Journal: eLife

Article Title: Molecular determinants of phase separation for Drosophila DNA replication licensing factors

doi: 10.7554/eLife.70535

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , 2Cc-T , QB3 Macrolab (UC Berkeley) , RRID: Addgene_37237 , Ligation independent cloning (LIC); E. coli expression vector.

Techniques: Recombinant, Ligation, Cloning, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Bacterial antiviral defense pathways encode eukaryotic-like ubiquitination systems

doi: 10.1101/2023.09.26.559546

Figure Lengend Snippet: (a) Domain schematic of E. aridi E1 BilD and the H. sapiens ubiquitin/FAT10 E1 UBA6 (PDB ID 7SOL) . IAD: inactive adenylation domain; AAD: active adenylation domain; CYS: catalytic cysteine-containing domain; FCCH: first catalytic cysteine half-domain; SCCH: second catalytic cysteine half-domain (equivalent to CYS); UFD: ubiquitin fold domain. (b) Structures of E. aridi E1 BilD (left) and H. sapiens UBA6 (PDB ID 7SOL) , with domains colored as in panel (a) and catalytic cysteines shown as spheres and colored yellow. The Cα r.m.s.d. is 3.5 Å over 401 residue pairs spanning the two proteins’ IAD and AAD domains. (c) Structures of E. aridi Ubl BilA (left), M. musculus ubiquitin (center; PDB ID 4NQL) , and S. cerevisiae SUMO/Smt3 (right; PDB ID 5JNE) . (d) Structures of E. aridi E2 BilB (left) and the H. sapiens E2 UBE2D2 (right; PDB ID 4DGG) . Each protein’s catalytic cysteine residue is shown as sticks and labeled (Cys85 is mutated to serine in the H. sapiens UBE2D2 structure. E. aridi E2 BilB His151 is also shown as sticks. See for an equivalent view showing all four conserved residues of the originally identified CEHH motif. For UBE2D2, the C-terminal α-helices not shared by E2 BilB are shown in white.

Article Snippet: To generate a model substrate for DUB BilC cleavage, Ubl BilA was cloned into a vector encoding a C-terminal GFP tag (UC Berkeley Macrolab vector H6-msfGFP, Addgene ID 29725) and purified as above.

Techniques: Residue, Labeling

Journal: bioRxiv

Article Title: Bacterial antiviral defense pathways encode eukaryotic-like ubiquitination systems

doi: 10.1101/2023.09.26.559546

Figure Lengend Snippet: (a) Unrooted evolutionary tree of all Ubl BilA proteins in Type II Bil systems ( Supplementary Table 1 ). Specific examples are labeled and their domain architectures notes. LLPS NTD: N-terminal domain predicted to undergo liquid-liquid phase separation; CC NTD: N-terminal domain predicted to form a coiled-coil. (b) Sequence logo from bacterial Type II Ubl BilA proteins ( Supplementary Table 1 ). Type II Ubl BilA homologs possess up to nine residues C-terminal to the highly conserved glycine (G97 in E. aridi BilA). (c) Operon architecture of Methylobacterium brachiatum DSM 19569 BilABCD (Ubl BilA IMG accession 2928979547). (d) Cartoon view of M. brachiatum Ubl BilA , with a full-length AlphaFold 2 model shown in gray and a 3.0 Å resolution crystal structure of β-grasp domains 1 and 2 ( Materials & Methods; Supplementary Table 1 ) shown in orange. The Cα r.m.s.d. of the two structures is 0.46 Å over 134 residue pairs. (e) Operon architecture of Hymenobacter coccineus CCM 8649 BilABCD (Ubl BilA IMG accession 2792394365). (f) catGRANULE profile plot showing that the N-terminus of H. coccineus Ubl BilA is strongly predicted to undergo liquid-liquid phase separation (LLPS). (g) AlphaFold 2 model of H. coccineus Ubl BilA , colored by confidence (pLDDT). The disordered N-terminal domain (residues 1-151) is enriched in glycine (Gly), asparagine (Asn), and serine (Ser) residues. (h) Operon architecture of Bradyrhizobium sp . TSA1 BilABCD (Ubl BilA NCBI accession 2881184429). (i) Paircoil2 plot showing that the N-terminal domain of B. nitroreducens Ubl BilA is predicted to form a coiled-coil. (j) AlphaFold 2 model of a homodimer of B. nitroreducens Ubl BilA , with one protomer colored dark orange and the second protomer colored light orange. The N-terminal domains of each protomer are arranged as an antiparallel coiled-coil dimer. (k) Predicted aligned error (PAE) plot for the AlphaFold 2 model shown in panel (h), showing that the N-terminal domains of each protomer are confidently predicted to interact with one another.

Article Snippet: To generate a model substrate for DUB BilC cleavage, Ubl BilA was cloned into a vector encoding a C-terminal GFP tag (UC Berkeley Macrolab vector H6-msfGFP, Addgene ID 29725) and purified as above.

Techniques: Labeling, Sequencing, Residue

Journal: bioRxiv

Article Title: Bacterial antiviral defense pathways encode eukaryotic-like ubiquitination systems

doi: 10.1101/2023.09.26.559546

Figure Lengend Snippet: (a) Biochemical analysis of E. aridi DUB BilC cleaving a Ubl BilA -GFP fusion protein. DUB BilC constructs used were wild-type (WT), E33A active site mutant, or D106A active site mutant. (b) Schematic of the cleavage reaction shown in panel (a) and analysis by N-terminal sequencing (Edman degradation) of the C-terminal fragment (marked with an asterisk in panel (a)). See for N-terminal sequencing data showing the sequence AGIGS, pinpointing the DUB BilC cleavage site as C-terminal to Ubl BilA Gly97. (c) Sequence logo from bacterial Type II Ubl BilA proteins (Supplementary Table 1). Type II Ubl BilA homologs possess up to nine residues C-terminal to the highly conserved glycine (Gly97 in E. aridi Ubl BilA ). (d) X-ray crystal structure of DUB BilC (E33A) (purple) bound to Ubl BilA (orange). A composite omit map shows that Ubl BilA is cleaved at Gly97. See for structural comparisons with eukaryotic JAMM-family peptidases. (e) Closeup view of the DUB BilC (E33A) active site with bound zinc ion (gray) and the Ubl BilA C-terminus (orange).

Article Snippet: To generate a model substrate for DUB BilC cleavage, Ubl BilA was cloned into a vector encoding a C-terminal GFP tag (UC Berkeley Macrolab vector H6-msfGFP, Addgene ID 29725) and purified as above.

Techniques: Construct, Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: Bacterial antiviral defense pathways encode eukaryotic-like ubiquitination systems

doi: 10.1101/2023.09.26.559546

Figure Lengend Snippet: (a) N-terminal sequencing (Edman degradation) of DUB BilC -cleaved Ubl BilA -GFP fusion (C-terminal fragment), showing the evaluated value from each of five cycles of degradation. The inferred N-terminal sequence of the fragment is AGIGS. (b) Closeup view of the DUB BilC (E33A):Ubl BilA (Form 1) active site, with proteins colored as in . Active site residues of DUB BilC and glycine 97 of Ubl BilA are labeled. (c) View equivalent to panel (b), showing 2 Fo-Fc composite omit map density at 1.5 σ. (d) Comparison of E. aridi DUB BilC :Ubl BilA (left) to two similar structures, Caldiarchaeum subterraneum Rpn11-homolog bound to ubiquitin-homolog (center) and Schizosaccharomyces pombe Sst2 bound to a ubiquitin K63-linked ubiquitin (right) . Overall Cα r.m.s.d. values for DUB BilC versus its homolog in each structure are shown.

Article Snippet: To generate a model substrate for DUB BilC cleavage, Ubl BilA was cloned into a vector encoding a C-terminal GFP tag (UC Berkeley Macrolab vector H6-msfGFP, Addgene ID 29725) and purified as above.

Techniques: Sequencing, Labeling, Comparison