Tsunehiro Mizushima

Structural Basis for Sorting Mechanism of p62 in Selective Autophagy

Impairment of autophagic degradation of the ubiquitin- and LC3-binding protein “p62” leads to the formation of cytoplasmic inclusion bodies. However, little is known about the sorting mechanism of p62 to autophagic degradation. Here we identified a motif of murine p62 consisting of 11 amino acids (Ser334-Ser344) containing conserved acidic and hydrophobic residues across species, as an LC3 recognition sequence (LRS). The crystal structure of the LC3-LRS complex at 1.56Å resolution revealed interaction of Trp340 and Leu343 of p62 with different hydrophobic pockets on the ubiquitin fold of LC3. In vivo analyses demonstrated that p62 mutants lacking LC3 binding ability accumulated without entrapping into autophagosomes in the cytoplasm and subsequently formed ubiquitin-positive inclusion bodies as in autophagy-deficient cells. These results demonstrate that the intracellular level of p62 is tightly regulated by autophagy through the direct interaction of LC3 with p62 and reveal that selective turnover of p62 via autophagy controls inclusion body formation.

The Structure of the Mammalian 20S Proteasome at 2.75 Å Resolution

The 20S proteasome is the catalytic portion of the 26S proteasome. Constitutively expressed mammalian 20S proteasomes have three active subunits, beta 1, beta 2, and beta 5, which are replaced in the immunoproteasome by interferon-gamma-inducible subunits beta 1i, beta 2i, and beta 5i, respectively. Here we determined the crystal structure of the bovine 20S proteasome at 2.75 A resolution. The structures of alpha 2, beta 1, beta 5, beta 6, and beta 7 subunits of the bovine enzyme were different from the yeast enzyme but enabled the bovine proteasome to accommodate either the constitutive or the inducible subunits. A novel N-terminal nucleophile hydrolase activity was proposed for the beta 7 subunit. We also determined the site of the nuclear localization signals in the molecule. A model of the immunoproteasome was predicted from this constitutive structure.

Structures and physiological roles of 13 integral lipids of bovine heart cytochrome c oxidase

All 13 lipids, including two cardiolipins, one phosphatidylcholine, three phosphatidylethanolamines, four phosphatidylglycerols and three triglycerides, were identified in a crystalline bovine heart cytochrome c oxidase (CcO) preparation. The chain lengths and unsaturated bond positions of the fatty acid moieties determined by mass spectrometry suggest that each lipid head group identifies its specific binding site within CcOs. The X‐ray structure demonstrates that the flexibility of the fatty acid tails facilitates their effective space‐filling functions and that the four phospholipids stabilize the CcO dimer. Binding of dicyclohexylcarbodiimide to the O2 transfer pathway of CcO causes two palmitate tails of phosphatidylglycerols to block the pathway, suggesting that the palmitates control the O2 transfer process.The phosphatidylglycerol with vaccenate (cis‐Δ11‐octadecenoate) was found in CcOs of bovine and Paracoccus denitrificans, the ancestor of mitochondrion, indicating that the vaccenate is conserved in bovine CcO in spite of the abundance of oleate (cis‐Δ9‐octadecenoate). The X‐ray structure indicates that the protein moiety selects cis‐vaccenate near the O2 transfer pathway against trans‐vaccenate. These results suggest that vaccenate plays a critical role in the O2 transfer mechanism.

Crystal structure of a chaperone complex that contributes to the assembly of yeast 20S proteasomes

Eukaryotic 20S proteasomes are composed of two α-rings and two β-rings, which form an αββα stacked structure. Here we describe a proteasome-specific chaperone complex, designated Dmp1–Dmp2, in budding yeast. Dmp1–Dmp2 directly bound to the α5 subunit to facilitate α-ring formation. In Δdmp1 cells, α-rings lacking α4 and decreased formation of 20S proteasomes were observed. Dmp1–Dmp2 interacted with proteasome precursors early during proteasome assembly and dissociated from the precursors before the formation of half-proteasomes. Notably, the crystallographic structures of Dmp1 and Dmp2 closely resemble that of PAC3—a mammalian proteasome-assembling chaperone; nonetheless, neither Dmp1 nor Dmp2 showed obvious sequence similarity to PAC3. The structure of the Dmp1–Dmp2–α5 complex reveals how this chaperone functions in proteasome assembly and why it dissociates from proteasome precursors before the β-rings are assembled.

Structural basis for the selection of glycosylated substrates by SCFFbs1 ubiquitin ligase

The ubiquitin ligase complex SCFFbs1, which contributes to the ubiquitination of glycoproteins, is involved in the endoplasmic reticulum-associated degradation pathway. In SCF ubiquitin ligases, a diverse array of F-box proteins confers substrate specificity. Fbs1/Fbx2, a member of the F-box protein family, recognizes high-mannose oligosaccharides. To elucidate the structural basis of SCFFbs1 function, we determined the crystal structures of the Skp1–Fbs1 complex and the sugar-binding domain (SBD) of the Fbs1–glycoprotein complex. The mechanistic model indicated by the structures appears to be well conserved among the SCF ubiquitin ligases. The structure of the SBD–glycoprotein complex indicates that the SBD primarily recognizes Man3GlcNAc2, thereby explaining the broad activity of the enzyme against various glycoproteins. Comparison of two crystal structures of the Skp1–Fbs1 complex revealed the relative motion of a linker segment between the F-box and the SBD domains, which might underlie the ability of the complex to recognize different acceptor lysine residues for ubiquitination.

Structural basis for the cooperative interplay between the two causative gene products of combined factor V and factor VIII deficiency

Combined deficiency of coagulation factors V and VIII (F5F8D), an autosomal recessive disorder characterized by coordinate reduction in the plasma levels of factor V (FV) and factor VIII (FVIII), is genetically linked to mutations in the transmembrane lectin ERGIC-53 and the soluble calcium-binding protein MCFD2. Growing evidence indicates that these two proteins form a complex recycling between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment and thereby function as a cargo receptor in the early secretory pathway of FV and FVIII. For better understanding of the mechanisms underlying the functional coordination of ERGIC-53 and MCFD2, we herein characterize their interaction by x-ray crystallographic analysis in conjunction with NMR and ultracentrifugation analyses. Inspection of the combined data reveals that ERGIC-53-CRD binds MCFD2 through its molecular surface remote from the sugar-binding site, giving rise to a 1∶1 complex in solution. The interaction is independent of sugar-binding of ERGIC-53 and involves most of the missense mutation sites of MCFD2 so far reported in F5F8D. Comparison with the previously reported uncomplexed structure of each protein indicates that MCFD2 but not ERGIC-53-CRD undergoes significant conformational alterations upon complex formation. Our findings provide a structural basis for the cooperative interplay between ERGIC-53 and MCFD2 in capturing FV and FVIII.

Crystallization and preliminary X-ray diffraction studies of a β-carbonic anhydrase from the red alga Porphyridium purpureum

The beta-carbonic anhydrase from the red alga Porphyridium purpureum was heterologously expressed, purified and crystallized. The crystals belong to space group P2(1) (unit-cell parameters a = 63.8, b = 113.9, c = 73.8 A, beta = 104.1 degrees) with two subunits per asymmetric unit and diffract to 2.5 A resolution.

Structure of ST0929, a putative glycosyl transferase from Sulfolobus tokodaii.

The Sulfolobus tokodaii protein ST0929 shares close structural homology with S. acidocaldarius maltooligosyl trehalose synthase (SaMTSase), suggesting that the two enzymes share a common enzymatic mechanism. MTSase is one of a pair of enzymes that catalyze trehalose biosynthesis. The relative geometries of the ST0929 and SaMTSase active sites were found to be essentially identical. ST0929 also includes the unique tyrosine cluster that encloses the reducing-end glucose subunit in Sulfolobus sp. MTSases. The current structure provides insight into the structural basis of the increase in the hydrolase side reaction that is observed for mutants in which a phenylalanine residue is replaced by a tyrosine residue in the subsite +1 tyrosine cluster of Sulfolobus sp.

Crystallization and preliminary X-ray characterization of the Skp1–Fbg3 complex

F-box proteins are the substrate-recognition components of Skp1-Cullin1-F-box protein-Rbx1 (SCF) ubiquitin ligase complexes. Fbs1, an F-box protein, binds specifically to proteins modified with high-mannose oligosaccharides. Fbg3, another F-box protein, has 51% sequence identity to Fbs1. Although the residues that are necessary for binding to oligosaccharides are conserved between Fbs1 and Fbg3, Fbg3 does not bind glycoproteins. Skp1 and Fbg3 were co-expressed in Escherichia coli and their complex was purified to homogeneity and crystallized. Microseeding combined with the sandwiched hanging-drop technique improved the quality of the resulting crystals. The plate-shaped crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 34.1, b = 76.6, c = 193.9 A and one molecule per asymmetric unit.

Structural analysis for lipid/protein interactions in bovine heart cytochrome c oxidase

4 inside surface is located at the D-pathway entrance. The zinc binding affinity for the second site suggests that the zinc site is tightly coupled with the proton-pumping site. Recently, we analyzed Zn/Cd-binding to monomeric CcO which gives crystal packing different from that in the dimeric CcO crystal. The x-ray structural analysis showed Znbinding to the Zn2, Zn3 and additional sites including the site near the K-pathway entrance. Several Zn-binding sites have been found on the outside surface. However none of them is located on the subunit I surface from which pumping protons exit.