Crystallization Strategy for the Glycoprotein-Receptor Complex Between Measles Virus Hemagglutinin and Its Cellular Receptor SLAM

Takao      Hashiguchi; Toyoyuki      Ose; Marie      Kubota; Nobuo      Maita; Jun      Kamishikiryo; Katsumi      Maenaka; Yusuke      Yanagi

Abstract

Measles virus (MV), one of the most contagious agents, infects immune cells using the signaling lymphocyte activation molecule (SLAM) on the cell surface. A complex of SLAM and the attachment protein, hemagglutinin (MVH), has remained elusive due to the intrinsic handling difficulty including glycosylation. Furthermore, crystals obtained of this complex are either nondiffracting or poorly-diffracting. To solve this problem, we designed a systematic approach using a combination of the following techniques; (1) a transient expression system in HEK293SGnTI(-) cells, (2) lysine methylation, (3) structure-guided mutagenesis directed at better crystal packing, (4) Endo H treatment, (5) single-chain formation for stable complex, and (6) floating-drop vapor diffusion. Using our approach, the receptor-binding head domain of MV-H covalently fused with SLAM was successfully crystallized and diffraction was improved from 4.5 Å to a final resolution of 3.15 Å . These combinational methods would be useful as crystallization strategies for complexes of glycoproteins and their receptors.

Keywords: SLAM, Crystallization, floating-drop vapor diffusion, glycoprotein, HEK293SGnTI(-) cells, lysine methylation, measles, virus, hemagglutinin, receptor