Journal: bioRxiv

Article Title: Non canonical scaffold-type ligase complex mediates protein UFMylation

doi: 10.1101/2022.01.31.478489

Figure Lengend Snippet: A. Schematic showing the workflow for substrate independent single turnover lysine discharge assays. B. Assay to monitor discharge of Ubiquitin from UBE2D3 and UBE2L3 in the presence of free amino acids. 0 indicates time 0. C. Assay to check for discharge of UFM1 from UFC1 in the presence of free amino acids. UBE2D3 is used as a positive control for lysine and cysteine discharge. D. Lysine discharge assays using UBE2D3 and UFC1 in the presence of increasing concentration of free Lysine. E. Time-dependent analysis of discharge of UFM1 from UFC1 in the presence of high concentration of free lysine (150 mM).

Article Snippet: Cys-UFM1 1–83 which contains a Cys residue upstream of M1 was purified and exchanged into a fresh buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM TCEP using CentriPure P10 columns (EMP Biotech).

Techniques: Positive Control, Concentration Assay

Journal: bioRxiv

Article Title: Non canonical scaffold-type ligase complex mediates protein UFMylation

doi: 10.1101/2022.01.31.478489

Figure Lengend Snippet: A. Size exclusion chromatography profile of UFL1 run on Superdex 200 Increase 10/300gl column (shown in red). Overlay of chromatogram of molecular weight standards of different sizes (shown in grey) run under same buffer conditions. B. Results obtained from ULTIMATE Y2H TM screening (Hybrigenics) for binary interactions with UFL1. Asterisk denotes protein hits obtained with low confidence. C. Schematic describing the strategy for co-expression and purification of UFL1/UFBP1 complex. D. SEC-MALS analysis of UFL1/UFBP1 complex. The theoretical and observed molecular weights are indicated. E. Analytical gel filtration chromatography analysis of UFL1/UFBP1 complex and its subunits run on Superdex 200 3.2/300. F. UFBP1 does not activate UFL1 when added exogenously. In vitro UFMylation assays to compare the E3 ligase activity of UFL1 and UFBP1 expressed alone and together as a complex. 0.25 µ M UBA5, 5 µ M UFC1 and 10 µ M UFM1 was incubated with 1 µ M UFL1 or 1 µ M UFBP1 or 1 µ M UFL1/UFBP1 complex for 1 h at 37°C in the presence of 50 mM HEPES 7.5, 0.5 mM DTT, 10 mM MgCl 2 and 10 mM ATP. The reaction was stopped by addition of 3x SDS loading dye and run on a 4-12% denaturing SDS PAGE gel under reducing conditions and immunoblotting was performed using indicated antibodies. G. MS 2 spectra showing the peptide derived from in vitro UFMylation assay showing the VG-remnant on K69 of UFM1.

Article Snippet: Cys-UFM1 1–83 which contains a Cys residue upstream of M1 was purified and exchanged into a fresh buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM TCEP using CentriPure P10 columns (EMP Biotech).

Techniques: Size-exclusion Chromatography, Molecular Weight, Expressing, Purification, Filtration, Chromatography, In Vitro, Activity Assay, Incubation, SDS Page, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Non canonical scaffold-type ligase complex mediates protein UFMylation

doi: 10.1101/2022.01.31.478489

Figure Lengend Snippet: A. Comparison of size exclusion chromatography profiles of UFL1 expressed alone (Red) and co-expressed UFL1/UFBP1 complex (Black) run under identical buffer conditions on a HiLoad TM Superdex 10/300 gl column. Molecular weight standards are shown in grey. Fractions corresponding to each peak were collected and analysed on 4-12% denaturing SDS-PAGE which is shown below. B. Mass photometry analysis of co-purified UFL1/UFBP1 complex. The theoretical and experimental molecular weights are indicated above. C. Immunoblot comparing UFL1 autoUFMylation (Top) and UFM1 chain synthesis (Middle) in the presence of UFL1 expressed alone and in complex with UFBP1. (Bottom) Assays to monitor UFMylation of substrates using purified TRIP4 in the presence of UFL1 alone and UFL1/UFBP1 complex. Reaction products were run on a 4-12% SDS-PAGE gel and analysed by immunoblotting using indicated antibodies. D. Immunoblot showing formation of free UFM1 chains in the presence and absence of UFL1/UFBP1. (Bottom) Schematic representation of the linkage composition of di-UFM1 chains obtained from LC-MS/MS analysis. E. In vitro UFMylation assay in the presence of Lys to Arg (K-R) and lysine less(K0) mutants of UFM1 to check for polyUFMylation (top) and UFL1 autoUFMylation (bottom) . F. In vitro UFMylation assay to check for formation of di-UFM1 chains in the presence of single Lys and Lys less mutants of UFM1.

Article Snippet: Cys-UFM1 1–83 which contains a Cys residue upstream of M1 was purified and exchanged into a fresh buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM TCEP using CentriPure P10 columns (EMP Biotech).

Techniques: Size-exclusion Chromatography, Molecular Weight, SDS Page, Purification, Western Blot, Liquid Chromatography with Mass Spectroscopy, In Vitro

Journal: bioRxiv

Article Title: Non canonical scaffold-type ligase complex mediates protein UFMylation

doi: 10.1101/2022.01.31.478489

Figure Lengend Snippet: A. Multiple sequence alignment of 1-410 a.a. region of UFL1 from various organisms to highlight the degree of conservation of cysteine residues in this region. B. Immunoblot showing in vitro UFMylation assays in the presence of C to A mutants of UFL1 to check for UFBP1 UFMylation (top) , UFL1 autoUFMylation (middle) and substrate UFMylation (bottom) . C. Coomassie stained SDS-PAGE gel showing discharge assays in the presence and absence of full length UFL1/UFBP1 complex. Charged UFC1 was incubated with lysine (50 mM) in the presence and absence of full length UFL1/UFBP1 complex and analysed for discharge on a 4-12% SDS PAGE gel under non-reducing conditions. D. Graphical representation showing the composition of linkage forms of di-UFM1 chains formed by minimal reconstitution of UBA5 and UFC1.

Article Snippet: Cys-UFM1 1–83 which contains a Cys residue upstream of M1 was purified and exchanged into a fresh buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM TCEP using CentriPure P10 columns (EMP Biotech).

Techniques: Sequencing, Western Blot, In Vitro, Staining, SDS Page, Incubation

Journal: bioRxiv

Article Title: Non canonical scaffold-type ligase complex mediates protein UFMylation

doi: 10.1101/2022.01.31.478489

Figure Lengend Snippet: A. Composite Foldindex profile of UFL1 showing folding propensity of different regions of UFL1 to aid in construct design for soluble protein expression. B. Size exclusion chromatography profile of MBP-UFL1 1-410 (dark blue) and His 6 -UFL1 1-410 /UFBP1 29-end (orange) run on Superdex 200 3.2/300 column (shown in red). Approximately 20 µ gs of sample was run on a Superdex 200 3.2/300 analytical gel filtration column and UV traces measured at 280 nm were recorded and analysed. (Right) Coomassie stained gel showing the purity of the proteins. C. In vitro UFMylation assay to check for E3 ligase activity of UFL1 1-410 /UFBP1 29-end . Full length UFL1/UFBP1 complex is used as a positive control. D. Time course assay to check for transthiolation activity of UFL1. Reaction products were analysed on a 4-12% SDS PAGE gel under reducing and non-reducing conditions. E. In vitro UFMylation assays to check for formation of di-UFM1 chains in the by minimal reconstitution in the presence UBA5 and UFC1 alone. F. Immunoblot to check for the presence of UFM1 chains with and without treatment of UFSP2. In vitro UFMylation products generated as shown in E was incubated with UFSP2 and analysed by immunoblotting using anti-UFM1 antibody to check for the disspearence of polyUFMylated products especially di-UFM1.

Article Snippet: Cys-UFM1 1–83 which contains a Cys residue upstream of M1 was purified and exchanged into a fresh buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM TCEP using CentriPure P10 columns (EMP Biotech).

Techniques: Construct, Expressing, Size-exclusion Chromatography, Filtration, Staining, In Vitro, Activity Assay, Positive Control, SDS Page, Western Blot, Generated, Incubation

Journal: bioRxiv

Article Title: Non canonical scaffold-type ligase complex mediates protein UFMylation

doi: 10.1101/2022.01.31.478489

Figure Lengend Snippet: A. Cartoon representation of predicted full length human UFL1/UFBP1 complex using Alphafold. UFL1 and UFBP1 are shown in blue and teal colors respectively. The dimeric interface of UFL1 and UFBP1 is highlighted in the dotted box. WH: Winged helix; NTR: N-terminal region; CTR: C-terminal region B. Enlarged view of the dimeric interface of UFL1/UFBP1 complex shown to highlight the formation of full WH domain bridged by two partial winged helix domains of UFL1 (blue) and UFBP1 (teal). C. Schematic representation of UFL1/UFBP1 constructs with different domain boundaries to identify the minimal catalytic region of UFL1/UFBP1 complex required for aminolysis. D. Quantitative representation of Lysine discharge assays to identify the minimal boundaries of UFL1 and UFBP1 required for activation of UFC1 (n=3, mean±SD). E. SEC elution profiles showing that full length UFL1/UFBP1 complex interacts specifically with charged E2. UFL1/UFBP1 complex was incubated with UFC1 -O-UFM1 at a 1:1 molar ratio for 20 min at 4°C and loaded on a Superdex 200 Increase 3.2/300 column. The fractions corresponding to each peak were collected and separated on a 4-12% SDS PAGE gel followed by Coomassie staining. F. Minimal catalytic region is sufficient for interaction with charged UFC1. UFL1 (1–, 179) /UFBP1 (1-116) complex was incubated with UFC1 -O-UFM1 at the molar ratio of 1:1 for 20 min at 4°C and analysed by analytical size exclusion chromatography as described in E). G. In vitro UFMylation assay to monitor formation of free di-UFM1 chains in the presence of UFL1/UFBP1 complexes bearing different domain boundaries. H. Substrate UFMylations assays to check for UFMylation of purified Histone H4 in the presence of different UFL1/UFBP1 truncations. I. Role of UFBP1 in substrate UFMylation. (Top) Comparison of UFMylation activities of UFL1/UFBP1 29-end and UFL1/UFBP1 207-end using Histone H4. (Bottom) Comparison of UFMylation activities of UFL1 1-179 /UFBP1 29-end and UFL1 1-179 /UFBP1 207-end using Histone H4.

Article Snippet: Cys-UFM1 1–83 which contains a Cys residue upstream of M1 was purified and exchanged into a fresh buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM TCEP using CentriPure P10 columns (EMP Biotech).

Techniques: Construct, Activation Assay, Incubation, SDS Page, Staining, Size-exclusion Chromatography, In Vitro, Purification

Journal: bioRxiv

Article Title: Non canonical scaffold-type ligase complex mediates protein UFMylation

doi: 10.1101/2022.01.31.478489

Figure Lengend Snippet: A. Lysine discharge assays to check for activation of UFC1 in the presence of different UFL1 truncations. Top gel: LiCOR scan of fluorescently labelled UFM1(UFM1*); bottom gel – Coomassie stained (Representative of three independent experiments). B. Lysine discharge assays as in A) in the absence of NTR of UFBP1(representative of 3 independent experiments). C. Coomassie stained gel showing analysis of in vitro UFMylation reaction products to check for the formation of stable oxy-ester linked UFC1-UFM1 conjugate (UFC1 -O-UFM1 ). D. Chromatogram obtained from SEC analysis using HiLoad TM Superdex 75 16/60pg column. (Bottom left) The fractions collected were run on an SDS-PAGE gel to identify fractions that contained pure UFC1 -O-UFM1 . (Bottom right) Coomassie stained gel showing analysis of purified UFC1 -O-UFM1 product to check for homogeneity. E. Quality check to analyse if UFC1-UFM1 conjugate is linked through an oxy-ester linkage by alkaline hydrolysis. F. Substrate UFMylations assays to check for UFMylation of MRE11 in the presence of different UFL1/UFBP1 truncations.

Article Snippet: Cys-UFM1 1–83 which contains a Cys residue upstream of M1 was purified and exchanged into a fresh buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM TCEP using CentriPure P10 columns (EMP Biotech).

Techniques: Activation Assay, Staining, In Vitro, SDS Page, Purification

Journal: bioRxiv

Article Title: Non canonical scaffold-type ligase complex mediates protein UFMylation

doi: 10.1101/2022.01.31.478489

Figure Lengend Snippet: A. CDK5RAP3 forms a complex with UFL1/UFBP1 in vitro . 30 µ g of UFL1/UFBP1 was mixed with 15 µ g of CDK5RAP3 and loaded on an analytical gel filtration column. (Bottom) Fractions were collected and analysed on 4-12% SDS PAGE and visualized by Coomassie staining. B. Mass photometry analysis showing the experimental molecular weight of UFL1/UFBP1/CDK5RAP3 complex. C. In vitro UFMylation assay in the presence of increasing concentrations of CDK5RAP3 to monitor the E3 ligase activity of UFL1/UFBP1 complex. The reaction products were run on a 4-12% SDS-PAGE gel and immunoblotting was performed using indicated antibodies. D. Lysine discharge assays to check for activation of UFC1 by UFL1/UFBP1 in the presence and absence of CDK5RAP3. The reaction products were run on a 4-12% SDS PAGE and visualized by Oriole staining. E. Quantification of discharge of UFM1 from UFC1 in the presence and absence of CDK5RAP3 as seen in E). n=3, mean ±SD. F. Substrate UFMylation assays using purified 60S Ribosomes in the presence of increasing concentration of CDK5RAP3. Reaction was performed for 10 min, stopped by addition of SDS Loading dye and analysed on an SDS-PAGE gel under reducing conditions followed by Immunobloting using indicated antibodies.

Article Snippet: Cys-UFM1 1–83 which contains a Cys residue upstream of M1 was purified and exchanged into a fresh buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM TCEP using CentriPure P10 columns (EMP Biotech).

Techniques: In Vitro, Filtration, SDS Page, Staining, Molecular Weight, Activity Assay, Western Blot, Activation Assay, Purification, Concentration Assay

Journal: bioRxiv

Article Title: Non canonical scaffold-type ligase complex mediates protein UFMylation

doi: 10.1101/2022.01.31.478489

Figure Lengend Snippet: A. In vitro UFMylation assay to compare the E3 ligase activity of UFL1/UFBP1 mixed with CDK5RAP3 and preassembled ternary E3 ligase complex containing UFL1/UFBP1/CDK5RAP3. Ponceau-stained membrane is showed below to indicate the amounts of reaction components used in the assay. B. UV traces of gel filtration chromatogram showing co-migration of (UFL1/UFBP1)/CDK5RAP3/UFC1 -O-UFM1 complex. Approximately, 200 µ l of sample containing (UFL1/UFBP1)/CDK5RAP3/UFC1 -O-UFM1 in the molar ratio of 1:1.5:3 was mixed and incubated at 4°C for 1 h and loaded onto a Superdex 200 10/300 gl column. The fractions were collected and analysed on a 4-12% SDS PAGE and visualized by Coomassie staining. C. Pulldown assay to check for interaction of UFL1/UFBP1 with charged UFC1 in the presence of absence of CDK5RAP3. Around 10 µ M of Untagged UFC1 and 10 µ M OF UFC1-O-UFM1 were mixed with 5 µ M of UFL1/UFBP1 complex in the presence and absence of CDK5RAP3. D. Lysine discharge assays to check for UFC1 discharge in the presence of UFL1/UFBP1 mixed with CDK5RAP3 and preassembled UFL1/UFBP1/CDK5RAP3 complex. The reaction products were run on a 4-12% SDS PAGE analysis and visualized by Coomassie staining. E. In vitro UFMylation assay to monitor UFMylation of purified substrates namely TRIP4, MRE11A and Histone H4.

Article Snippet: Cys-UFM1 1–83 which contains a Cys residue upstream of M1 was purified and exchanged into a fresh buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM TCEP using CentriPure P10 columns (EMP Biotech).

Techniques: In Vitro, Activity Assay, Staining, Filtration, Migration, Incubation, SDS Page, Purification

Journal: bioRxiv

Article Title: Non canonical scaffold-type ligase complex mediates protein UFMylation

doi: 10.1101/2022.01.31.478489

Figure Lengend Snippet: A. Multiple sequence alignment of UFC1 homologs from various organisms. A graphical representation of the secondary structure is extracted from the crystal structure of UFC1 (PDB ID:2Z6O) B. Crystal structures of UBE2D3 (PDB ID:5EGG) and UFC1 (PDB ID:2Z6O) are shown in cartoon representation. (Inset) Catalytic cysteines and critical residues required for catalytic activity of UBE2D3 (green) and UFC1(dark red) are labelled and shown in ball and stick representation. C. In vitro UFMylation assays in the presence of Wild type UFC1 (UFC1 WT ) and UFC1 lacking the N-terminal helix (UFC1 ΔN ). Schematic representation of domain features of UFC1 WT and UFC1 ΔN is shown above. D. Assay to compare Lys discharge activities of UFC1 WT and UFC1 ΔN on its own, in the presence of UFL1/UFBP1 or in the presence of preassembled UFL1/UFBP1/CDK5RAP3 complex. Top gel: LiCOR scan of fluorescently labelled UFM1(UFM1*); bottom gel – Coomassie stained (Representative of three independent experiments). E. Quantitative analysis of the intrinsic Lys reactivity (Lysine 25 mM) of UFC1 WT and in UFC1 ΔN in the absence of E3 ligase. F. Quantitative analysis of Lys reactivity of UFC1 WT and in UFC1 ΔN in the presence of UFL1/UFBP1. G. Quantitative analysis of Lys reactivity of UFC1 WT and in UFC1 ΔN in the the presence of preassembled UFL1/UFBP1/CDK5RAP3. E – G: n=3, mean ±SD.

Article Snippet: Cys-UFM1 1–83 which contains a Cys residue upstream of M1 was purified and exchanged into a fresh buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM TCEP using CentriPure P10 columns (EMP Biotech).

Techniques: Sequencing, Activity Assay, In Vitro, Staining