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.

Keywords: methanogenic archaea; proteasome; protein ligation; sortase; transpeptidase.

Fig. 1.

Connectase (Cnt) binds and modifies MtrA. (A) Polyacrylamide gel showing purified MtrA^Δ219–240 and His₆-tagged Connectase (Cnt) proteins from M. mazei. When a mix of both (Load; 20 µM each) is applied on an Ni-NTA column, MtrA is not found in the flow-through (FT) but coelutes with Connectase (Elu). Furthermore, MtrA forms a specific reaction product (MtrA^N-Connectase; ∼5 µM) with wild-type Connectase (Cnt^WT) but not with its active site mutant (Cnt^T1A). (B and C) Newly generated masses in a M. mazei Connectase-MtrA reaction, as identified via LC-MS. These are interpreted as MtrA^C (residues 155 to 218; theoretical mass: 6,844.7 Da/detected mass: 6,843.5 Da) and as MtrA^N-Connectase conjugate (39,669.4 Da/39,665.8 Da). (D) MS/MS spectrum of the fusion peptide DTLVIAFIGK resulting from amide bond formation between the M. mazei Connectase N terminus (TLVIAFIGK...) and the MtrA KDPGA motif. The sample was digested with trypsin and free amino groups were dimethylated (C₂H₄). The threonine amino group is not modified, suggesting its involvement in an amide bond.

Fig. 2.

MtrA interacts with Connectase via a short amino acid sequence, forming a heterodimer. (A) Gel filtration and light-scattering analyses of M. jannaschii MtrA^Δ225–245 and Connectase^S1A proteins. While Connectase and MtrA alone show a comparable elution behavior, the mixture of both elutes at a lower volume, indicating complex formation (thin lines, plotted on the primary y axis). This interpretation is supported by light-scattering measurements (thick lines, secondary y axis). The determined masses (table, Right) closely resemble the theoretical monomeric masses for Connectase and MtrA alone and for the MtrA–Connectase heterodimer. The values were determined in n = 3 independent experiments, with ± signifying the SD. (B) Binding curve visualizing the formation of a complex between the MtrA-derived labeled peptide (5)KDPGA(10) and M. jannaschii Connectase^S1A, as determined by MST. Analogous experiments with other substrates (table, Right) show that the 15-amino-acid peptide (0)KDPGA(10) is sufficient for this high-affinity interaction. The values were determined in n = 3 independent experiments (red, green, and blue dots), with ± signifying the SD. (C) Crystal structures of M. jannaschii Connectase^S1A in complex with (15)KDPGA(10) (Left) and the proteasome beta subunit [Right, PDB ID code 3H4P (57)]. Both proteins share a common NTN core domain (red and green) and an N-terminal active site residue (pink) but diverge in three protein-specific elements (cyan). A model based on an alignment with (15)KDPGA(10) shows how MtrA [colorless, PDB ID code 5L8X (23)] could potentially bind to Connectase.

Fig. 3.

Connectase (Cnt) modifications are reversible and allow the recombination of substrates. (A) Schematic representation of a Connectase-mediated ligation of two protein substrates, R₁-(5)KDPGA(10) and PGA(10)-R₂. (B) Polyacrylamide gel showing the incubation of M. mazei Connectase with various proteins bearing the Connectase recognition sequence. A constant fraction of Ub-(5)KDPGA(10) forms a conjugate with Connectase [Ub-(5)KD-Connectase], suggesting a reversible reaction between the two (lanes 1 to 4). In place of PGA(10), PGA(10)-sdAb/CyP/GST can be used as alternative substrates for the “reverse reaction,” resulting in an equilibrium of educts and Ub-(5)KDPGA(10)-sdAb/CyP/GST products (lanes 5 to 10). A time course of these ligations in shown in SI Appendix, Fig. S7. (C) The proposed Connectase reaction mechanism (green) as a combination of the first steps of two known proteasomal reactions: proteolysis (orange) and autolysis (i.e., propeptide processing, cyan). A substrate, R₁-R₂ (Left), is cleaved (Middle) and the N-terminal fragment R₁ transferred on the Connectase Thr-1/Ser-1 N terminus (Right; see Fig. 1D). The reaction is reversible, allowing substrate recombination with an alternative R₂ substrate (A). It differs from proteolysis/autolysis by avoiding the irreversible hydrolysis step (Bottom). Some Connectase variants use Ser-1 instead of Thr-1 (shown) as active site residue (Dataset S2).

Fig. 4.

Connectase (Cnt) catalyzes specific and efficient ligations without side reactions. (A) The time course of M. mazei Connectase and SrtA5*-mediated ligations of two ubiquitin molecules at various molar enzyme:substrate (1:1, 1:10, and 1:400) concentrations. Connectase is used at very low concentrations and therefore not visible on the gel. (B) Ligation rates for Connectase reactions with various peptide and protein substrate pairs at 25 µM. The values were determined in n = 3 independent experiments, with ± signifying the SD. (C) Michaelis–Menten plot of a M. mazei Connectase ligation using Ub-(5)KDPGA(15) and PGA(15)-Ub substrates. Although the Connectase reaction can be expected to follow more complex mathematics, the experimental data are well described by the Michaelis–Menten model (gray line). The values were determined in n = 3 independent experiments. (D) Polyacrylamide gel showing a test for M. mazei Connectase hydrolase activity with Ub-(5)KDPGA(15) and PGA(10) substrates. At an enzyme:substrate concentration of 1:1,000 (1×), Connectase catalyzes ∼50% product formation [i.e., Ub-(5)KDPGA(10)] in 30 min without forming the putative hydolysis product Ub-(5)KD. Even at 1,000× increased enzyme concentrations and prolonged incubation times, no hydrolysis product can be detected. (E) Polyacrylamide gel showing the ligation of two recombinantly expressed substrates [Strep-Ub-(5)KDPGA(10)-His₆ and PGA(15)-Ub] in cell lysates and the single-step purification of the respective ligation product [Strep-Ub-(5)KDPGA(15)-Ub] using an Ni-NTA column in series with a Streptavidin column.