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Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis

DOI: 10.7717/peerj.9408 DOI Help

Authors: Shanshan Wu (University of Copenhagen) , Tam T. T. N. Nguyen (University of Copenhagen) , Olga V. Moroz (University of York) , Johan P. Turkenburg (University of York) , Jens E. Nielsen (Novozymes A/S) , Keith S. Wilson (University of York) , Kasper D. Rand (University of Copenhagen) , Kaare Teilum (University of Copenhagen)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Peerj , VOL 8

State: Published (Approved)
Published: June 2020
Diamond Proposal Number(s): 9948

Open Access Open Access

Abstract: Background: Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown. Methods: Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals. Results: The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.

Journal Keywords: Protein dynamics; Hydrogen exchange; Mass spectrometry; NMR spectroscopy; X-ray crystallography

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography