Photon Factory Activity Report 2014 #32 (2015) B

BL-17A/2012G042

Crystal structure of the yeast condensin hinge with long coiled coils
1Young-Min Soh1, Byung-Ha Oh1
Department of Biological Sciences, KAIST Institute for the Biocentury, Korea Advanced Institute ofScience and Technology, Daejeon 305-701, Korea

1 Introduction
During cell division, the cell is given the daunting taskof packaging enormous amount of DNA into a limited
amount of space as well as proper segregation ofreplicated DNA into daughter cells. The condensin
complex is essential for both processes. The eukaryotic
condensin complex consists of Smc2, Smc4, and threeother subunits [2]. The Smc subunits are part of a large
family of proteins called the Structural Maintenance ofChromosomes. The N-terminal and C-terminal fold toform an ABC ATPase on one end while the opposite end
consists of the hinge domain responsible for heterodimerization. The two globular domains are separated by
a 50 nm long coiled coil domain [1]. Here we solved thecrystal structure of the yeast Smc2 and Smc4 hinge
heterodimer with long stretches of coiled coils. Contrary
to expectation, the crystal structure revealed that thecoiled coils are juxtaposed onto each other forming aclosed structure rather than open structure. This was alsoobserved in solution through SAXS analysis.
2 Experiment
The DNA fragments encoding Smc2 residues 396-792(Smc2H-LCC) and Smc4 residues 555-951 (Smc4HLCC) were inserted into pProExHTa (Invitrogen) andpET30a (Novagen) by standard PCR-based cloning
methods. SelMet-substituted Smc4H-LCC was expressedin E.coli B834(DE3) RIL (Novagen), and native Smc2HLCC was expressed in E. coli BL21(DE3) RIPL strain.
The cells obtained from the two different cultures wereco-sonicated and purified using affinity chromatography,
ion exchange chromatography, and size exclusion
chromatography. The final buffer contained 100 mM
NaCl, 20 mM Tris-HCl pH 7.5, and 2 mM DTT. The
Smc2H-LCC–Smc4H-LCC complex (20 mg/ml) was
crystallized using the hanging-drop vapor diffusion
technique at 20 °C in a precipitant solution containing
16% PEG 300, 0.1 M Na/K phosphate pH 6.0, 8%
glycerol and 10 mM dithiothreitol (DTT). X-ray
diffraction data were collected at the beamline BL17A at
the Photon Factory, Japan. A single-wavelength
anomalous dispersion (SAD) data set was collected with a
SelMet-substituted Smc2H-LCC–Smc4H-LCC crystal at
the Se absorption peak. Diffraction data were processedwith HKL2000. The Smc2H-LCC–Smc4H-LCC structure
was solved by the single isormorphous replacement withanomalous scattering (SIR-AS) method. The modelbuilding and structure refinement were carried out using
the programs COOT and CNS. The final model of
Smc2H-LCC–Smc4H-LCC does not include residues
671-673 of Smc2 and 836-841 of Smc4, which aredisordered in the crystals. The structure factor andcoordinates of the structure are deposited in the Protein
Data Bank (www.rcsb.org, PDB ID:4RSI).
3 Results and Discussion
The overall structure of yeast Smc2/4 hinge
heterodimer is depicted in Figure 1. It contains about 50
residues of coiled coil stretches for the Smc2 hinge andmore than 100 residues for the Smc4 hinge.
Similar to previous Smc hinge domain structures, theyeast Smc2/4 hinge forms a toroid structure whichcontains a central hole. However, because the coiled coils
are oriented differently in Smc2 and Smc4, uponheterodimerization, the coiled coils form closed structures
unlike the open structures depicted in previous bacterial
Smc hinge structures.
In order to know whether or not the coiled coils form
closed structures in solution, the same construct wasanalyzed using SAXS. The distance distribution function
P(r) indicated a maximum dimension of 200 Å, which is
close to the measured length (190 Å) of the complex in
the crystal structure. The molecular envelope derived
from SAXS analysis fits well with the crystal structure(Fig. 2). Hence, the coiled coils of yeast Smc2/4 hinge
form closed structures also in solution.
In summary, these results provide new insight into theconformation of the coiled coils of Smc proteins.

Fig. 1: Overall structure of yeast Smc2/4 hinge with longcoiled coil stretches. In depth view of dimerizationinterface is detailed in the box.

Fig. 2: crystal structure superimposed to SAXS envelope.
The distance distribution function P(r) is boxed.

Photon Factory Activity Report 2014 #32 (2015) B

References[1] S. Nolivos, D. Sherratt, FEMS Microbiol. Rev. (2013).
[2] R. Thadani, F. Uhlmann, S. Heeger, Curr. Biol. 22:
R1012-1021 (2012).
*youngminsoh@gmail.com