Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 488, Issue 1, Pages 129-135Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2017.05.021
Keywords
ERMES; Membrane contact sites; Mdm12/Mmm1 complex reconstitution; SMP domain; Crystal structure; SAXS
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Funding
- UCLA Geffen School of Medicine
- Stein-Oppenheimer Seed Grant Award
- Alexander and Renee Kolin Endowed Chair in Molecular Biology and Biophysics
- Gates Millennium Fellowship
- UCLA Dissertation of the Year Fellowship
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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 angstrom resolution and a new structure of Mdm12 solved at 4.1 angstrom resolution. Our structure reveals a monomeric form of Mdm12 with a conformationally dynamic N-terminal beta-strand; it differs from a previously reported homodimeric structure where the N-terminal beta 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. (C) 2017 Elsevier Inc. All rights reserved.
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