Crystal structure of Mdm12 and combinatorial reconstitution of Mdm12/Mmm1 ERMES complexes for structural studies

Abstract

Membrane contact sites between organelles serve as molecular hubs for the exchange of metabolites and signals. In yeast, the Endoplasmic Reticulum − Mitochondrion Encounter Structure (ERMES) tethers these two organelles likely to facilitate the non-vesicular exchange of essential phospholipids. Present in Fungi and Amoebas but not in Metazoans, ERMES is composed of five distinct subunits; among those, Mdm12, Mmm1 and Mdm34 each contain an SMP domain functioning as a lipid transfer module. We previously showed that the SMP domains of Mdm12 and Mmm1 form a hetero-tetramer. Here we describe our strategy to diversify the number of Mdm12/Mmm1 complexes suited for structural studies. We use sequence analysis of orthologues combined to protein engineering of disordered regions to guide the design of protein constructs and expand the repertoire of Mdm12/Mmm1 complexes more likely to crystallize. Using this combinatorial approach we report crystals of Mdm12/Mmm1 ERMES complexes currently diffracting to 4.5 Å resolution and a new structure of Mdm12 solved at 4.1 Å resolution. Our structure reveals a monomeric form of Mdm12 with a conformationally dynamic N-terminal β-strand; it differs from a previously reported homodimeric structure where the N-terminal β strands where swapped to promote dimerization. Based on our electron microscopy data, we propose a refined pseudo-atomic model of the Mdm12/Mmm1 complex that agrees with our crystallographic and small-angle X-ray scattering (SAXS) solution data.

Lipid transfer proteins: the lipid commute via shuttles, bridges and tubes

“…Many years after the extracellular TULIPs were discovered, they were shown to have intracellular counterparts in the Synaptotagmin-like Mitochondrial-lipid-binding Protein (SMP) domain family [55][56][57][58] . Like extracellular TULIPs, SMP domains mostly dimerise head-to-head ( Fig.…”

Crystal structures of Mmm1 and Mdm12–Mmm1 reveal mechanistic insight into phospholipid trafficking at ER-mitochondria contact sites

“…Previously, we determined the crystal structure of Saccharomyces cerevisiae Mdm12 at 3.1 Å resolution and revealed that Mdm12 forms a dimeric SMP structure that binds phospholipids inside a hydrophobic channel, with a preference for glycerophospholipids harboring a positively charged head group (20). Another study determined a 17 Å resolution electron microscopy (EM) structure of the Mdm12-Mmm1 (SMP domain) complex, revealing an elongated tubular structure with an Mdm12-Mmm1-Mmm1-Mdm12 arrangement (19,35). Despite these structure studies, the molecular-level mechanism by which the SMP domains of Mdm12, Mmm1, and Mdm34 are directly organized and facilitate phospholipid trafficking without consuming energy at the ER-mitochondria contact site remains unknown.…”

Determining the Lipid-Binding Specificity of SMP Domains: An ERMES Subunit as a Case Study

“…3b) and the amounts of protein obtained are sufficient to perform the lipid identification by HPTLC (but also by MS) described in Subheading 3.3. Yeast Mdm12 overexpressed and purified from bacterial cultures is also suited for crystallization trials [19]. …”

“…Two variable insertions (VI1 and VI2) and the N and C termini are indicated. ( c ) Crystal structures of different SMP domains bound to phospholipids as seen in human E-SYT2 [17] and the fungal Mdm12 ERMES subunit from Saccharomyces cerevisiae (Sce) [18, 19] and Kluyveromyces lactis (Kla) [20]. Notice the different positions occupied by the PL molecules along the lateral seam…”