The Architecture of the Anbu Complex Reflects an Evolutionary Intermediate at the Origin of the Proteasome System

Abstract

SummaryProteasomes are self-compartmentalizing proteases that function at the core of the cellular protein degradation machinery in eukaryotes, archaea, and some bacteria. Although their evolutionary history is under debate, it is thought to be linked to that of the bacterial protease HslV and the hypothetical bacterial protease Anbu (ancestral beta subunit). Here, together with an extensive bioinformatic analysis, we present the first biophysical characterization of Anbu. Anbu forms a dodecameric complex with a unique architecture that was only accessible through the combination of X-ray crystallography and small-angle X-ray scattering. While forming continuous helices in crystals and electron microscopy preparations, refinement of sections from the crystal structure against the scattering data revealed a helical open-ring structure in solution, contrasting the ring-shaped structures of proteasome and HslV. Based on this primordial architecture and exhaustive sequence comparisons, we propose that Anbu represents an ancestral precursor at the origin of self-compartmentalization.

On the Origins of Symmetry and Modularity in the Proteasome Family

“…Our studies suggest that this twofold (C2) symmetry predates the additional six‐ or sevenfold symmetry of the closed rings in the 20S, HslV, and BPH. Our recent bioinformatic analyses suggest that Anbu and the proteasomal β subunit have a shared ancestry in the last universal common ancestor, while structural analysis suggests that the architecture of Anbu resembles that of their common precursor (Figure ) . In all characterized Anbu proteins, the fundamental structural building blocks are dimers of opposing protomers, which represent the first oligomerization step in the evolution of the proteasome family.…”

“…Interestingly, Anbu is found in different oligomeric states in different organisms. So far, both dodecameric assemblies consisting of six and tetradecameric assemblies consisting of seven dimers have been observed, which underlines the apparent evolutionary ease of oligomerization transitions in absence of dihedral symmetry constraints. One key element of the helical assembly is a coiled coil that forms between the long C‐terminal α helices of the opposing protomers, spanning the dimer interface and stabilizing the dimer significantly .…”

“…The evolutionary history of the proteasome family, especially of the 20S proteasome and HslV, has long been controversial. However, in a comprehensive sequence‐based bioinformatic study including also the newly characterized members of the family, we distilled a clear and parsimonious evolutionary scenario, which is substantiated by structural studies . Therein, Anbu resembles the architecture of a precursor of the proteasome family.…”

“…The recent identification and characterization of two further bacterial self‐compartmentalizing NTN‐hydrolases, ancestral β subunit (Anbu), and betaproteobacterial proteasome homolog (BPH), added more diversity to this family (Figure ). While BPH was found to form tetradecamers of D7 symmetry similar to the D6‐symmetric HslV, Anbu revealed an unexpected helical oligomerization architecture of two stacked open rings without dihedral symmetry, resembling a split lock washer with C2 symmetry . The evolutionary history of the proteasome family, especially of the 20S proteasome and HslV, has long been controversial.…”

Target highlights in CASP13: Experimental target structures through the eyes of their authors

“…As we did not expect a fast breakthrough in crystallization experiments, we started to equip ourselves with experimental restraints for an in silico structure modeling. To this aim, we collected small angle X‐ray scattering (SAXS) data and, in collaboration with Alexander Leitner from ETH Zürich, cross‐linking mass‐spectrometry (XL‐MS) data, which we aimed to combine for a rigid‐body modeling and refinement approach based on the known structures of the individual enzymatic domains (see also). However, we indeed obtained well‐diffracting crystals quite early on and thus focused on crystallographic structure solution.…”