Abstract
Ancestral β-subunit (Anbu) is homologous to HslV and 20S proteasomes. Based on its phylogenetic distribution and sequence clustering, Anbu has been proposed as the “ancestral” form of proteasomes. Here, we report biochemical data, small-angle X-ray scattering results, negative-stain electron microscopy micrographs and a crystal structure of the Anbu particle from Yersinia bercovieri (YbAnbu). All data are consistent with YbAnbu forming defined 12–14 subunit multimers that differ in shape from both HslV and 20S proteasomes. The crystal structure reveals that YbAnbu subunits form tight dimers, held together in part by the Anbu specific C-terminal helices. These dimers (“protomers”) further assemble into a low-rise left-handed staircase. The lock-washer shape of YbAnbu is consistent with the presence of defined multimers, X-ray diffraction data in solution and negative-stain electron microscopy images. The presented structure suggests a possible evolutionary pathway from helical filaments to highly symmetric or pseudosymmetric multimer structures. YbAnbu subunits have the Ntn-hydrolase fold, a putative S1 pocket and conserved candidate catalytic residues Thr1, Asp17 and Lys32(33). Nevertheless, we did not detect any YbAnbu peptidase or amidase activity. However, we could document orthophosphate production from ATP catalyzed by the ATP-grasp protein encoded in the Y. bercovieri Anbu operon.
Highlights
Proteasomes are large macromolecular machineries that occur in all eukaryotes and carry out degradation of cytosolic and nuclear proteins that have been marked for degradation by ubiquitination [1,2]
Ancestral β-subunit (Anbu) (HyAnbu) was expressed and purified as described by Vielberg et al Among the tested proteins, only Hyphomicrobium sp. Anbu (HyAnbu) and YbAnbu assembled into multi-subunit particles in our hands
We refer to Vielberg et al for the characterization of HyAnbu and concentrate on YbAnbu
Summary
Proteasomes are large macromolecular machineries that occur in all eukaryotes and carry out degradation of cytosolic and nuclear proteins that have been marked for degradation by ubiquitination [1,2]. Proteasomes are labile complexes of one or two 19S cap particles and a 20S core particle [2]. The cap is involved in specific recognition of ubiquitinated proteins, their deubiquitination, unfolding and translocation. The cap contains multiple ATPases of the AAA+ type thought to be involved in substrate unfolding and translocation. The 20S proteasome is responsible for subsequent degradation of proteins. It is a 28-mer complex of at least 14 or more different subunits that together build a particle of pseudo-7-fold symmetry [3]. All 20S proteasomal subunits are paralogous and phylogenetically cluster into two groups, termed α and β. The paralogy of 20S proteasomal subunits suggests evolution from simpler precursor particles. 20S proteasomes made from just one type of α- and (http://creativecommons.org/licenses/by/4.0/)
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