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

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.

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

“…HDX-MS is an adequate technique for detecting structural changes induced by ligand binding as well as localizing allosteric regulatory sites [ 16 , 20 , 126 ]. Since structural alterations due to direct/local effects and allostery are cumbersome to distinguish, expression of the relevant binding domains as well as complementary methods, such as Förster resonance energy transfer (FRET), chemical cross-linking [ 10 , 127 , 128 , 129 ] and mutagenesis [ 130 ], should often be also employed. A good example of this principle was reported by Masson et al in 2016 [ 131 ], where the authors studied the protein called Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), an important tumor suppressor.…”

Geometric Algebra Attention Networks for Small Point Clouds

“…• 3X2L (Nakamura et al, 2015) • 3X32 (Hirano et al, 2015) • 4TKJ (Matsuoka et al, 2015) • 3X0J (Furuike et al, 2016) • 5WQR (Ohno et al, 2017) • 6EQE (Austin et al, 2018) • 6ETK (Vergara et al, 2018) • 6FJN (Baker et al, 2019) • 6JGJ (Takaba et al, 2019) • 6RYG (Paterson et al, 2019) • 6IIP (Tian et al, 2019) • 6Q01 (Schellenberg et al, 2020) • 6XVM (Plaza-Garrido et al, 2020) • 6Y5S (Wu et al, 2020) • 6YP6 (Franke et al, 2020) • 7A5M (Barone et al, 2020) • 7K4T (Otten et al, 2020) • 6YK4 (Frydenvang et al, 2020) • 6SAY (Glöckner et al, 2020)…”

Proteolytic Performance is Dependent on Binding Efficiency, Processivity and Turnover: Single Protease Insights

“…Proteases hydrolyse the peptide bonds in proteins 1,2 , making them important catalysts in living organisms and invaluable in biotechnological industries [3][4][5][6] . Savinase is an alkaline serine endoprotease belonging to the subtilisin superfamily [7][8][9] . It plays a paramount role in biotechnology due to its high stability and broad substrate specificity and is widely applied for protein-based stainremoval in detergency 10 as well as in, e.g., protein extraction 11 and biocatalysis 12 .…”

“…While we have an overall understanding of protease reactions 13,14 , the precise steps and interactions involved in substrate binding and degradation are still subject to ongoing research 7,[15][16][17] . The reactions of biotechnological interest are almost exclusively interfacial, and adsorption of the protease to the substrate surface is a fundamental requirement for activity.…”

“…The impact of the surface environment is also evident for Savinase that shows a different activity profile in heterogeneous versus homogeneous reaction conditions 10 . Curiously, Savinase has been shown to exhibit conformational dynamics in the region around the active site which may have functional significance on substrate binding 20,21 . The evidence emphasizes the importance of further studies to elucidate protease function in relevant interfacial contexts 10,18,19 but also the need for new tools to cope with spatiotemporal complexities of molecular mechanisms.…”