Structural insight into the TRIAP1/PRELI-like domain family of mitochondrial phospholipid transfer complexes.

The composition of the mitochondrial membrane is important for its architecture and proper function. Mitochondria depend on a tightly regulated supply of phospholipid via intra-mitochondrial synthesis and by direct import from the endoplasmic reticulum. The Ups1/PRELI-like family together with its mitochondrial chaperones (TRIAP1/Mdm35) represent a unique heterodimeric lipid transfer system that is evolutionary conserved from yeast to man. Work presented here provides new atomic resolution insight into the function of a human member of this system. Crystal structures of free TRIAP1 and the TRIAP1-SLMO1 complex reveal how the PRELI domain is chaperoned during import into the intermembrane mitochondrial space. The structural resemblance of PRELI-like domain of SLMO1 with that of mammalian phoshatidylinositol transfer proteins (PITPs) suggest that they share similar lipid transfer mechanisms, in which access to a buried phospholipid-binding cavity is regulated by conformationally adaptable loops.

Structural comparison of yeast and human intra-mitochondrial lipid transport systems.

Mitochondria depend on a tightly regulated supply of phospholipids. The protein of relevant evolutionary and lymphoid interest (PRELI)/Ups1 family together with its mitochondrial chaperones [TP53-regulated inhibitor of apoptosis 1 (TRIAP1)/Mdm35] represents a unique heterodimeric lipid-transfer system that is evolutionary conserved from yeast to man. Recent X-ray crystal structures of the human and yeast systems are compared and discuss here and shed new insight into the mechanism of the PRELI/Ups1 system.

An intermolecular hydrogen bonded network in the PRELID-TRIAP protein family plays a role in lipid sensing.

The PRELID-TRIAP1 family of proteins is responsible for lipid transfer in mitochondria. Multiple structures have been resolved of apo and lipid substrate bound forms, allowing us to begin to piece together the molecular level details of the full lipid transfer cycle. Here, we used molecular dynamics simulations to demonstrate that the lipid binding is mediated by an extended, water-mediated hydrogen bonding network. A key mutation, R53E, was found to disrupt this network, causing lipid to be released from the complex. The X-ray crystal structure of R53E was captured in a fully closed and apo state. Lipid transfer assays and molecular simulations allow us to interpret the observed conformation in the context of the biological role. Together, our work provides further understanding of the mechanistic control of lipid transport by PRELID-TRIAP1 in mitochondria.

The protease associated (PA) domain in ScpA from Streptococcus pyogenes plays a role in substrate recruitment.

Annually, over 18 million disease cases and half a million deaths worldwide are estimated to be caused by Group A Streptococcus. ScpA (or C5a peptidase) is a well characterised member of the cell enveleope protease family, which possess a S8 subtilisin-like catalytic domain and a shared multi-domain architecture. ScpA cleaves complement factors C5a and C3a, impairing the function of these critical anaphylatoxins and disrupts complement-mediated innate immunity. Although the high resolution structure of ScpA is known, the details of how it recognises its substrate are only just emerging. Previous studies have identified a distant exosite on the 2nd fibronectin domain that plays an important role in recruitment via an interaction with the substrate core. Here, using a combination of solution NMR spectroscopy, mutagenesis with functional assays and computational approaches we identify a second exosite within the protease-associated (PA) domain. We propose a model in which the PA domain assists optimal delivery of the substrate's C terminus to the active site for cleavage.

Structural determinants of lipid specificity within Ups/PRELI lipid transfer proteins.

Conserved lipid transfer proteins of the Ups/PRELI family regulate lipid accumulation in mitochondria by shuttling phospholipids in a lipid-specific manner across the intermembrane space. Here, we combine structural analysis, unbiased genetic approaches in yeast and molecular dynamics simulations to unravel determinants of lipid specificity within the conserved Ups/PRELI family. We present structures of human PRELID1-TRIAP1 and PRELID3b-TRIAP1 complexes, which exert lipid transfer activity for phosphatidic acid and phosphatidylserine, respectively. Reverse yeast genetic screens identify critical amino acid exchanges that broaden and swap their lipid specificities. We find that amino acids involved in head group recognition and the hydrophobicity of flexible loops regulate lipid entry into the binding cavity. Molecular dynamics simulations reveal different membrane orientations of PRELID1 and PRELID3b during the stepwise release of lipids. Our experiments thus define the structural determinants of lipid specificity and the dynamics of lipid interactions by Ups/PRELI proteins.