Molecular motion regulates the activity of the mitochondrial serine protease HtrA2
HtrA2 (high-temperature requirement 2) is a human mitochondrial protease that has a role in apoptosis and Parkinson's disease. The structure of HtrA2 with an intact catalytic triad was determined, revealing a conformational change in the active site loops, involving mainly the regulatory LD loop, which resulted in burial of the catalytic serine relative to the previously reported structure of the proteolytically inactive mutant. Mutations in the loops surrounding the active site that significantly restricted their mobility, reduced proteolytic activity both in vitro and in cells, suggesting that regulation of HtrA2 activity cannot be explained by a simple transition to an activated conformational state with enhanced active site accessibility. Manipulation of solvent viscosity highlighted an unusual bi-phasic behavior of the enzymatic activity, which together with MD calculations supports the importance of motion in the regulation of the activity of HtrA2. HtrA2 is an unusually thermostable enzyme (TM=97.3 °C), a trait often associated with structural rigidity, not dynamic motion. We suggest that this thermostability functions to provide a stable scaffold for the observed loop motions, allowing them a relatively free conformational search within a rather restricted volume. ; We would like to thank Professor Margarida Bastos and Dr. Frederico Silva for assistance with calorimetry, Professor Manuel J.B. Marques with refractometry, Professor Eduardo Marques and Dr. João Neves with viscosometry, Dr. Trent Balius with hierarchical clustering, Professor Brian K. Shoichet with interpreting protein stability measurements, Dr. Hélder Maiato, Dr. Cristina Ferrás, Dr. Emma Commas-Casellas and Luísa Ferreira for help with cell culture. Funding to Biomolecular Structure and Function lab was provided by the project Norte-01-0145-FEDER-000008 – Porto Neurosciences and Neurologic Disease Research Initiative at I3S, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). CM, MJR and PAF acknowledge the financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007728) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/MULTI/04378/2013. C.M. is a recipient of a FCT PhD fellowship (SFRH/BD/84016/2012). M.M. thanks the Fundação para a Ciência e a Tecnologia (FCT) for its support through the Ciência 2008 program. ; info:eu-repo/semantics/publishedVersion
