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

Abstract

MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats-Trypanosoma brucei MORN1. Surprisingly, no evidence for binding to membranes or lipid vesicles by TbMORN1 could be obtained either in vivo or in vitro. Conversely, TbMORN1 did interact with individual phospholipids. High- and low-resolution structures of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. This enabled a first structure-based definition of the MORN repeat itself. Furthermore, all three structures dimerised via their C-termini in an antiparallel configuration. The dimers could form extended or V-shaped quaternary structures depending on the presence of specific interface residues. This work provides a new perspective on MORN repeats, showing that they are protein-protein interaction modules capable of mediating both dimerisation and oligomerisation.

Fig 1. TbMORN1 primary structure and dimerisation.

(A) Primary structure of TbMORN1, with individual MORN repeats shown in alignment, and coloured according to amino acid conservation. A schematic of the predicted secondary structure of each repeat is shown above the alignment. A consensus amino acid sequence of the individual MORN repeats from TbMORN1 based on the alignment is shown in the sequence logo below. (B) TbMORN1 dimerises via its C-terminus. SEC-MALS profiles of TbMORN1(1–15), TbMORN1(2–15), and TbMORN1(1–14). Schematics are shown underneath. TbMORN1(1–15) tended to form high-order assemblies, whereas removal of the first MORN repeat resulted in a monodisperse dimer. Removal of the last MORN repeat in TbMORN1(1–14) resulted in a polydisperse mixture of monomers, dimers, and other species. Chromatographic separation was done using a Superdex 200 Increase 10/300 GL column, void volume 7.2 ml.

Fig 2. Recombinant TbMORN1 co-purifies with PE and increases E. coli PE levels.

Negative ion mode survey scan (600–900 m/z) of lipid extracts from the indicated conditions. (A,B) Lipid extracts from purified recombinant TbMORN1(1–15) (A) and TbMORN1(10–15) (B). A large amount of PE co-purified with TbMORN1(1–15) but very little was associated with TbMORN1(10–15). (C,D) Lipid extracts from E. coli cells expressing the indicated constructs. (C) Cells expressing TbMORN1(1–15) had elevated PE levels. (D) Cells expressing TbMORN1(10–15) showed no changes to cellular lipid ratios compared to wild-type (empty vector control). In all cases, phospholipid identity was confirmed by daughter fragmentation and reported here. Schematics of the recombinant TbMORN1 constructs are shown above the traces.

Fig 3. Overexpression of Ty1-tagged TbMORN1 causes a dominant negative phenotype.

(A) Overexpression of Ty1-TbMORN1 is deleterious. Growth curves of control (-Tet) cells, and cells inducibly expressing Ty1-TbMORN1(1–15) (+Tet). Population density was measured every 24h, and the cultures split and reseeded at 48h. Data were compiled from 3 separate clones, each induced in 3 independent experiments; bars show mean +/- SD. (B) Overexpression of Ty1-TbMORN1 produces a BigEye phenotype. The incidence of BigEye cells was counted in control (-Tet) and Ty1-TbMORN1-expressing cells at the indicated timepoints. Data were compiled from 3 separate clones, each induced in 3 independent experiments; bars show mean + SD. The inset shows an example BigEye cell. Scale bar, 2 μm. (C) Tight induction of Ty1-TbMORN1 expression. Whole-cell lysates were harvested from control (-Tet) and Ty1-TbMORN1-expressing cells (+Tet) at the indicated timepoints and probed with anti-TbMORN1 and anti-Ty1 antibodies. PFR1,2 were used as a loading control. At least three independent inductions were carried out for each clone; an exemplary blot is shown. (D) Quantification of overexpression. The levels of endogenous TbMORN1 and ectopic Ty1-TbMORN1 in immunoblots were normalised relative to the PFR1,2 signal. Data were compiled using 3 separate clones, each induced in at least two independent experiments; bars show mean + SD. (E) Ty1-TbMORN1 can localise correctly to the cytoskeleton. Whole cells or detergent-extracted cytoskeletons were fixed and labelled with anti-TbMORN1 or anti-Ty1 antibodies. The fluorescence signal is shown with the transmitted light image of the cell overlaid; inset shows the fluorescence signal from the antibody in greyscale. Results confirmed for 3 separate clones, exemplary images are shown. Scale bars, 2 μm. (F) Schematic of the fractionation protocol. (G) Overexpression of Ty1-TbMORN1 displaces the endogenous protein from the cytoskeleton. Control and Ty1-TbMORN1-expressing cells were fractionated as shown in panel F and the I, SN, and P fractions were blotted. PFR1,2 was used as a marker for the cytoskeleton. Expression of Ty1-TbMORN1 was accompanied by a displacement of endogenous TbMORN1 from the insoluble (P) fraction into the soluble (SN) fraction (arrows 1,2). Equal fractions (5%) were loaded in each lane. 3 independent experiments using 3 separate clones were carried out; an exemplary blot is shown. (H) Quantification of the fractionation data. Bars show mean + SD.

Fig 4. Overexpressed Ty1-TbMORN1 is predominantly cytosolic.

(A) Schematic of the two-step fractionation scheme. (B) Immunoblots of fractions taken from control and Ty1-TbMORN1 overexpressing cells, using anti-BiP, anti-TbMORN1 and anti-GFP antibodies. Note that the membrane was cut into three strips for the immunoblot. Equal fractions (5%) were loaded in each lane. The overexpressed Ty1-TbMORN1 was predominantly extracted by digitonin and partitioned with the cytosolic GFP into the SN1 fraction (arrows 1). Very little of the remainder was subsequently extracted with non-ionic detergent into the SN2 fraction (arrows 2), with most partitioning into the cytoskeleton-associated P2 fraction. Three independent experiments were carried out using cells from three clones pooled together; an exemplary blot is shown. (C,D) Quantification of the immunoblots of the two-step fractionation. Data were compiled from three independent experiments, each using cells pooled from three separate clones. Bars show mean values + SD.

Fig 5. High-resolution structures of MORN repeat proteins and a structural redefinition of the MORN repeat.

(A) Schematic depiction and crystal structure of the TbMORN1(7–15) dimer in its P2₁ crystal form. Amino acid numbers and N- and C-termini are indicated in the schematic. The crystal structure is shown in two orientations, with main dimensions indicated in Å. The structure contains 2 x 9 MORN repeats and is an antiparallel homodimer with the subunits arranged in a splayed tail-to-tail configuration. The secondary structure consists of exclusively antiparallel beta-strands and peripherally positioned loops, which together form a longitudinal groove through the middle of the protein. (B) Crystal structure of TgMORN1(7–15) shown in two orientations. The number of MORN repeats and the configuration is the same as for TbMORN1(7–15) in panel A. (C) Crystal structure of PfMORN1(7–15) shown in two orientations. The bound Zn²⁺ ion is labelled. The structure contains 2 x 9 MORN repeats, again in tail-to-tail configuration but with an overall V-shaped arrangement. (D) Alignment of all 9 TbMORN1(7–15) MORN repeats in the crystal structure reveals a high level of structural conservation. (E) A revised consensus MORN repeat sequence, based on the crystal structures. A new alignment of the MORN repeats in TbMORN1 is shown. Repeats 7–15 are present in the crystal structure; repeats 1–6 are inferred. Conservation of sequence identity is indicated by colour intensity. Each MORN repeat consists of a β-hairpin, built up of two 6-residue β-strands connected by a 5-residue loop. The β-hairpin is followed by a 6-residue loop that connects to the next MORN repeat. The new 23-residues long consensus MORN repeat starts with the GxG motif. (F) The tertiary structure of individual MORN repeats is stabilised by hydrogen bonds between the first G of the GxG motif and the W from the YEGEW motif. MORN repeat arrays are further stabilised by aromatic stacking between the highly conserved aromatic residues in the YEGEW and LxY motifs, and by T-shaped π-stacking interactions of the highly conserved Y of the YEGEW motif, which is sandwiched between the W residue of its own motif, and the W residue in the next YEGEW motif. (G) A single TbMORN1(7–15) subunit viewed at an oblique angle. The residues of the YEGEW and LxY motifs involved in aromatic stacking line the surface of the longitudinal groove running through the middle of the protein.

Fig 6. Dimerisation interfaces of TbMORN1(7–15) and PfMORN1(7–15).

(A) The dimerisation interface of TbMORN1(7–15) P2₁ involves residues from MORN repeats 12–15, which stabilise the dimer interface via aromatic π-stacking (Tyr330 and Phe345 from the respective subunits), hydrophobic interactions (Leu301, Leu347, Ile339, Ile322, Leu324), and additionally via hydrogen bonding interactions at the edges of the dimer interface. In comparison to the TbMORN1(7–15) C2 crystal structure, there are no disulphide bridges stabilising the dimerisation interface of TbMORN1(7–15) P2₁. (B) The dimerisation interface of the V-shaped PfMORN1(7–15) dimer is smaller and is additionally stabilised by the incorporation of a structural Zn²⁺ ion, which is tetrahedrally coordinated by Cys306 and Asp309 residues from each respective subunit. Thr311 holds the side chain of Asp309 in the appropriate orientation. The dimer interface is additionally stabilised by symmetric hydrogen bonding between the Thr311 pair, aromatic stacking between the Phe330 pair, a hydrophobic cluster formed by Leu328, Val331 and Leu336, two salt bridges between Lys322 and Glu308 from the respective subunits, anion-π interaction of a side chain of Glu308 with Phe304, and a combination of aromatic stacking (His334 pair) and polar interactions (His334, Asn332) at the vertex of the dimer. (C) An electrostatic map calculated for a single sub-unit of TbMORN1(7–15). Vacuum electrostatics were calculated in PyMOL, where red denotes -65.7 kT and blue denotes +65.7 kT. The structure on the right shows the predicted effect of two point mutations, R293A from MORN repeat 13, and K316A from MORN repeat 14. The mutations are expected to result in the loss of a positively-charged patch close to the dimer interface, and consequently disrupt the dimerisation of the TbMORN1(2–15) constructs.

Fig 7. MORN1 proteins form extended dimers in solution as assessed by SAXS and EM experiments.

(A-D) SAXS experiments on: (A) TbMORN1(7–15), (B) TgMORN1(7–15), (C) PfMORN1(7–15), and (D) TbMORN1(2–15). For each respective protein, the results include a P(r) plot with derived experimental D_max value compared with a D_max value derived from the structure, an experimental SAXS scattering data with a fit calculated by the Crysol programme, and a SAXS-based ab initio molecular envelope. In the case of TbMORN1(2–15), the theoretical D_max value was derived from a structural model, which was generated by spiking the TbMORN1(7–15) structure with additional structures of individual TbMORN1(7–15) subunits. (E-G) EM with rotary shadowing of TbMORN1(2–15) and full-length TbMORN1. (E) TbMORN1(2–15) forms a homogenous population of extended dimers of approximately 25 nm in length (see insets for individual examples). (F) Full-length TbMORN1 is heterogeneous and includes rare filaments of 175–200 nm in length (first inset) and individual dimers (second and third inset). (G) Higher-order assemblies of full-length TbMORN1 assembled in a mesh-like structure. Magnification, 71,000x; scale bars, 25 nm, 50 nm as indicated. n (independent replicates) = 2, n (biological replicates) = 2.