Heidi L. Schubert

Ubiquitin-binding domains

Ubiquitin-binding domains (UBDs) are a collection of modular protein domains that non-covalently bind to ubiquitin. These recently discovered motifs interpret and transmit information conferred by protein ubiquitylation to control various cellular events. Detailed molecular structures are known for a number of UBDs, but to understand their mechanism of action, we also need to know how binding specificity is determined, how ubiquitin binding is regulated, and the function of UBDs in the context of full-length proteins. Such knowledge will be key to our understanding of how ubiquitin regulates cellular proteins and processes.

Many paths to methyltransfer: a chronicle of convergence

S-adenosyl-L-methionine (AdoMet) dependent methyltransferases (MTases) are involved in biosynthesis, signal transduction, protein repair, chromatin regulation and gene silencing. Five different structural folds (I-V) have been described that bind AdoMet and catalyze methyltransfer to diverse substrates, although the great majority of known MTases have the Class I fold. Even within a particular MTase class the amino-acid sequence similarity can be as low as 10%. Thus, the structural and catalytic requirements for methyltransfer from AdoMet appear to be remarkably flexible.

Deficiency of glutaredoxin 5 reveals Fe-S clusters are required for vertebrate haem synthesis.

Iron is required to produce haem and iron–sulphur (Fe–S) clusters, processes thought to occur independently. Here we show that the hypochromic anaemia in shiraz (sir) zebrafish mutants is caused by deficiency of glutaredoxin 5 (grx5), a gene required in yeast for Fe–S cluster assembly. We found that grx5 was expressed in erythroid cells of zebrafish and mice. Zebrafish grx5 rescued the assembly of Δgrx5 yeast Fe–S, showing that the biochemical function of grx5 is evolutionarily conserved. In contrast to yeast, vertebrates use iron regulatory protein 1 (IRP1) to sense intracellular iron and regulate mRNA stability or the translation of iron metabolism genes. We found that loss of Fe–S cluster assembly in sir animals activated IRP1 and blocked haem biosynthesis catalysed by aminolaevulinate synthase 2 (ALAS2). Overexpression of ALAS2 RNA without the 5′ iron response element that binds IRP1 rescued sir embryos, whereas overexpression of ALAS2 including the iron response element did not. Further, antisense knockdown of IRP1 restored sir embryo haemoglobin synthesis. These findings uncover a connection between haem biosynthesis and Fe–S clusters, indicating that haemoglobin production in the differentiating red cell is regulated through Fe–S cluster assembly.

The biosynthesis of adenosylcobalamin (vitamin B12)

Vitamin B12, or cobalamin, is one of the most structurally complex small molecules made in Nature. Major progress has been made over the past decade in understanding how this synthesis is accomplished. This review covers some of the most important findings that have been made and provides the reader with a complete description of the transformation of uroporphyrinogen III into adenosylcobalamin (AdoCbl). 183 references are cited.

Ubiquitin recognition by the human TSG101 protein

The UEV domain of the TSG101 protein functions in both HIV-1 budding and the vacuolar protein sorting (VPS) pathway, where it binds ubiquitylated proteins as they are sorted into vesicles that bud into late endosomal compartments called multivesicular bodies (MVBs). TSG101 UEV-ubiquitin interactions are therefore important for delivery of both substrates and hydrolytic enzymes to lysosomes, which receive proteins via fusion with MVBs. Here, we report the crystal structure of the TSG101 UEV domain in complex with ubiquitin at 2.0 A resolution. TSG101 UEV contacts the Ile44 surface and an adjacent loop of ubiquitin through a highly solvated interface. Mutations that disrupt the interface inhibit MVB sorting, and the structure also explains how the TSG101 UEV can independently bind its ubiquitin and Pro-Thr/Ser-Ala-Pro peptide ligands. Remarkably, comparison with mapping data from other UEV and related E2 proteins indicates that although the different E2/UEV domains share the same structure and have conserved ubiquitin binding activity, they bind through very different interfaces.

Biosynthesis of cobalamin (vitamin B12): a bacterial conundrum.

Abstract. The biosynthesis of cobalamin (vitamin B12) is described, revealing how the concerted action of around 30 enzyme-mediated steps results in the synthesis of one of Natures most structurally complex ‘small molecules’. The plethora of genome sequences has meant that bacteria capable of cobalamin synthesis can be easily identified and their biosynthetic genes compared. Whereas only a few years ago cobalamin synthesis was thought to occur by one of two routes, there are apparently a number of variations on these two pathways, where the major differences seem to be concerned with the process of ring contraction. A comparison of what is currently known about these pathways is presented. Finally, the process of cobalt chelation is discussed and the structure/function of the cobalt chelatase associated with the oxygen-independent pathway (CbiK) is described.

Structural Basis for ESCRT-III Protein Autoinhibition

Endosomal sorting complexes required for transport-III (ESCRT-III) subunits cycle between two states: soluble monomers and higher-order assemblies that bind and remodel membranes during endosomal vesicle formation, midbody abscission and enveloped virus budding. Here we show that the N-terminal core domains of increased sodium tolerance-1 (IST1) and charged multivesicular body protein-3 (CHMP3) form equivalent four-helix bundles, revealing that IST1 is a previously unrecognized ESCRT-III family member. IST1 and its ESCRT-III binding partner, CHMP1B, both form higher-order helical structures in vitro, and IST1-CHMP1 interactions are required for abscission. The IST1 and CHMP3 structures also reveal that equivalent downstream α5 helices can fold back against the core domains. Mutations within the CHMP3 core–α5 interface stimulate the proteins in vitro assembly and HIV-inhibition activities, indicating that dissociation of the autoinhibitory α5 helix from the core activates ESCRT-III proteins for assembly at membranes.

Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium

In Bacillus megaterium, the hemAXBCDL genes were isolated and were found to be highly similar to the genes from Bacillus subtilis that are required for the conversion of glutamyl-tRNA into uroporphyrinogen III. Overproduction and purification of HemC (porphobilinogen deaminase) and -D (uroporphyrinogen III synthase) allowed these enzymes to be used for the in vitro synthesis of uroporphyrinogen III from porphobilinogen. A second smaller cluster of three genes (termed sirABC) was also isolated and found to encode the enzymes that catalyse the transformation of uroporphyrinogen III into sirohaem on the basis of their ability to complement a defined Escherichia coli (cysG) mutant. The functions of SirC and -B were investigated by direct enzyme assay, where SirC was found to act as a precorrin-2 dehydrogenase, generating sirohydrochlorin, and SirB was found to act as a ferrochelatase responsible for the final step in sirohaem synthesis. CbiX, a protein found encoded within the main B. megaterium cobalamin biosynthetic operon, shares a high degree of similarity with SirB and acts as the cobaltochelatase associated with cobalamin biosynthesis by inserting cobalt into sirohydrochlorin. CbiX contains an unusual histidine-rich region in the C-terminal portion of the protein, which was not found to be essential in the chelation process. Sequence alignments suggest that SirB and CbiX share a similar active site to the cobaltochelatase, CbiK, from Salmonella enterica.

Structural and functional studies on the extracellular domain of BST2/tetherin in reduced and oxidized conformations

HIV-1 and other enveloped viruses can be restricted by a host cellular protein called BST2/tetherin that prevents release of budded viruses from the cell surface. Mature BST2 contains a small cytosolic region, a predicted transmembrane helix, and an extracellular domain with a C-terminal GPI anchor. To advance understanding of BST2 function, we have determined a 2.6 Å crystal structure of the extracellular domain of the bacterially expressed recombinant human protein, residues 47–152, under reducing conditions. The structure forms a single long helix that associates as a parallel dimeric coiled coil over its C-terminal two-thirds, while the N-terminal third forms an antiparallel four-helix bundle with another dimer, creating a global tetramer. We also report the 3.45 Å resolution structure of BST2(51-151) prepared by expression as a secreted protein in HEK293T cells. This oxidized construct forms a dimer in the crystal that is superimposable with the reduced protein over the C-terminal two-thirds of the molecule, and its N terminus suggests pronounced flexibility. Hydrodynamic data demonstrated that BST2 formed a stable tetramer under reducing conditions and a dimer when oxidized to form disulfide bonds. A mutation that selectively disrupted the tetramer (L70D) increased protein expression modestly but only reduced antiviral activity by approximately threefold. Our data raise the possibility that BST2 may function as a tetramer at some stage, such as during trafficking, and strongly support a model in which the primary functional state of BST2 is a parallel disulfide-bound coiled coil that displays flexibility toward its N terminus.

Crystal structure of human uroporphyrinogen III synthase.

Uroporphyrinogen III synthase, U3S, the fourth enzyme in the porphyrin biosynthetic pathway, catalyzes cyclization of the linear tetrapyrrole, hydroxymethylbilane, to the macrocyclic uroporphyrino gen III, which is used in several different pathways to form heme, siroheme, chlorophyll, F430 and vitamin B12. U3S activity is essential in all organisms, and decreased activity in humans leads to the autosomal recessive disorder congenital erythropoetic porphyria. We have determined the crystal structure of recombinant human U3S at 1.85 Å resolution. The protein folds into two α/β domains connected by a β‐ladder. The active site appears to be located between the domains, and variations in relative domain positions observed between crystallographically independent molecules indicates the presence of flexibility that may be important in the catalytic cycle. Possible mechanisms of catalysis were probed by mutating each of the four invariant residues in the protein that have titratable side chains. Additionally, six other highly conserved and titratable side chains were also mutated. In no case, however, did one of these mutations abolish enzyme activity, suggesting that the mechanism does not require acid/base catalysis.