Photon Factory Activity Report 2016 #34 (2017)

BL-10C/2016G680

Solution structure of MBP-fused MDV1 for determining coiled-coil orientation
Byeong-Won Kim, Yang Ouk Jung and Hyun Kyu Song*Department of Life Sciences, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Korea

1 Introduction
A coiled-coil domain (CCD) consists of two or more
α-helices that are twisted around each other to form asuper-helical structure. Coiled-coils (CCs) have a heptad
repeat pattern (a-b-c-d-e-f-g)n, where the a and dpositions are usually occupied by hydrophobic residues[1-2]. CCs are easily detected by prediction programs orservers but they are very versatile structural domains that
can adopt many different structures. They can form
different oligomeric complexes, and the helix orientation
can either be parallel or antiparallel. Because of thesecharacteristics, CCs typically participate in diverse
protein-protein interactions that are involved in many
biological functions and components. To understand thedetailed molecular functions of CC-containing proteins, it
is absolutely necessary to determine the relativeorientation of each coil because the functional domain
outside a parallel CCD dimer must be in close proximity,
but that outside an antiparallel CCD is located furtheraway. So far, many programs or servers have beendeveloped to determine the oligomeric state of CCDs but
none of these programs provide adequate discrimination
between parallel and antiparallel orientations. To
overcome the above limitations, we have developed a
new biochemical technique using a fusion tag, which isan appropriately spaced and oriented molecule that also
aids in enhancing the solubility of CC proteins, to assessthe orientation of the CC dimer [3].
2 Experiment
The gene fragment of yeast MDV1 (mitochondrial
division protein 1; 231-300) was ligated into a modified
pMAL vector with an N-terminal hexa-histidine sequence.
The overexpressed fusion protein was loaded to a column
containing amylose resin and collected by gravity flow
followed by anion exchange and size exclusion
chromatography. The concentration of MBP (MaltoseBinding Protein)-MDV1 was 9.8 mg/ml. Scattering dataof this protein was collected at BL-10C. The scattering
images from the proteins were reduced to 2D data by
circular integration. Preliminary analysis of these 2D datausing PRIMUS (ATSAS program suite) provided theradius of gyration (Rg), Porod volume, and experimental
molecular weight.
3 Results
CCD of MDV1 was observed as a clear antiparallel
dimer [4]. To confirm the directionality of the CCs of
MDV1 in solution, we used SAXS (small angle X-rayscattering). The theoretical scattering curve calculatedusing DAMMIF fits well with the scattering intensity offusion protein (Fig. 1A). This fusion protein has a P(r)function with a two peaks and Dmax is about 150 (Fig. 1B).
The experimental scattering data and the patterncalculated from the atomic co-ordinates match well using
the program CRYSOL (Fig. 1C). These results clearly
showed that it has a dumbbell-shaped envelop (Fig. 1D).
The crystal structure was fitted into the SAXS envelop.

AC

B

DFig. 1: SAXS curves of MBP-MDV1. Scattering curve
calculated with the DAMMIF (A) and distance
distribution function, P(r) function (B). Comparison ofI(q) calculated by CRYSOL from the atom structure (C).
Molecular envelop fitted the high-resolution crystal
structure (D).

References[1] A.N. Lupas et al. Adv. Protein Chem. 70, 37 (2005).
[2] A.N. Lupas et al. Science 252, 1162 (1991).
[3] B.W. Kim et al. Sci. Rep. 7, 43318 (2017).
[4] S. Koirala et al. J. Cell Biol. 191, 1127 (2010).

* hksong@korea.ac.kr