Research Article: Protein–ligand complex structure from serial femtosecond crystallography using soaked thermolysin microcrystals and comparison with structures from synchrotron radiation

Date Published: August 01, 2017

Publisher: International Union of Crystallography

Author(s): Hisashi Naitow, Yoshinori Matsuura, Kensuke Tono, Yasumasa Joti, Takashi Kameshima, Takaki Hatsui, Makina Yabashi, Rie Tanaka, Tomoyuki Tanaka, Michihiro Sugahara, Jun Kobayashi, Eriko Nango, So Iwata, Naoki Kunishima.

http://doi.org/10.1107/S2059798317008919

Abstract

The applicability of the ligand-soaking method in serial femtosecond crystallography has been examined to examine the feasibility of pharmaceutical applications of X-ray free-electron lasers.

Partial Text

X-ray free-electron lasers (XFELs) generate very short/intense pulses that enable the collection of diffraction data before the destruction of the specimen (Neutze et al., 2000 ▸). This ‘diffraction-before-destruction’ principle of XFELs has successfully been applied in serial femtosecond crystallo­graphy (SFX), in which hundreds of thousands of single-shot diffraction images from randomly oriented microcrystals at room temperature are merged to determine a crystal structure (Chapman et al., 2011 ▸; Boutet et al., 2012 ▸). To date, a substantial number of SFX structures have been reported, including those of natively inhibited trypanosome protease from in vivo-grown microcrystals (Redecke et al., 2013 ▸), of membrane proteins from microcrystals grown in lipidic cubic phase (Zhang et al., 2015 ▸; Kang et al., 2015 ▸) and of the photoactive yellow protein in a time-resolved pump–probe experiment (Pande et al., 2016 ▸). Because SFX provides crystal structures at room temperature without radiation damage, it has the potential to be a useful tool in structural biology, which requires structural information under physiological conditions. For instance, a damage-free structure from SFX could account for the proton-transfer mechanism of nitrite reductase (Fukuda et al., 2016 ▸). From this point of view, structure-based drug design (SBDD) is expected to be a likely application of SFX (Zhang et al., 2015 ▸; Hol, 2015 ▸). In SBDD, a small-molecule ligand is designed so as to improve its affinity for the target protein based on the structure of protein–ligand complex crystals, which are typically prepared by soaking protein crystals into a solution containing the ligand (Hol, 1986 ▸; Klebe, 2000 ▸). However, the applicability of soaked crystals in SFX has not fully been examined to date. Here, we present a ligand-soaking experiment in SFX using microcrystals of thermolysin, which has recently been demonstrated as a model system (Hattne et al., 2014 ▸). From a comparison of the SFX structures with those of a conventional experiment using synchrotron radiation at low temperatures, the applicability of SFX to SBDD will be discussed.

In this work, the feasibility of ligand screening in SFX has been examined using thermolysin as a model system. As a result, a ligand-soaking experiment using SFX successfully provided untreated protein–ligand complex structures at room temperature. From a structural comparison between SFX and SR, clear structural differences in the ligand-binding mode were observed. Notably, the SFX structures were highly reproducible regardless of the type of crystal carrier used, whereas the SR structures showed substantial differences depending on the cryoprotection procedure that was used; Cα superposition between the liganded SR1 form and the liganded SR2 form provided a distribution with significantly higher values of the Cα deviation when compared with any superposition between a pair of SFX structures (Table 2 ▸ and Supplementary Table S1). In conclusion, ligand screening in SFX may be useful for the design of small-molecule ligands for SBDD in the near future, because it provides structural information without factors that may affect the physiological structure of proteins.

 

Source:

http://doi.org/10.1107/S2059798317008919

 

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