Search

New developments in analytical technologies and biophysical methods have advanced the characterization of increasingly complex biomolecular assemblies using native mass spectrometry (MS). Ion mobility methods, in particular, have enabled a new dimension of structural information and analysis of proteins, allowing separation of conformations and providing size and shape insights based on collision cross sections (CCSs). Based on the concepts of absorption-mode Fourier transform (aFT) multiplexing ion mobility spectrometry (IMS), here, a modular drift tube design proves capable of separating native-like proteins up to 148 kDa with resolution up to 45. Coupled with high-resolution Orbitrap MS, binding of small ligands and cofactors can be resolved in the mass domain and correlated to changes in structural heterogeneity observed in the ion-neutral CCS distributions. We also demonstrate the ability to rapidly determine accurate CCSs for proteins with 1-min aFT-IMS-MS sweeps without the need for calibrants or correction factors.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is an intrinsically disordered DNA-binding protein expressed during embryogenesis. In the present work, the conformational and binding dynamics of HMGA2 and HMGA2 in complex with a 22-nt (DNA(22)) and a 50-nt (DNA(50)) AT-rich DNA hairpin were investigated using trapped ion mobility spectrometry–mass spectrometry (TIMS–MS) under native starting solvent conditions (e.g., 100 mM aqueous NH(4)Ac) and collision-induced unfolding/dissociation (CIU/CID) as well as solution fluorescence anisotropy to assess the role of the DNA ligand when binding to the HMGA2 protein. CIU-TIMS–CID-MS/MS experiments showed a significant reduction of the conformational space and charge-state distribution accompanied by an energy stability increase of the native HMGA2 upon DNA binding. Fluorescence anisotropy experiments and CIU-TIMS–CID-MS/MS demonstrated for the first time that HMGA2 binds with high affinity to the minor groove of AT-rich DNA oligomers and with lower affinity to the major groove of AT-rich DNA oligomers (minor groove occupied by a minor groove binder Hoechst 33258). The HMGA2·DNA22 complex (18.2 kDa) 1:1 and 1:2 stoichiometry suggests that two of the AT-hook sites are accessible for DNA binding, while the other AT-hook site is probably coordinated by the C-terminal motif peptide (CTMP). The HMGA2 transition from disordered to ordered upon DNA binding is driven by the interaction of the three basic AT-hook residues with the minor and/or major grooves of AT-rich DNA oligomers.

Although it is widely accepted that protein function is largely dependent on its structure, intrinsically disordered proteins (IDPs) lack defined structure but are essential in proper cellular processes. Mammalian high mobility group proteins (HMGA) are one such example of IDPs that perform a number of crucial nuclear activities and have been highly studied due to their involvement in the proliferation of a variety of disease and cancers. Traditional structural characterization methods have had limited success in understanding HMGA proteins and their ability to coordinate to DNA. Ion mobility spectrometry and mass spectrometry provide insights into the diversity and heterogeneity of structures adopted by IDPs and are employed here to interrogate HMGA2 in its unbound states and bound to two DNA hairpins. The broad distribution of collision cross sections observed for the apo-protein are restricted when HMGA2 is bound to DNA, suggesting that increased protein organization is promoted in the holo-form. Ultraviolet photodissociation was utilized to probe the changes in structures for the compact and elongated structures of HMGA2 by analyzing backbone cleavage propensities and solvent accessibility based on charge-site analysis, which revealed a spectrum of conformational possibilities. Namely, preferential binding of the DNA hairpins with the second of three AT-hooks of HMGA2 is suggested based on the suppression of backbone fragmentation and distribution of DNA-containing protein fragments.

Analysis of native-like protein structures in the gas phase via native mass spectrometry and auxiliary techniques has become a powerful tool for structural biology applications. In combination with ultraviolet photodissociation (UVPD), native top-down mass spectrometry informs backbone flexibility, topology, hydrogen bonding networks, and conformational changes in protein structure. Although it is known that the primary structure affects dissociation of peptides and proteins in the gas phase, its effect on the types and locations of backbone cleavages promoted by UVPD and concomitant influence on structural characterization of native-like proteins is not well understood. Here, trends in the fragmentation of native-like proteins were evaluated by tracking the propensity of 10 fragment types (a, a+1, b, c, x, x+1, y, y-1, Y, and z) in relation to primary structure in a native-top down UVPD data set encompassing >9600 fragment ions. Differing fragmentation trends are reported for the production of distinct fragment types, attributed to a combination of both direct dissociation pathways from excited electronic states and those surmised to involve intramolecular vibrational energy redistribution after internal conversion. The latter pathways were systematically evaluated to evince the role of proton mobility in the generation of "CID-like" fragments through UVPD, providing pertinent insight into the characterization of native-like proteins. Fragmentation trends presented here are envisioned to enhance analysis of the protein higher-order structure or augment scoring algorithms in the high-throughput analysis of intact proteins.

Development of chemical chaperones to solubilize membrane protein complexes in aqueous solutions has allowed for gas-phase analysis of their native-like assemblies, including rapid evaluation of stability and interacting partners. Characterization of protein primary sequence, however, has thus far been limited. Ultraviolet photodissociation (UVPD) generates a multitude of sequence ions for the E. coli ammonia channel (AmtB), provides improved localization of a possible post-translational modification of aquaporin Z (AqpZ), and surpasses previous reports of sequence coverage for mechanosensitive channel of large conductance (MscL). Variations in UVPD sequence ion abundance have been shown to correspond to structural changes induced upon some perturbation. Preliminary results are reported here for elucidating increased rigidity or flexibility of MscL when bound to various phospholipids.

As applications in mass spectrometry continue to expand into the field of structural biology, there have been an increasing number of studies on noncovalent biological assemblies. Ensuring that protein complexes maintain native-like conformations and architectures during the transition from solution to the gas phase is a key aim. Probing composition and arrangement of subunits of multi-charged complexes via tandem mass spectrometry (MS/MS) may lead to protein unfolding and the redistribution of charges on the constituent subunits, leading to asymmetric charge partitioning and ejection of a high-charged monomer. Additionally, the overall dissociation efficiency of many ion activation methods is often suppressed for low charge states, hindering the effectiveness of MS/MS for complexes that have low charge density. Ultraviolet photodissociation (UVPD) of proteins using 193-nm photons is a high-energy alternative to collisional activation and demonstrates little to no charge state dependence. Here the symmetry of charge partitioning upon UVPD is evaluated for an array of multimeric protein complexes as a function of initial charge state. The results demonstrate that high laser energies (3 mJ) for UVPD induces more symmetric charge partitioning and ejection of low-charged, presumably compact monomers than higher-energy collisional dissociation (HCD).

The C-terminal domain (CTD) of the largest subunit in eukaryotic RNA polymerase II has a repetitive heptad sequence of Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 which is responsible for recruiting transcriptional regulatory factors. The seventh heptad residues in mammals are less conserved and subject to various post-translational modifications, but the consequences of such variations are not well understood. In this study, we use ultraviolet photodissociation mass spectrometry, kinetic assays, and structural analyses to dissect how different residues or modifications at the seventh heptad position alter Tyr1 phosphorylation. We found that negatively charged residues in this position promote phosphorylation of adjacent Tyr1 sites, whereas positively charged residues discriminate against it. Modifications that alter the charges on seventh heptad residues such as arginine citrullination negate such distinctions. Such specificity can be explained by conserved, positively charged pockets near the active sites of ABL1 and its homologues. Our results reveal a novel mechanism for variations or modifications in the seventh heptad position directing subsequent phosphorylation of other CTD sites, which can contribute to the formation of various modification combinations that likely impact transcriptional regulation.

SummaryThe Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of RNA polymerase II’s C-terminal domain (CTD) and is essential for the transition from transcription initiation to elongationin vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activityin vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Throughin vitroreconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes a reduction of phosphorylated Ser2 and accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD’s coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of the C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes an accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD’s coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes., eLife digest DNA contains the instructions for making proteins, which build and maintain our cells. So that the information encoded in DNA can be used, a molecular machine called RNA polymerase II makes copies of specific genes. These copies, in the form of a molecule called RNA, convey the instructions for making proteins to the rest of the cell. To ensure that RNA polymerase II copies the correct genes at the correct time, a group of regulatory proteins are needed to control its activity. Many of these proteins interact with RNA polymerase II at a region known as the C-terminal domain, or CTD for short. For example, before RNA polymerase can make a full copy of a gene, a small molecule called a phosphate group must first be added to CTD at specific units known as Ser2. The regulatory protein P-TEFb was thought to be responsible for phosphorylating Ser2. However, it was previously not known how P-TEFb added this phosphate group, and why it did not also add phosphate groups to other positions in the CTD domain that are structurally similar to Ser2. To investigate this, Mayfield, Irani et al. mixed the CTD domain with different regulatory proteins, and used various biochemical approaches to examine which specific positions of the domain had phosphate groups attached. These experiments revealed a previously unknown aspect of P-TEFb activity: its specificity for Ser2 increased dramatically if a different regulatory protein first added a phosphate group to a nearby location in CTD. This additional phosphate group directed P-TEFb to then add its phosphate specifically at Ser2. To confirm the activity of this mechanism in living human cells, Mayfield, Irani et al. used a drug that prevented the first phosphate from being added. In the drug treated cells, RNA polymerase II was found more frequently ‘stalled’ at positions on the DNA just before a gene starts. This suggests that living cells needs this two-phosphate code system in order for RNA polymerase II to progress and make copies of specific genes. These results are a step forward in understanding the complex control mechanisms cells use to make proteins from their DNA. Moreover, the model presented here – one phosphate addition priming a second specific phosphate addition – provides a template that may underlie similar regulatory processes.

The C-terminal domain (CTD) of RNA polymerase II contains a repetitive heptad sequence (YSPTSPS) whose phosphorylation states coordinate eukaryotic transcription by recruiting protein regulators. The precise placement and removal of phosphate groups on specific residues of the CTD are critical for the fidelity and effectiveness of RNA polymerase II–mediated transcription. During transcriptional elongation, phosphoryl-Ser(5) (pSer(5)) is gradually dephosphorylated by CTD phosphatases, whereas Ser(2) phosphorylation accumulates. Using MS, X-ray crystallography, protein engineering, and immunoblotting analyses, here we investigated the structure and function of SSU72 homolog, RNA polymerase II CTD phosphatase (Ssu72, from Drosophila melanogaster), an essential CTD phosphatase that dephosphorylates pSer(5) at the transition from elongation to termination, to determine the mechanism by which Ssu72 distinguishes the highly similar pSer(2) and pSer(5) CTDs. We found that Ssu72 dephosphorylates pSer(5) effectively but only has low activities toward pSer(7) and pSer(2). The structural analysis revealed that Ssu72 requires that the proline residue in the substrate's SP motif is in the cis configuration, forming a tight β-turn for recognition by Ssu72. We also noted that residues flanking the SP motif, such as the bulky Tyr(1) next to Ser(2), prevent the formation of such configuration and enable Ssu72 to distinguish among the different SP motifs. The phosphorylation of Tyr(1) further prohibited Ssu72 binding to pSer(2) and thereby prevented untimely Ser(2) dephosphorylation. Our results reveal critical roles for Tyr(1) in differentiating the phosphorylation states of Ser(2)/Ser(5) of CTD in RNA polymerase II that occur at different stages of transcription.