Structure of the ALS Mutation Target Annexin A11 Reveals a Stabilising N-Terminal Segment

Abstract

The functions of the annexin family of proteins involve binding to Ca2+, lipid membranes, other proteins, and RNA, and the annexins share a common folded core structure at the C terminus. Annexin A11 (AnxA11) has a long N-terminal region, which is predicted to be disordered, binds RNA, and forms membraneless organelles involved in neuronal transport. Mutations in AnxA11 have been linked to amyotrophic lateral sclerosis (ALS). We studied the structure and stability of AnxA11 and identified a short stabilising segment in the N-terminal end of the folded core, which links domains I and IV. The crystal structure of the AnxA11 core highlights main-chain hydrogen bonding interactions formed through this bridging segment, which are likely conserved in most annexins. The structure was also used to study the currently known ALS mutations in AnxA11. Three of these mutations correspond to buried Arg residues highly conserved in the annexin family, indicating central roles in annexin folding. The structural data provide starting points for detailed structure–function studies of both full-length AnxA11 and the disease variants being identified in ALS.

RNA-binding is an ancient trait of the Annexin family

“…All restriction enzymes used were FastDigest Restriction Enzymes (Thermo Scientific; Waltham, United States). N-terminally truncated versions of AnxA7 (Δ142AnxA7) and AnxA11 (Δ188AnxA11) (Lillebostad et al, 2020) were generated by PCR amplification using the pETM10 plasmid harboring rat full-length anxA7 and anxA11 cDNA, respectively, as templates. This resulted in AnxA7 and AnxA11 lacking 142 amino and 188 amino acids, respectively, at the N-terminally end.…”

“…Purification on Co2+ 2+ instead of Ni 2+ resin resulted in less impurities in the AnxA7, AnxA9 and AnxA11 protein samples. Purification of the Anxs was performed essentially as described (Lillebostad et al, 2020). All Anxs were subjected to size exclusion chromatography using a Superdex 75 or 200 Increase 10/300 GL column (GE Healthcare, Chicago, IL, United States), except for wt AnxA7, wt AnxA9 and wt AnxA11 due to their instability and low yield.…”

“…Based on the circular dichroism scanning spectra obtained (not shown), all Anx family members showed an overall α-helical content, except wt AnxA7 and wt AnxA11, which possess long unstructured N-termini. Since these two Anxs display a high tendency for aggregation and degradation, we also cloned and purified their N-terminally truncated versions, which are more stable and soluble (Lillebostad et al, 2020). The purified Anxs were spotted in their native form onto nitrocellulose membranes, with the three spots containing increasing amounts of protein (5, 10 and 20 μM) in a total volume of 3 μL (see Figure 2A, showing an outline of the spotted proteins).…”

Investigating Open Reading Frames in Known and Novel Transcripts using ORFanage

“…Proteins in this family contain two major domains, one at the C-terminus for the Ca 2+ binding effect, and the other at the N-terminus responsible for the membrane interactions. While the core domain at the C-terminus is highly conserved across the gene family, the N-terminus is variable 47 , allowing for tissue-specific regulation 48, 49 and localization 50 .The two known forms of the gene differ only in the length of the last helical structure, where the incorporation of additional peptides allows for an extension of the first helix.…”

Genetic analysis of Indian sporadic young onset patient with Amyotrophic Lateral Sclerosis

“…ANXA11 which is ubiquitously expressed in different cell types encodes Annexin A11, which is a calcium-dependent phospholipid-binding protein [12], [13] with a molecular weight of 56 kDa and consisting of 504 amino acids [14]. It has an stabilising N terminal low complexity domain which is rich in Proline, Glycine, and tyrosine residues and are involved in liquid-liquid phase separation (LLPS) [15]. It has a binding site for a calcium-binding protein called calcyclin which is encoded by S100A6 and also for programmed cell death protein 6 encoded by PDCD6 respectively, whereas the C terminal region with 4 homologous Conserved Annexin domains of approximately 70 amino acid residues each [12], are responsible for phospholipid binding in a calcium-dependent manner and are also involved in Ca 2+ signalling, cell division, apoptosis, and vesicle trafficking [11], [16].…”