Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

Abstract

38MORN (membrane occupation and recognition nexus) repeat proteins have a wide taxonomic 39 distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain 40 poorly characterised at both a structural and a functional level compared to other common 41 repeat motifs such as leucine-rich repeats, armadillo repeats, WD40 repeats, and ankyrin 42 repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate 43 membrane targeting, but direct evidence for this role is actually lacking. This putative activity 44 was addressed by focusing on a protein composed solely of MORN repeats -Trypanosoma 45 brucei MORN1. No evidence for binding to membranes or lipid vesicles by TbMORN1 either 46 in vivo or in vitro could be obtained. TbMORN1 did interact with individual phospholipids, but 47 it remains unclear if this was physiological or an artefact. High-and low-resolution structures 48 of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites 49 Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of 50 macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. The 51 structures indicated that MORN repeats can mediate homotypic interactions, and can function 52 as both dimerisation and oligomerisation devices.53 54 93 al., 2006, Im, Davis et al., 2007, Camacho, Smertenko et al., 2009). It therefore remains 94 unclear what role(s) this ubiquitous class of repeat motifs actually have (Mikami, Saavedra et 95 al., 2010). 97Coupled to this lack of unambiguous functional data is a lack of high-resolution structural 98 information, exemplified by the ongoing lack of consensus as to whether a single repeat is 14 99 4 or 23 amino acids. This contrasts sharply with the considerable amount of information 100 available on other classes of protein repeat motifs such as ankyrin repeats, leucine-rich 101 repeats, or WD40 repeats (Andrade, Perez-Iratxeta et al., 2001). Until very recently, the 102 structure of the SETD7 histone methyltransferase was the sole representative of the MORN 103 repeat protein family in the protein data bank (PDB) (Jacobs, Harp et al., 2002, Wilson et al., 104 2002, Xiao, Jing et al., 2003. Even here, the structure of the N-terminal domain containing 105 the repeats is incomplete, and the level of sequence similarity of the repeats to those of 106 junctophilins and other family members makes assignment difficult. Each repeat appears to 107 form a β-hairpin with an acidic surface, but it remains unclear if this is a general property of 108 MORN repeats. The SETD7 structure has not been analysed in this context, with more work 109 focusing on its catalytic methyltransferase domain. In 2019, and while this manuscript was in 110 preparation, Li et al. published the first high-resolution structure of a canonical MORN repeat 111 protein, specifically the MORN4/retinophilin protein in complex with its Myo3a binding partner 112 (Li, Liu et al., 2019). This structure contains ...

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“…7a-e). The recent report that MORN1 dimers can attain two different confirmations, either exhibiting an extended or a V-shape conformation, provides further support for a stretchable MORN1 ring (Sajko et al, 2020).…”

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“…2e ). As predicted from their sequences, the latter two proteins are composed of multiple beta-strands with the configuration of membrane occupation and recognition nexus (MORN) repeats, forming elongated curved beta-sheets abundant in aromatic residues 26 . The concave surfaces of RSP1 and RSP10 accommodate the proline-rich C-termini of RSP4 and RSP6, respectively, through a network of hydrogen bonds, hydrophobic and electrostatic interactions ( Fig.…”

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“…S2 [ https://doi.org/10.6084/m9.figshare.13286486 ]). MORN-repeats have been shown to bind to phospholipids ( 18 , 19 ), promoting stable interactions with plasma membranes ( 16 ) and also function as protein-protein interaction modules involved in di- and oligomerization ( 20 ). They are expected to be intracellular and provide a large putative interaction surface (either with other MORN-HeRs or other proteins).…”