Structural mass spectrometry decodes domain interaction and dynamics of the full-length Human Histone Deacetylase 2.

Human Histone Deacetylase 2 (HDAC2) belongs to a conserved enzyme superfamily that regulates deacetylation inside cells. HDAC2 is a drug target as it is known to be upregulated in cancers and neurodegenerative disorders. It consists of globular deacetylase and C-terminus intrinsically-disordered domains [1-3]. To date, there is no full-length structure of HDAC2 available due to the high intrinsic flexibility of its C-terminal domain. The intrinsically-disordered domain, however, is known to be important for the enzymatic function of HDAC2 [1, 4]. Here we combine several structural Mass Spectrometry (MS) methodologies such as denaturing, native, ion mobility and chemical crosslinking, alongside biochemical assays and molecular modelling to study the structure and dynamics of the full-length HDAC2 for the first time. We show that MS can easily dissect heterogeneity inherent within the protein sample and at the same time probe the structural arrangement of the different conformers present. Activity assays combined with data from MS and molecular modelling suggest how the structural dynamics of the C-terminal domain, and its interactions with the catalytic domain, regulate the activity of this enzyme.

Optimization Workflow for the Analysis of Cross-Linked Peptides Using a Quadrupole Time-of-Flight Mass Spectrometer.

Cross-linking mass spectrometry is an emerging structural biology technique. Almost exclusively, the analyzer of choice for such an experiment has been the Orbitrap. We present an optimized protocol for the use of a Synapt G2-Si for the analysis of cross-linked peptides. We first tested six different energy ramps and analyzed the fragmentation behavior of cross-linked peptides identified by xQuest. By combining the most successful energy ramps, cross-link yield can be increased by up to 40%. When compared to previously published Orbitrap data, the Synapt G2-Si also offers improved fragmentation of the ß peptide. In order to improve cross-link quality control we have also developed ValidateXL, a programmatic solution that works with existing cross-linking software to improve cross-link quality control.