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

Abstract

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.

An astonishing wealth of new proteasome homologs

“…Many of these proteins only have few representatives or show a narrow phylogenetic distribution, indicating either a less essential or a phylum-specific function for them. However, such proteins can still be of considerable biological interest, as demonstrated by the study of connectase, a protein ligase that occurs only in some methanogenic archaea ( Fuchs et al , 2021 ). We note that there are also some proteins with a higher number of representatives (e.g.…”

“…A second insertion site is found between β strands 3 and 4 (S3/S4). This site is particularly interesting because it harbors the signature element of connectase ( Fuchs et al , 2021 ), a protein ligase that may prove useful in biotechnological applications. Here, a mobile, two-helical insertion covers the active site and contributes to the high substrate specificity of connectase.…”

“…While heat shock locus V (HslV) ( Rohrwild et al , 1996 ), which is found in bacteria and some eukaryotic organelles of endosymbiotic origin, was the only known proteasome homolog for many years, we and others have recently characterized three further proteasome homologs: the bacterial complexes Anbu ( Fuchs et al , 2017 ; Gille et al , 2003 ; Piasecka et al , 2018 ; Valas and Bourne, 2008 ; Vielberg et al , 2018 ) and BPH ( Fuchs et al , 2018a ), and the monomeric archaeal connectase ( Fuchs et al , 2021 ). In the Pfam database ( Finn et al , 2014 ), the proteasome subunits, HslV, BPH and Anbu are classified into the ‘Proteasome subunit’ family within the ‘NTN hydrolase’ clan (N-terminal nucleophile).…”

“…Likewise, no function is known for the Anbu complex, but its unique split-ring architecture suggests a different mode of action ( Fuchs et al , 2017 ; Piasecka et al , 2018 ; Vielberg et al , 2018 ). Lastly, unlike the other proteasome-like homologs, connectase is not a protease, but instead acts as a highly specific protein ligase ( Fuchs et al , 2021 ).…”

“…At their N-terminus, proteasome α subunits have an additional helical extension that enables them to regulate substrate access to the proteolytic channel. By contrast, monomeric connectase does not possess the first two helices (H1–H2) and instead uses a two-helical insertion (H ins1 /H ins2 ) at a different position to control substrate specificity ( Fuchs et al , 2021 ). Finally, Anbu features a C-terminal helical extension (H5) that mediates a coiled-coil interaction between the two ring-layers and thereby stabilizes the split-ring architecture ( Fuchs et al , 2017 ; Piasecka et al , 2018 ; Vielberg et al , 2018 ).…”