole treated HeLaS3 cells stably expressing either the empty (Flag) or Zw10 containing
vector (Flag-Zw10). The correspondence of each number indicated band is explained in the
Table. B. Western blot of anti-GFP immunoprecipitation from HeLaS3 cells stably expressing
either GFP or GFP-Nag.
NAG is a novel Zw10 interactor:
Besides Zw10, Zwilch and ROD, we also identified Rint-1 (Rad50-interactor-1) and NAG
(Neuroblastoma Amplified Protein) as very abundant components of our Flag-Zw10 pull-downs
(Figure 4A). Interphase interaction of Zw10 with Rint-1 is well known to be important in Golgi
trafficking (Hirose, Arasaki et al. 2004; Arasaki, Taniguchi et al. 2006). However, this
interaction has not been characterized in mitosis. The second protein, NAG, is a novel Zw10
interactor which is confirmed by reciprocal immunoprecipitation (Figure 4B). The knowledge on
NAG protein is concentrated on its co-amplification with myc in neuroblastomas (Scott, Elsden
et al. 2003) and recently about its role in mRNA decay (Longman, Plasterk et al. 2007).
Similarity of NAG and ROD
NAG and ROD are remarkably similar in their size. A psi-BLAST run for NAG identified ROD
as a possible NAG homologue (Figure 5). At a more detailed analysis, ROD and NAG show very
high structural similarity. Both proteins have an N-terminal beta-propeller domain (a proteinprotein interaction motif), which is followed by ARM (Armadillo) repeats (a protein-protein
interaction motif) (Figure 6) (by Federico Giorgi).
Figure 5. PSI-BLAST scores. Stars indicate Rod (KIAA0166/ Kinetochore associated-1).
The similarity between NAG and ROD is particularly strong in a domain, adjacent to the betapropeller region, of about 150 aminoacids. Based on the identification of this homologous region
between ROD and NAG, we speculated that this homologous region could be putative Zw10
binding site. To test this idea we sub-cloned these regions either alone or in a bi-cistronic vector
with Zw10. Unfortunately; we have so far failed to get the NAG or ROD regions in a soluble
form from single expression. We have also failed to convert the insolubility of the NAG and
ROD segments by Zw10 co-expression.
1
NAG
1
ROD
420/460
725
Rod
homolog
y
521
709
ß-propeller
830
1374
ARM
repeat
Sec39
1800
2371
ARM
repeat
1330
390
595
Nag
ß-propeller homology
2209
ARM repeats
Figure 6. Structural analysis of Rod and Nag sequences (by Federico Giorgi)
NAG is not a member of RZZ complex:
Based on the similarities between NAG and ROD, we speculated that NAG could play a role as a
mitotic partner of ZW10. Moreover, NAG could be a binding partner of RZZ complex. In order
to check this possibility, we immunoprecipitated the RZZ complex by using a polyclonal antiZwilch antibody from mitotic HeLaS3 cells but we failed to detect NAG in Zwilch
immunoprecipitates (Figure 7A). So we concluded that NAG is not a RZZ member.
Figure 7 A. Colloidal Blue Staining of anti-Zwilch immuno-precipitation from Nocodazole
treated HeLaS3 cells. The correspondence of each number indicated band is explained in the
Table. B. Western blot of anti-Zw10 or anti-GFP immunoprecipitation respectively from
HeLaS3 cells or from HeLaS3 cells stably expressing either GFP or GFP-Nag.
Zw10 is a protein with both mitotic and interphase functions. The latter is well associated with
its Rint-1 interaction (Hirose, Arasaki et al. 2004; Arasaki, Taniguchi et al. 2006) and Rint-1 was
one of the proteins that we had observed in our Flag-Zw10 immunoprecipitates. Considering this
information and the presence of Rint-1 and Zw10 in GFP-NAG immunoprecipitations while
Zwilch is absent (Figure 7B) we confirmed the existence of two different Zw10 including
complexes; one with ROD and the other with NAG proteins.
Two distinct Zw10 complexes:
In order to show the existence of two distinct pools of Zw10 in two different complexes namely;
RZZ or NAG-Rint-1-Zw10 (NRZ) we have fractionated the HeLaS3 cell lysates in Superose 6
column and followed RZZ with anti-Zwilch and NRZ with anti-NAG and anti-Rint-1 antibodies
by western blotting. And NRZ complex eluted slightly before RZZ complex while Zw10 was
present in both peaks (Figure 8).
Figure 8. Size exclusion chromatography of
HeLaS3 cell lysates on Superose 6 and
western blotting of the black line indicated
region with with mentioned antibodies.
(BioRad Satndards: Thyroglobulin (85A),
Gamma-globulin (52.5), Ovalbumin (30.5A),
Myoglobulin (19A))
Hydrodynamic analysis of RZZ and NRZ complexes:
To calculate the actual size of the complexes by using Monty-Siegel equation we have further
analyzed the HeLaS3 cell lysates to determine the sedimentation coefficients of RZZ and NRZ
complexes. Both complexes appeared in the same fraction of the sedimentation experiment
(Figure 9).
Figure 9. Western blotting of fractions from sedimentation
analysis of HeLaS3 cell lysates by glycerol gradient with
indicated antibodies. (GE Healthcare Gel Filtration Calibration
Kit HMW: Thyroglobulin (19.1S), Catalase (11.3S), Aldolase
(7.45S), BSA (4.22S))
The Stokes radii of Zwilch/ ROD or NAG containing complexes were derived from a calibration
curve of size exclusion chromatography elution volume and determined to be 99 Å and 124 Å,
respectively. The sedimentation behaviors of Zwilch/ ROD or NAG containing complexes were
calculated using glycerol density gradients (10–40%). This experiment returned a sedimentation
coefficient of 19.3 S for both complexes.
By applying Monty-Siegel equation we calculated the molecular weight of the Zwilch/ ROD
containing Zw10 complex to be approximately 800 kDa and NAG containing Zw10 complex to
be approximately 1 MDa. We suggest that NAG replaces ROD in a separate but structurally
analogous complex of Zw10, and that also include RINT-1 (Figure 10). In future we will try to
confirm this hypothesis and will try to assess the architecture of the two assemblies.
Figure 10. Model for RZZ and NRZ complexes.
NAG localizes to Endoplasmic Reticulum (ER) as Zw10:
We have used polyclonal peptide anti-NAG antibodies to localize NAG in the cell. Our
preliminary results suggest the presence of NAG in ER structure similar to Zw10 (Figure 11).
However, our peptide antibodies recognize many unspecific bands on western blotting and in
order to prove the localization experiment we would like to couple it with siRNA depletion of
the gene and also the localization signal. Moreover, we aim to show the depletion of NAG
protein from the cells causes Golgi dispersal as in the case of Zw10 or Rint-1 siRNA depletions
(Vallee, 2006, Hirose 2004, Tagaya 2006, Storrie 2007) due to the collapse in the membrane
trafficking between ER and Golgi.
Figure 11. Nag and Zw10 (Green) localization in HeLa cells with two different polyclonal
peptide anti-NAG antibodies or with anti-Zw10, respectively.
Characterization and Crystallization of Zwilch
The bacterial expression of Zwilch yields around 2 mg of pure soluble protein per liter of
bacterial culture. The soluble full-length Zwilch elutes as a single peak at the expected molecular
weight (~ 67 kDa) in size exclusion chromatography (Figure 12) indicating a monomeric
globular protein. Analytical ultracentrifugation sedimentation velocity experiments also
confirmed that Zwilch is monomeric and showed that its preparations are homogeneous.
158kDa
17kDa
670kDa
44kDa
Figure 12. Size Exclusion Chromatography of
full-length Zwilch. Blue curve: Zwilch, Red curve:
Bio-Rad molecular weight markers (sizes indicated
on the figure) Below, the Coomassie blue stained
SDS-PAGE of the gel filtration fractions.
Full-length Zwilch was concentrated to 15 mg/ml and tested in crystallization screens. Although
we obtained preliminary encouraging pre-crystalline states our optimizations failed to provide us
with crystals.
The failure in crystallization might be due to disordered or floppy regions in the protein structure
so we characterized Zwilch by using limited proteolysis to look for smaller constructs to work
with. This characterization revealed that the protein is made of two stable domains with an
apparently flexible linker that is heavily susceptible to proteolytic cleavage (Figure 13).
Moreover, the full-length protein degrades to two similar stable fragments if left at 4°C for a few
days. These results immediately suggested a strategy for improving the chances of crystallizing
the protein and its domains using different constructs that were designed based on the segments
identified by limited proteolysis. We have created several constructs in which the N-terminal and
the C-terminal domains of the protein and cloned to the first or the second cassette of the pGEX6p-2rbs bi-cistronic vector, respectively and co-purified from the preparations.
Figure 13. Trypsin digestion of full-length Zwilch.
10 ug of Zwilch was digested in indicated trypsin
concentration at room temperature for 15 min.
The two domains of Zwilch tightly interact with each other because they elute together from the
size-exclusion chromatography column at a molecular weight already observed for the full length
protein (Figure 14). We refer to these complex as Zwilch2D. Among seven different complexes
with different N-terminal domain endings and C-terminal domain beginnings we got crystals of
one of the constructs which gave promising results from initial diffraction analysis at the
synchrotron. Recently, we concentrated on optimizing the size of the crystals for further
diffraction studies.
A.
B.
Figure 14. A. Size Exclusion Chromatography of full-length Zwilch (ZwilchFL) and Zwilch2D .
Below, the Coomassie blue stained SDS-PAGE of the gel filtration fractions of Zwilch 2D. B.
Zwilch2D crystals.