Crystal structure of human dynein light chain Dnlc2A: Structural insights into the interaction with IC74

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Abstract

The human light chain of the motor protein dynein, Dnlc2A, is also a novel TGF-β-signaling component, which is altered with high frequency in epithelial ovarian cancer. It is an important mediator of dynein and the development of cancer, owing to its ability to bind to the dynein intermediate light chain (DIC) IC74 and to regulate TGF-β-dependent transcriptional events. Here we report the 2.1-Å crystal structure of Dnlc2A using single anomalous diffraction. The proteins form a homodimer in solution and interact mainly through the helix α2, strand β3, and the loop following this strand in each protein to generate a 10-stranded β-sheet core. The surface of the β-sheet core is mainly positively charged and predicted (by software PPI-Pred) to be the site that interacts with other partners. At the same time, the residues 79–82, 88, and 90 of each molecule formed two holes in the core. Residue 89 of each molecule, which is crucial for the DIC binding function of Dnlc2A, is within the holes. On the basis of these observations, we propose that the homodimer is the structural and functional unit maintained by hydrogen bonding interactions and hydrophobic packing, and that the patch of the surface of the β-sheet core is the main area of interaction with other partners. Furthermore, the two holes would be the key sites to interact with IC74.

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Materials and methods

Cloning, expression, and purification. The expression vector pET-Dnlc2A was constructed by the Shanghai Institute of Hematology. The protein was produced in an Escherichia coli strain, BL21 (DE3), that was transformed with the expression vector. Cells were grown on LB media containing 100-μg/μl ampicillin at 37 °C to an A600 of 0.6 and then induced with 1 mM isopropyl β-d-thiogalactoside for 3 h. Cells were harvested by centrifugation, resuspended in buffer (500 mM NaCl, 50 mM Tris–HCl, pH 8.0), and

Description of the structure

The crystal structure of Dnlc2A reveals a homodimer with a non-crystallographic twofold axis of symmetry (Fig. 1). Each of the monomers of Dnlc2A folds as a compact domain and consists of a central five-stranded antiparallel β-sheet, β1 (18–23), β2 (30–32), β3 (67–73), β4 (77–81), and β5 (86–92) sandwiched between α-helix α2 (36–60) on one side and the terminal helices α1 (6–13) on the other side. The strand order is 2 1 5 4 3. The electron density for the N terminal (the His tag, residues A1–8

Characterization of the dimer interface

The two molecules in the asymmetric unit are related by a non-crystallographic twofold axis and are superimposable with a root mean square deviation of 0.41 Å over all C atoms of residues 8–95.

The dimer of Dnlc2A associates to form a continuous, antiparallel 10-stranded sheet with strand order 2 1 5 4 3 3* 4* 5* 1* 2* (the asterisk denotes molecule B). One side of the central β-sheet is covered by α2-helix bundles, α2 and α2′, whereas the other side is covered by the α1/α1′ two-helix bundle (

Acknowledgments

This work was supported by the National Natural Science Foundation of China (NSFC) Nos. 30130080, 30200046; 973 project No. 2002CB713801; Chinese High Tech Research and Development (863) Program No. 2001AA233021, and KSCX1-SW-17 from the Ministry of Science and Technology of China (MOST) as well as fund from the Chinese Academy of Science (CAS). We wish to thank Mr. Han Yi, for his help during diffraction data collection. We also wish to thank Dr. Qiuhua Huang of the Shanghai Institute of

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