1. Volume 25, Issue 6, Pages 823-833.e6 (June 2017)
Structural Insight into Ubiquitin-Like Protein Recognition and Oligomeric States of JAMM/MPN+ Proteases 
Shiyun Cao, Sylvain Engilberge, Eric Girard, Frank Gabel, Bruno Franzetti, Julie A. Maupin-Furlow 
Structure 
Volume 25, Issue 6, Pages e6 (June 2017)
DOI: /j.str
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2. Structure 2017 25, 823-833.e6DOI: (10.1016/j.str.2017.04.002)
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3. Figure 1 PfJAMM1 Cleaves Covalently Linked SAMP2 Dimer
(A and B) Dimeric PfJAMM1 (8 μM) was incubated with 66 μM SAMP2 dimer or SAMP2 dimer∗ (where ∗ means -Val66-Ser67-Gly68-Gly69- residues have been deleted, rendering the SAMP2 dimer∗ uncleavable) at 37°C–100°C for 4 hr (A) or at 100°C for 10 min to 4 hr (B) as indicated. EDTA (50 mM) was used to chelate the catalytic zinc ion from the active site of PfJAMM1.
(C) PfJAMM1 Δ2–11 (deletion of Pro2 to Phe11) and C93S were similarly assayed for 4 hr at 100°C with enzyme monomer (m, 16 μM) and dimer (d, 8 μM) as indicated.
For demonstration of PfJAMM1 cleavage accuracy by mass spectrometry analysis, see Figure S1. For PfJAMM1 cleavage of Lys48- and Lys63-linked Ub dimers and Ubl/Ub-modified archaeal proteome, see Figures S2 and S3.
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4. Figure 2
PfJAMM1 Subunit Configuration and Association with SAMP2 Dimer∗
(A–F) Gel filtration (Superdex 75 10/300 GL) chromatographs. For simplicity, the chromatographs are presented as an overlay in panels (B) and (C) and (E) and (F), with the colors red, blue, and black representing each independent profile. See inset for details on the protein peaks detected for each chromatograph. Samples applied to the gel filtration columns included ion-exchange fractions (A and D) and purified proteins incubated separately (B and E) or as a mixture (C and F) for 4 hr at 37°C. Mixtures included PfJAMM1 (wt and α2v4) dimers and monomers incubated in a 1:2 and 1:1 molar ratio with SAMP2 dimer∗, respectively.
(G) Reducing SDS-PAGE analysis of peak fractions as indicated.
For complexes formed in vivo, see Figure S4. For comparison of PfJAMM1 monomer and dimer kinetics, see Table 1.
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5. Figure 3 PfJAMM1 Homodimer
(A) The structure of the PfJAMM1 dimer is presented in ribbon diagram with key residues of the active site and intersubunit interface in stick form. The H-bonding residues and Cys93-Cys93 disulfide bridge of the interface are highlighted.
(B) Dimer interface H bonds.
(C) Electron-density map of Cys93 region (2Fo-Fc contoured at 1σ in blue, and Fo-Fc contoured at 3σ in green). The two Cys93 of two monomeric units have been modeled in two different conformation, as two free Cys and as a disulfide bond with an equal occupancy. The B factors of these Cys93 residues are in agreement with those of neighboring residues.
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6. Figure 4
PfJAMM1 Is Related to Characterized JAMM Homologs in 3D Structure, Not Primary Amino Acid Sequence
Ribbon diagram of the PfJAMM1 monomeric unit (light blue) and its overlay with JAMM homologs (gold): AfJAMM (PDB: 1R5X), AMSH-LP (PDB: 2ZNR), CSN5 (PDB: 4F7O), Rpn11 (PDB: 4O8X), and BRCC36 (PDB: 5CW3). The β3-α2 loop, α2-helix, and catalytic active site (boxed with the zinc ion [gray sphere], water molecule [small red sphere], and amino acid side chains) are highlighted. For structure-based sequence alignment, see Figure S9.
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7. Figure 5
Archaeal JAMM Domains Cluster into Two Distinct Groups, JAMM1 and JAMM2, with the α2-Helix Region of PfJAMM1 Missing from the Group 2 Proteins
(A) Multiple amino acid sequence alignment of archaeal JAMM1 and JAMM2 proteins. Yellow highlight: α2-helix region, present in JAMM1 but not JAMM2 proteins. PfJAMM1 residues (Met69, Leu70, and Leu73) that mediate hydrophobic interaction with the SAMP2 Ile40 patch and the H-bonding Glu74 are indicated. Blue highlight: PfJAMM1 region in close proximity to the SAMP2 C-terminal tail region with residues that support multiple H bonds indicated. Red/pink highlight: residues related to the PfJAMM1 Trp108, which binds a hydrophobic pocket of SAMP2. Green highlight: residues related to PfJAMM1 Cys93, which forms a disulfide bond at the homodimer interface. Other residues discussed in the text are also highlighted. Sequence UniProt number follows the name of each JAMM protein. Secondary structure of AfJAMM and PfJAMM1 are indicated above the multiple sequence alignments of group 2 and group 1 proteins, respectively.
(B) Zoomed-in image of the sequence alignment which highlights the PfJAMM1 α2-helix region.
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8. Figure 6
Interaction Features of PfJAMM1:SAMP2 Compared with AMSH-LP:Ub
X-ray crystal structure of the PfJAMM1:SAMP2 complex (A, left) including zoomed-in views of the SAMP2 Ile40 patch (B, left), hydrophobic pocket (C, left), and H-bond interactions (D). The AMSH-LP:distal Ub complex (PDB: 2ZNV) is shown for comparison (A–C, right). The proximal Ub, responsible for Lys63 linkage-specific interaction, is not shown. The PfJAMM1 (gold), SAMP2 (light blue), AMSH-LP (gray), and Ub (dark blue) structures are displayed in ribbon diagram with residues of interest highlighted in sticks. For the simulated annealing omit 2Fo-Fc map and 2Fo-Fc map of the PfJAMM1:SAMP2 complex, see Figure S8.
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9. Figure 7
Archaeal PfJAMM1 Associated with Ub and the Interaction Features of this Complex
(A) Structural model of the PfJAMM1:Ub complex, which was generated by replacing PfJAMM1 into AMSH-LP:Ub complex (mentioned in Figure 6).
(B) Zoomed-in view of the Ub Ile44 hydrophobic patch.
(C) Hydrophobic pocket accommodation of Ub for PfJAMM1 Trp108. Ribbon diagrams of the PfJAMM1 (gold) and Ub (dark blue) are presented with residues of interest highlighted in stick.
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10. Figure 8
SAMP2 Is More Closely Related to Ub in the PfJAMM1 Bound State than in the Free Form
SAMP2free (PfSAMP2; PDB: 1RWS, NMR structure), SAMP2complex (PfSAMP2 in complex with PfJAMM1; this study, crystal structure and supported by SAXS analysis of the complex solution structure), and Ub (the distal Ub of AMSH-LP in complex with the Lys63 linked Ub2, PDB: 2ZNV, crystal structure) are indicated.
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