The goal of this project is to elucidate structure-function relationships in macromolecular machines. During FY14, our studies focussed on: membrane remodeling by alfa-synuclein;and characterization of a novel bacterial nano compartment. (1) All cells must be capable of degrading aberrant and foreign proteins that would otherwise pollute them. These activities are carried out by energy-dependent proteolytic machines, which consist of two subcomplexes - a protease and an ATPase/unfoldase. Since 1995, we have studied the Clp complexes of E. coli, considered as a model system. We described the structures of the two sub complexes and characterized the interactions between them and with bound substrate proteins. In FY14, activity on this project was minor but it is still in our portfolio. (2) Membrane Remodeling. Remodeling, a process in which lipid bilayer structures are reconfigured by interacting proteins, is central to the functioning and metabolism of cells. We are investigating this phenomenon by cryo-electron microscopy (EM) and cryo-electron tomography (ET) applied to several systems. In FY14, our main effort was directed towards characterizing the effects of the protein alpha-synuclein (aS) on lipid vesicles and the influence of this interaction on the oligomeric/polyeric state of aS (1). Alfa-Synuclein (aS) is a membrane-binding protein with sequence similarity to apolipoproteins and other lipid-carrying proteins, which are capable of forming lipid-containing nanoparticles, sometimes referred to as discs. Hitherto it has been unclear whether aS also possesses this property. Using cryo-electron microscopy and light scattering, we found that aS can remodel phosphatidyl glycerol vesicles into nanoparticles whose shape (ellipsoidal) and dimensions (in the 7-10 nm range) resemble those formed by apolipoproteins. Their molar ratio of aS to lipid is approximately 1 : 20 and aS is oligomeric (including trimers and tetramers). Similar nanoparticles form when aS is added to vesicles of mitochondrial lipids. This observation suggests a mechanism for the previously reported disruption of mitochondrial membranes by aS. Circular dichroism and 4-pulse DEER experiments reveal that in nanoparticles aS assumes a broken helical conformation distinct from the extended helical conformation adopted when aS is bound to intact vesicles or membrane tubules. We also observed aS-dependent tubule and nanoparticle formation in the presence of oleic acid, implying that αS can interact with fatty acids and lipids in similar manners. aS-related nanoparticles might play a role in lipid and fatty acid transport functions previously attributed to this protein. 3) Recently we have participated in two projects aimed at determining the struures of protein complexes involved in genetic transposition. 3a) The first concerns MuB, an enzyme encoded by bacteriophage Mu (2). Mechanistic understanding of MuB function had previously been hindered by its poor solubility. We found that MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition. To do so, we combined bioinformatic, mutagenic, biochemical and electron microscopy. We demonstrated that MuB forms ATP-dependent filaments with or without DNA. We also identified critical residues for its ATPase, DNA binding, protein polymerization and MuA interaction activities. By EM, we showed that DNA binds in the axial hole of the MuB filament. These findings, together with the influence of MuB-filament size on strand-transfer efficiency, led to a model in which MuB-imposed symmetry transiently deforms the DNA at the boundary of the MuB filament and results in a bent DNA favored for transposition. 3b) The Hermes protein is a member of the hAT transposon superfamily which has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. Our colleagues determined the crystal structure of the Hermes transposase-DNA complex which revealed that Hermes forms an octamer, and that each monomer has bound a cleaved transposon end. We contributed EM analyses that localized the BED domains what were invisible, i.e. poorly ordered, in the crystal (3). The overall picture is that the catalytic unit is a dimer: however, only octamers are active in vivo. This suggests that they provide crucial multiple specific DNA binding domains that recognize repeated subterminal sequences and non-specific DNA binding surfaces for target capture. The unusual structure explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and demonstrates how an octamer could provide multiple sites of interaction to allow the transposase to locate its transposon ends amidst a sea of chromosomal DNA. 3) A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress. Living cells compartmentalize materials and enzymatic reactions to increase their metabolic efficiency. While eukaryotes use membrane-bound organelles, bacteria and archaea rely primarily on protein-bound nanocompartments. Encapsulins constitute a recently discovered class of nanocompartments that are widespread in bacteria and archaea. Hitherto, their functions have been unclear. We have characterized the structure of the encapsulin nanocompartment from Myxococcus xanthus and shown that its role is to sequester cytosolic iron, thereby to protect the cells from oxidative stress (4). Inasmuch as this project relates to bacterial management of iron resources, this project dovetails with the one reporting molecular machine involved in the uptake of iron by Neisseria that we reported last year. The nanocompartment of M. xanthus consists of a protein shell with internal contents. It has a shell protein (EncA, 32.5 kDa) and three internal proteins (EncB, 17 kDa;EncC, 13 kDa;EncD, 11 kDa). Using cryo-electron microscopy, we determined that EncA expressed in E. coli self-assembles into an icosahedral shell 32 nm in diameter (26 nm internal diameter), built from 180 subunits with the fold first observed in bacteriophage HK97 capsid. The internal proteins, of which EncB and EncC have ferritin-like domains, attach to its inner surface. Native nanocompartments have dense iron-rich cores. Functionally, they resemble ferritins, cage-like iron storage proteins, but with a massively greater capacity (30,000 Fe atoms vs. 3,000 in ferritin). Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase five-fold upon starvation, protecting cells from oxidative stress through iron sequestration. Further investigation of this system is ongoing. 4) Molecular modeling of mutated STING residues in clinically affected children. In a multi-participant multi-faceted study by a consortium led by Dr Raphaela Goldbach-Mansky (NIAMS), a cohort of 6 patients was identified with early onset systemic inflammation, vasculitis, and pulmonary inflammation. These patients were found to have de novo gain-of-function mutations in TMEM173, which encodes STimulator of Interferon Genes (STING). We participated in this effort by using molecular graphics and modeling to map the mutated residues on a homology model of a STING dimer. Intriguingly, the mutations tend to map at or near the dimer interface (5).
| Tolun, Gökhan; Vijayasarathy, Camasamudram; Huang, Rick et al. (2016) Paired octamer rings of retinoschisin suggest a junctional model for cell-cell adhesion in the retina. Proc Natl Acad Sci U S A 113:5287-92 |
| Marabini, Roberto; Ludtke, Steven J; Murray, Stephen C et al. (2016) The Electron Microscopy eXchange (EMX) initiative. J Struct Biol 194:156-63 |
| Heymann, J Bernard (2015) Validation of 3D EM Reconstructions: The Phantom in the Noise. AIMS Biophys 2:21-35 |
| McHugh, Colleen A; Fontana, Juan; Nemecek, Daniel et al. (2014) A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress. EMBO J 33:1896-911 |
| Hickman, Alison B; Ewis, Hosam E; Li, Xianghong et al. (2014) Structural basis of hAT transposon end recognition by Hermes, an octameric DNA transposase from Musca domestica. Cell 158:353-367 |
| Liu, Y; Jesus, A A; Marrero, B et al. (2014) Activated STING in a vascular and pulmonary syndrome. N Engl J Med 371:507-518 |
| Mizuno, Naoko; Dramicanin, Marija; Mizuuchi, Michiyo et al. (2013) MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition. Proc Natl Acad Sci U S A 110:E2441-50 |
| Cardone, Giovanni; Heymann, J Bernard; Steven, Alasdair C (2013) One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions. J Struct Biol 184:226-36 |
| Varkey, Jobin; Mizuno, Naoko; Hegde, Balachandra G et al. (2013) ýý-Synuclein oligomers with broken helical conformation form lipoprotein nanoparticles. J Biol Chem 288:17620-30 |
| Heymann, J Bernard; Winkler, Dennis C; Yim, Yang-In et al. (2013) Clathrin-coated vesicles from brain have small payloads: a cryo-electron tomographic study. J Struct Biol 184:43-51 |
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