Louise M. Sternicki

The binding of PROTACs to their partner ubiquitin E3 ligase (E3) and a protein of interest (POI) is critical for PROTAC development and validation. Characterization of PROTAC complexes by cryo-electron microscopy and X-ray crystallography is not always feasible, especially where species may be transient and protein structures may not resolve due to flexible domains or intrinsically disordered regions or where the air-water interface poses risks such as dissociation. More routine biophysical methods with broader applicability to varied samples are essential to support the rapidly expanding targeted protein degradation field. The majority of PROTACs in development and in the clinic act through a Cullin-RING E3 ligase (CRL), of which the pentameric von Hippel-Lindau (VHL) Cullin 2 RING E3 complex (CRL2^VHL) is the premier example. Native mass spectrometry (nMS) can be used to characterize protein complexes but has not previously been used to characterize any full E3 or any E3-E2 interactions. Here, we show for the first time that CRL2^VHL (a full E3) is amenable to characterization by nMS and, notably, that all its key interactions with the other protein components integral to the targeted protein degradation mechanism can be observed. Specifically, we characterize binary, ternary, and higher-order complexes that comprise CRL2^VHL, including the multiprotein systems of POI-PROTAC-CRL2^VHL, CRL2^VHL-E2-Ub, and POI-PROTAC-CRL2^VHL-E2-Ub, all of which are essential in facilitating productive POI ubiquitination and degradation. We benchmarked the nMS with two POI examples (BRD4^BD2 and KRAS) with their respective PROTACs (MZ1 and ACBI3) and were able to observe all of the relevant complexes across both systems. We anticipate that our findings will open avenues for nMS to integrate as an alternative experimental method enabling scalable characterization of the intricate high-mass multiprotein interactions central to biological mechanisms of action.

Carbonic anhydrase 3 (CA3) exhibits low enzymatic activity compared to other CA isoforms but contains two surface-exposed cysteine residues that undergo glutathionylation under oxidative stress. Highly expressed in muscle tissue, CA3 has been implicated in cellular protection, particularly through interactions with Bcl2-Associated Athanogene 3 (BAG3), modulating autophagy, while CA3 overexpression decreased hypoxia-induced apoptosis in cardiomyocytes. In this study, we investigated the impact of CA3 overexpression on cellular pathways in HEK293T, MDA-MB-231, and SVCT cells using RNA sequencing and proteomics. Gene Set Enrichment Analysis (GSEA) in HEK293T cells revealed the down-regulation of pathways related to protein synthesis, RNA processing, Roundabout signalling, selenocysteine-metabolism, and suppression of neurodegenerative disease-associated pathways. Human breast epithelial cell lines under normoxia and hypoxia showed down-regulation of similar pathways, although notably, hypoxic conditions also suppressed interferon α/β signalling. Proteomic analysis in HEK293T cells using HaloTag pull-down experiments identified putative novel CA3 binding partners, including heat shock 70 kDa proteins 1 and 8, and ribosomal protein S2 (RPS2). RANBP2 protein was consistently up-regulated after CA3 overexpression, irrespective of the presence of CA3 surface-exposed cysteines and HaloTag orientation. These findings suggest that CA3 modulates key cellular processes beyond its enzymatic role, contributing to stress resilience through pathway-level regulation and protein interactions, potentially impacting autophagy and neurodegenerative disease.

The binding of PROTACs to their partner ubiquitin E3 ligase (E3) and a protein of interest (POI) is critical for PROTAC development and validation. Characterisation of PROTAC complexes by cryo-electron microscopy and X-ray crystallography is not always feasible, especially where species may be transient and protein structures may not resolve due to flexible domains or intrinsically disordered regions. More routine biophysical methods with broader applicability to varied samples is desirable to support the rapidly expanding targeted protein degradation field. The majority of PROTACs in development and in the clinic act through a Cullin RING E3 Ligase (CRL) of which the pentameric von Hippel-Lindau (VHL) Cullin 2 RING E3 complex (CRL2 ^VHL ) is the foundational example. Native mass spectrometry (nMS) can be used to characterise protein complexes but has not previously been used to characterise a full E3 or any E3-E2 interactions. Here, we show that CRL2 ^VHL is amenable to characterisation by nMS and its interactions with the other protein components integral to the targeted protein degradation mechanism can be observed. Specifically, we characterise binary, ternary and higher order complexes that comprise CRL2 ^VHL , including the multiprotein systems of POI-PROTAC-CRL2 ^VHL , CRL2 ^VHL -E2-Ub, and POI-PROTAC-CRL2 ^VHL -E2-Ub, all of which are essential in facilitating productive POI ubiquitination and degradation. We benchmarked the nMS with two POI examples (BRD4 ^BD2 and KRAS) with their respective PROTACs (MZ1 and ACBI3) and were able to observe all relevant complexes across both systems. We anticipate that our findings will open avenues for nMS to integrate as an alternative experimental method enabling scalable characterisation of the intricate high mass multiprotein interactions central to the PROTAC mechanism of action.

Biotin protein ligase (BPL) catalyzes the covalent attachment of biotin onto biotin‐dependent enzymes, where it functions as an essential cofactor. Eukaryotic BPLs are distinct due to the presence of a large N‐terminal extension to the conserved catalytic domain and C‐terminal cap. No high‐resolution structures of a eukaryotic BPL have been solved; however, previous functional studies revealed the N‐terminal extension interacts with the biotinylation substrate. Mass spectrometry (MS) and complementary techniques were utilized to investigate the structure of the yeast Saccharomyces cerevisiae BPL ( Sc BPL). Lower resolution techniques suggested holo‐ Sc BPL had a more compact structure and sampled fewer conformational states. In addition, solution‐phase and a charge state dependent gas‐phase stabilization was observed. Hydrogen‐deuterium exchange (HDX) MS provided experimental validation of the AlphaFold predicted structure of Sc BPL, with a folded domain structurally homologous to a glutamine amidotransferase identified in the N‐terminal extension, and a mostly homologous catalytic domain to that of other species’ BPLs. Further HDX analyses identified localized conformational changes in the Sc BPL active site and N‐terminal domain that occur concomitantly with ligand binding. These data provide novel insights into the unique structure of a class III BPL and how ligands influence this structure for catalysis of protein biotinylation.

Covalent chemical probes form a covalent bond with a target protein of interest to elicit an effect and methods to identify and characterise them are needed. We developed a native mass spectrometry (nMS) method to screen an electrophilic covalent fragment library and identified specific novel binders for the surface exposed cysteine residues of carbonic anhydrase III (CA III). The nMS method was extended to determine the site of protein modification and measure simultaneous binding of an active site noncovalent inhibitor and covalent fragment hit, which is not possible with intact denaturing MS. This study demonstrates the utility of using nMS and the advantages when compared to intact denaturing MS for the discovery and characterisation of new covalent ligands.

We discuss how native mass spectrometry (nMS) is contributing important analytical insights to guide contemporary drug discovery and development. We focus on diverse modalities including targeted protein degradation (TPD), fragments (FBDD) and mRNA.

The intentional targeting of RNA with small molecules is recognized as a viable pathway to new therapeutics with potential to vastly expand the proportion of the human genome considered druggable. The practical considerations to deliberately target RNA are largely under development, including optimal screening methods to identify small molecules for binding to RNA. Native mass spectrometry (nMS) is established as a valuable biophysical method for identifying small molecule hits against diverse biomolecular targets, most frequently proteins and protein-protein interactions. Herein we assess nMS for studying the binding of small molecules with RNA aptamers as a model for nMS screening of RNA as a drug target. We first develop workflows for characterizing the binding of cognate RNA aptamer ligands and then establish a nMS method to screen a small molecule library against the RNA aptamers. nMS analysis permitted identification of binders, quantitation of binding strength and generation of structure-activity relationships with some dependence on the aptamer class. This work demonstrates the utility of nMS as a complementary and efficient target-based biophysical screening method that can characterize RNA-small molecule interactions and the potential of nMS becoming a powerful enabling tool in RNA-targeting drug discovery.

Carbonic anhydrases (CAs) are a family of enzymes that play an important pH regulatory role in health and disease. While different CA isozymes have a high degree of structural similarity, they have variable enzymatic activity, with CA III being the least active and having less than 1% of the activity of CA II, the most active. Furthermore, ligand binding studies for CA III are limited, and a resulting lack of chemical probes impedes understanding of this CA isozyme in comparison to other CA family members where studies are abundant. Therefore, we employed native mass spectrometry (nMS), also known as intact mass spectrometry, to assess ligand binding to CA II and CA III and discovered two novel compounds that for the first time display strong binding to CA III. We present a new data visualization and quantification tool developed to display native mass spectra as an intuitive stacked heat map representation for rapidly interpreting the results of ligand-protein binding from nMS screening.