Exploring the Conformations and Binding Location of HMGA2·DNA Complexes Using Ion Mobility Spectrometry and 193 nm Ultraviolet Photodissociation Mass Spectrometry

Abstract

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.

Up-regulated transcriptional regulators in mutant RAS gene signatures: a time-resolved multi-omics study in generic epithelial cell models

“…The AT hooks preferentially recognize AT-rich double-stranded DNA in the minor groove and can DNA segments in cis and trans (Chen et al, 2010;Cui et al, 2005;Pfannkuche et al, 2009;Reeves and Nissen, 1990). The C-terminal domain is engaged in protein-protein interactions (Gaudreau-Lapierre et al, 2023) and coordinates intramolecular interactions with individual AT-hook domains (Sgarra et al, 2009;Sipe et al, 2022). As a consequence of its architectural function, HMGA2 forms specialized nucleoprotein complexes at enhancers, promoters or branched DNA (Pfannkuche et al, 2009) and invokes heterochromatin structures (Divisato et al, 2022).…”

Exploiting ion‐mobility mass spectrometry for unraveling proteome complexity

“…Fouque et al. explored the conformational and binding dynamics of an intrinsically disordered DNA‐binding protein expressed during embryogenesis [91]. Upon DNA binding, a reduction of the conformational space and charge‐state distribution was observed.…”

Fragment Ion Abundance Reveals Information about Structure and Charge Localization in Highly Charged Proteins

“…To interpret the data obtained in any of these dissociation-based experiments, the presence or absence of certain fragment ions is often attributed to specific structural features. Although less commonly used, differences in the abundance of fragment ions between two systems have also been interpreted as the result of structural dissimilarities. ,,,− …”

“…There are a variety of methods capable of fragmenting intact proteins, each with potential strengths and weaknesses in terms of structural interrogation. Dissociation based on electron capture or transfer (ECD/ETD) yields high sequence coverage and does not require heating of the ion to initiate dissociation. − Ultraviolet photodissociation (UVPD) is also useful for protein characterization, yielding high sequence coverage through a variety of dissociation mechanisms (including some that heat the protein). − Collisional activation can also be used with intact proteins by way of many low energy collisions (as occurs in an ion trap) or by fewer but higher energy collisions (as occurs in beam-type arrangements). Although it might be reasonable to expect that the heating process preceding dissociation by collisional activation might erase any memory of solution phase structure, recent results obtained by the Loo lab suggest otherwise .…”