On the Origins of Symmetry and Modularity in the Proteasome Family

Sequence-specific protein ligations are widely used to produce customized proteins “on demand.” Such chimeric, immobilized, fluorophore-conjugated or segmentally labeled proteins are generated using a range of chemical, (split) intein, split domain, or enzymatic methods. Where short ligation motifs and good chemoselectivity are required, ligase enzymes are often chosen, although they have a number of disadvantages, for example poor catalytic efficiency, low substrate specificity, and side reactions. Here, we describe a sequence-specific protein ligase with more favorable characteristics. This ligase, Connectase, is a monomeric homolog of 20S proteasome subunits in methanogenic archaea. In pulldown experiments with Methanosarcina mazei cell extract, we identify a physiological substrate in methyltransferase A (MtrA), a key enzyme of archaeal methanogenesis. Using microscale thermophoresis and X-ray crystallography, we show that only a short sequence of about 20 residues derived from MtrA and containing a highly conserved KDPGA motif is required for this high-affinity interaction. Finally, in quantitative activity assays, we demonstrate that this recognition tag can be repurposed to allow the ligation of two unrelated proteins. Connectase catalyzes such ligations at substantially higher rates, with higher yields, but without detectable side reactions when compared with a reference enzyme. It thus presents an attractive tool for the development of new methods, for example in the preparation of selectively labeled proteins for NMR, the covalent and geometrically defined attachment of proteins on surfaces for cryo-electron microscopy, or the generation of multispecific antibodies.

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An astonishing wealth of new proteasome homologs

Fuchs

Alva

Lupas

2021

Bioinformatics

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Motivation The proteasome is the main proteolytic machine for targeted protein degradation in archaea and eukaryotes. While some bacteria also possess the proteasome, most of them contain a simpler and more specialized homolog, the HslV protease. In recent years, three further homologs of the proteasome core subunits have been characterized in prokaryotes: Anbu, BPH, and connectase. With the inclusion of these members, the family of proteasome-like proteins now exhibits a range of architectural and functional forms, from the canonical proteasome, a barrel-shaped protease without pronounced intrinsic substrate specificity, to the monomeric connectase, a highly specific protein ligase. Results We employed systematic sequence searches to show that we have only seen the tip of the iceberg so far and that beyond the hitherto known proteasome homologs lies a wealth of distantly related, uncharacterized homologs. We describe a total of 22 novel proteasome homologs in bacteria and archaea. Using sequence and structure analysis, we analyze their evolutionary history and assess structural differences that may modulate their function. With this initial description, we aim to stimulate the experimental investigation of these novel proteasome-like family members. Availability The protein sequences in this study are searchable in the MPI Bioinformatics Toolkit (https://toolkit.tuebingen.mpg.de) with ProtBLAST/PSI-BLAST and with HHpred (database "proteasome_homologs"). The following data are available at https://data.mendeley.com/datasets/t48yhff7hs/3: (I) sequence alignments for each proteasome-like homolog, (II) the coordinates for their structural models, and (III) a cluster-map file, which can be navigated interactively in CLANS and gives direct access to all the sequences in this study. Supplementary information Supplementary data are available at Bioinformatics online.

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On the Origins of Symmetry and Modularity in the Proteasome Family

Cited by 4 publications

References 36 publications

The Proteasome System in Health and Disease

The Proteasome System in Health and Disease

Archaeal Connectase is a specific and efficient protein ligase related to proteasome β subunits

An astonishing wealth of new proteasome homologs

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