No abstract

ISWI is a member of the SWI2/SNF2 family of chromatin remodellers, which also includes Snf2, Chd1, and Ino80. ISWI is the catalytic subunit of several chromatin remodelling complexes, which mobilize nucleosomes along genomic DNA, promoting replication progression, transcription repression, heterochromatin formation, and many other nuclear processes. The ATPase motor of ISWI is an autonomous remodelling machine, whereas its carboxy (C)-terminal HAND-SAND-SLIDE (HSS) domain functions in binding extranucleosomal linker DNA. The activity of the catalytic core of ISWI is inhibited by the regulatory AutoN and NegC domains, which are in turn antagonized by the H4 tail and extranucleosomal DNA, respectively, to ensure the appropriate chromatin landscape in cells. How AutoN and NegC inhibit ISWI and regulate its nucleosome-centring activity remains elusive. Here we report the crystal structures of ISWI from the thermophilic yeast Myceliophthora thermophila and its complex with a histone H4 peptide. Our data show the amino (N)-terminal AutoN domain contains two inhibitory elements, which collectively bind the second RecA-like domain (core2), holding the enzyme in an inactive conformation. The H4 peptide binds to the core2 domain coincident with one of the AutoN-binding sites, explaining the ISWI activation by H4. The H4-binding surface is conserved in Snf2 and functions beyond AutoN regulation. The C-terminal NegC domain is involved in binding to the core2 domain and functions as an allosteric element for ISWI to respond to the extranucleosomal DNA length.

The cytoskeleton of red blood cell (RBC) is anchored to cell membrane by the ankyrin complex. This complex is assembled during RBC genesis and comprises primarily band 3, protein 4.2 and ankyrin, whose mutations contribute to numerous human inherited diseases. Highresolution structures of the ankyrin complex have been long sought-after to understand its assembly and disease-causing mutations. Here, we analyzed native complexes on human RBC membrane by stepwise fractionation. Cryo-electron microscopy structures of nine band 3associated complexes reveal that protein 4.2 stabilizes the cytoplasmic domain of band 3 dimer.In turn, the superhelix-shaped ankyrin binds to this protein 4.2 via ankyrin repeats (ARs) 6-13 and to another band 3 dimer via ARs 17-20, bridging two band 3 dimers in the ankyrin complex.Integration of these structures with both prior and our biochemical data supports a model of ankyrin complex assembly during erythropoiesis and identifies interactions essential for mechanical stability of RBC. MainThe human red blood cell (RBC, or the erythrocyte) is the most abundant cell in our blood and the principal gas exchanger between O2 and CO2 in our bodies. Devoid of nucleus, RBC has been engineered for a wide range of medical applications 1 . RBC exhibits unusual biconcave disc shape and remarkable membrane mechanical stability, both of which are essential for cycling through the vasculature for O2-CO2 exchange. These properties are endowed by the RBC cytoskeleton, which is bridged to the RBC membrane by junctional complex and ankyrin complex 2 . The ankyrin complex, which primarily contains band 3, protein 4.2, ankyrin and Rh subcomplex, connects the cytoskeleton to the membrane through the cytoplasmic domain of band 3 (refs. 3,4 ). Defects in these cytoskeleton and cytoskeleton-associated proteins are associated with numerous human hereditary diseases, such as hereditary spherocytosis, South Asian ovalocytosis and hereditary stomatocytosis 5,6 ..

The α-keto acid dehydrogenase complex family catalyzes the essential oxidative decarboxylation of α-keto acids to yield acyl-CoA and NADH. Despite performing the same overarching reaction, members of the family have different component structures and structural organization between each other and across phylogenetic species. While native structures of α-keto acid dehydrogenase complexes from bacteria and fungi became available recently, the atomic structure and organization of their mammalian counterparts in native states remain unknown. Here, we report the cryo-electron microscopy structures of the endogenous cubic 2-oxoglutarate dehydrogenase complex (OGDC) and icosahedral pyruvate dehydrogenase complex (PDC) cores from bovine kidney determined at resolutions of 3.5 Å and 3.8 Å, respectively. The structures of multiple proteins were reconstructed from a single lysate sample, allowing direct structural comparison without the concerns of differences arising from sample preparation and structure determination. Although native and recombinant E2 core scaffold structures are similar, the native structures are decorated with their peripheral E1 and E3 subunits. Asymmetric sub-particle reconstructions support heterogeneity in the arrangements of these peripheral subunits. In addition, despite sharing a similar monomeric fold, OGDC and PDC E2 cores have distinct interdomain and intertrimer interactions, which suggests a means of modulating self-assembly to mitigate heterologous binding between mismatched E2 species. The lipoyl moiety lies near a mobile gatekeeper within the interdomain active site of OGDC E2 and PDC E2. Analysis of the twofold related intertrimer interface identified secondary structural differences and chemical interactions between icosahedral and cubic geometries of the core. Taken together, our study provides a direct structural comparison of OGDC and PDC from the same source and offers new insights into determinants of interdomain interactions and of architecture diversity among α-keto acid dehydrogenase complexes.