Improved Peak Detection and Deconvolution of Native Electrospray Mass Spectra from Large Protein Complexes.

Native electrospray-ionization mass spectrometry (native MS) measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact protein assemblies. However, native spectra derived from these assemblies are often partially obscured by low signal-to-noise as well as broad peak shapes because of residual solvation and adduction after the electrospray process. The wide peak widths together with the fact that sequential charge state series from highly charged ions are closely spaced means that native spectra containing multiple species often suffer from high degrees of peak overlap or else contain highly interleaved charge envelopes. This situation presents a challenge for peak detection, correct charge state and charge envelope assignment, and ultimately extraction of the relevant underlying mass values of the noncovalent assemblages being investigated. In this report, we describe a comprehensive algorithm developed for addressing peak detection, peak overlap, and charge state assignment in native mass spectra, called PeakSeeker. Overlapped peaks are detected by examination of the second derivative of the raw mass spectrum. Charge state distributions of the molecular species are determined by fitting linear combinations of charge envelopes to the overall experimental mass spectrum. This software is capable of deconvoluting heterogeneous, complex, and noisy native mass spectra of large protein assemblies as demonstrated by analysis of (1) synthetic mononucleosomes containing severely overlapping peaks, (2) an RNA polymerase II/α-amanitin complex with many closely interleaved ion signals, and (3) human TriC complex containing high levels of background noise. Graphical Abstract ᅟ.

Reconstitution of nucleosome demethylation and catalytic properties of a Jumonji histone demethylase.

Jumonji histone demethylases catalyze removal of methyl marks from lysine residues in histone proteins within nucleosomes. Here, we show that the catalytic domain of demethylase JMJD2A (cJMJD2A) utilizes a distributive mechanism to remove the histone H3 lysine 9 trimethyl mark. By developing a method to assess demethylation of homogeneous, site-specifically methylated nucleosomes, we determined that the kinetic parameters for demethylation of nucleosomes by cJMJD2A are comparable to those of peptide substrates. These findings imply that other domains of the demethylase or its protein partners may contribute to nucleosome recognition in vivo and, in this way, may further regulate demethylation activity and processivity. The quantitative assays of nucleosome demethylation developed in our work provide a platform for future work with complex chromatin substrates and full-length demethylases.

3'-Phosphoadenosine-5'-phosphosulfate reductase in complex with thioredoxin: a structural snapshot in the catalytic cycle.

The crystal structure of Escherichia coli 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase in complex with E. coli thioredoxin 1 (Trx1) has been determined to 3.0 A resolution. The two proteins are covalently linked via a mixed disulfide that forms during nucleophilic attack of Trx's N-terminal cysteine on the Sgamma atom of the PAPS reductase S-sulfocysteine (E-Cys-Sgamma-SO3-), a central intermediate in the catalytic cycle. For the first time in a crystal structure, residues 235-244 in the PAPS reductase C-terminus are observed, depicting an array of interprotein salt bridges between Trx and the strictly conserved glutathione-like sequence, Glu238Cys239Gly240Leu241His242. The structure also reveals a Trx-binding surface adjacent to the active site cleft and regions of PAPS reductase associated with conformational change. Interaction at this site strategically positions Trx to bind the S-sulfated C-terminus and addresses the mechanism for requisite structural rearrangement of this domain. An apparent sulfite-binding pocket at the protein-protein interface explicitly orients the S-sulfocysteine Sgamma atom for nucleophilic attack in a subsequent step. Taken together, the structure of PAPS reductase in complex with Trx highlights the large structural rearrangement required to accomplish sulfonucleotide reduction and suggests a role for Trx in catalysis beyond the paradigm of disulfide reduction.

Substrate recognition, protein dynamics, and iron-sulfur cluster in Pseudomonas aeruginosa adenosine 5'-phosphosulfate reductase.

APS reductase catalyzes the first committed step of reductive sulfate assimilation in pathogenic bacteria, including Mycobacterium tuberculosis, and is a promising target for drug development. We report the 2.7 A resolution crystal structure of Pseudomonas aeruginosa APS reductase in the thiosulfonate intermediate form of the catalytic cycle and with substrate bound. The structure, high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, and quantitative kinetic analysis, establish that the two chemically discrete steps of the overall reaction take place at distinct sites on the enzyme, mediated via conformational flexibility of the C-terminal 18 residues. The results address the mechanism by which sulfonucleotide reductases protect the covalent but labile enzyme-intermediate before release of sulfite by the protein cofactor thioredoxin. P. aeruginosa APS reductase contains an [4Fe-4S] cluster that is essential for catalysis. The structure reveals an unusual mode of cluster coordination by tandem cysteine residues and suggests how this arrangement might facilitate conformational change and cluster interaction with the substrate. Assimilatory 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductases are evolutionarily related, homologous enzymes that catalyze the same overall reaction, but do so in the absence of an [Fe-S] cluster. The APS reductase structure reveals adaptive use of a phosphate-binding loop for recognition of the APS O3' hydroxyl group, or the PAPS 3'-phosphate group.