TY - JOUR

T1 - Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis

AU - Wu, Shanshan

AU - Nguyen, Tam T. T. N.

AU - Moroz, Olga V.

AU - Turkenburg, Johan P.

AU - Nielsen, Jens E.

AU - Wilson, Keith S.

AU - Rand, Kasper D.

AU - Teilum, Kaare

PY - 2020

Y1 - 2020

N2 - 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.

AB - 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.

KW - Protein dynamics

KW - Hydrogen exchange

KW - Mass spectrometry

KW - NMR spectroscopy

KW - X-ray crystallography

KW - SERINE-PROTEASE PB92

KW - BACILLUS-LENTUS

KW - TEMPERATURE-DEPENDENCE

KW - SECONDARY STRUCTURE

KW - DYNAMICS

KW - ASSIGNMENTS

KW - REVEALS

KW - MOTIONS

KW - STATES

KW - SITE

U2 - 10.7717/peerj.9408

DO - 10.7717/peerj.9408

M3 - Journal article

C2 - 32617193

VL - 8

JO - PeerJ

JF - PeerJ

SN - 2167-8359

M1 - e9408

ER -