1. Ulp1-SUMO Crystal Structure and Genetic Analysis Reveal Conserved Interactions and a Regulatory Element Essential for Cell Growth in Yeast 
Elena Mossessova, Christopher D Lima 
Molecular Cell 
Volume 5, Issue 5, Pages (May 2000)
DOI: /S (00)

2. Figure 1 Structure-Based Sequence Alignment of Ulp1 and Smt3
(A) Sequence alignment of S. cerevisiae Ulp1(403–621)p (ScUlp1) and other Ulp1 orthologs from H. sapiens (HsUlp1), S. pombe (SpUlp1), D. melanogaster (DmUlp1), and A. thaliana (AtUlp1). Gaps are denoted by periods. Numbering with respect to full-length ScUlp1. The ScUlp1 secondary structure is shown above the sequence with β strands numbered, helices lettered, and coil depicted as a line. Side chain identity or similarity is denoted by bold capital letters or capital letters, respectively. Catalytic residues are capitalized in bold green. Ulp1 motifs interacting with Smt3 are coded pink (motif 1), blue (motif 2), red (motif 3), green (motif 4), yellow (motif 5), and magenta (motif 6). Asterisks (*) indicate residues in direct contact with Smt3. Results of site-directed mutagenesis denoted by circles: blue (no growth defect), yellow (temperature sensitive), and red (lethal). Deletion mutants are denoted by an arrow with plus or minus signs at positions 338, 348, 358, 368, 388, and 403. The AVP protease sequence is structurally aligned to Ulp1.
(B) Sequence- and structure-based alignment of S. cerevisiae Smt3 and other SUMO orthologs (as above) that include HsSumo, SpSmt3, DmSmt3, and AtSmt3. S. cerevisiae Rub1 is structurally aligned to ScSmt3. ScSmt3 secondary structure, contact residues (*), and motifs as described for Ulp1.
Molecular Cell 2000 5, DOI: ( /S (00) )

3. Figure 2 Proteolytic Cleavage of SUMO Substrates by Ulp1(403–621)p
Lanes alternate with (+) and without (−) Ulp1(403–621)p in the reaction. Lanes 1 and 2 (SDS-PAGE): cleavage reactions with a C-terminally His-tagged human Sumo-1 protein. Lanes 3 and 4 (SDS-PAGE): cleavage reactions with a His6-ubiquitin-Smt3-HA fusion protein. Lanes 5 and 6 (native PAGE): cleavage reactions with a large α-amine-linked Smt3-GFP protein conjugate (see Experimental Procedures).
Molecular Cell 2000 5, DOI: ( /S (00) )

4. Figure 3 Structure of the Ulp1-Smt3 Complex
Ulp1 is colored blue; Smt3 is colored red.
(A) View looking into a side of the complex.
(B) A perpendicular view of the complex looking onto the active site. β strands are numbered; α helices are lettered. The C-terminal Smt3 Gly-Gly motif is located above Ulp1 helix F.
(C) Stereo representation of the Ulp1-Smt3 complex in an orientation approximately 45° from (A) or (B). Cα positions are numbered every ten residues with Smt3 denoted by a thicker line. Graphics prepared using SETOR unless otherwise noted (Evans 1993).
Molecular Cell 2000 5, DOI: ( /S (00) )

5. Figure 4 Surface Views of the Ulp1-Smt3 Interface
The views were generated by pulling the Ulp1-Smt3 complex apart and rotating each protein by 90°.
(A) Buried surface area (green) between Ulp1 and Smt3 as calculated by a 3.0 Å distance matrix cutoff.
(B) Ulp1 motifs color coded as in Figure 1: pink (motif 1), blue (motif 2), red (motif 3), green (motif 4), yellow (motif 5), and magenta (motif 6). Smt3 motifs colored as in Figure 1.
(C) Surfaces color coded based on Ulp1 site–directed mutant viability. Red, lethal; yellow, temperature sensitive; and blue, no growth defect (as in Figure 1). Smt3 color coding derived by mapping Ulp1 mutant residue contacts and colors onto the Smt3 surface. Figure prepared with GRASP (Nicholls et al. 1991).
Molecular Cell 2000 5, DOI: ( /S (00) )

6. Figure 5 Ulp1 Motifs and the Smt3 Interface
The Ulp1 polypeptide backbone is depicted in ribbon representation, while the Smt3 peptide is depicted in stick representation. Ulp1 color-coded motifs as in Figure 1. (A) Ulp1 motif 1 (pink); (B) motif 2 (blue); (C) motif 3 (red); and (D) motif 4 (green). Views approximate a close-up for each motif as it appears in Figure 4B. Hydrogen bonds are denoted by black spheres. Waters are depicted by red spheres. Amino acid residues discussed in the text are numbered.
Molecular Cell 2000 5, DOI: ( /S (00) )

7. Figure 6 The Active Site and Catalytic Intermediate
(A) Stereo image of the Ulp1 active site (motifs 2 [blue], 5 [yellow], and 6 [magenta]) shown with residues 94–98 from Smt3 β strand 5. Hydrogen bonds are depicted as black spheres. Cys-580, His-514, and Asp-531 are residues in the catalytic triad. The Gly-98 carbonyl is an OH in the thiohemiacetal transition state analog. To illustrate tetrahedral geometry, the Gly-98 hydrogen has been modeled as a short grey bond off the carbon covalently bonded to Cys-580 Sγ.
(B) Experimental electron density at 1.8 Å obtained from the MAD data set at 1.25σ. The covalent bond between the Smt3 C-terminal glycine (Gly-98) and the active site cysteine (Cys-580) is located in the center of the image.
Molecular Cell 2000 5, DOI: ( /S (00) )

8. Figure 7 Functional Analysis of Ulp1 in Yeast
(A) Complementation of ulpΔ by wild-type ULP1, vector alone, and mutant ULP1 alleles containing the mutations W490A, D451A, W515H, D451N, F474A, and R489A grown on 5FOA media at 30°C and 37°C. Mutations listed in the text that had no growth defect resemble the R489A.
(B) Comparison of strains carrying extrachromosomal ULP1 alleles on 2% raffinose ± 0.01% galactose. Mutants that disrupt full-length ULP1 complementation (A) also block the dominant-negative effect observed for NΔ403.
(C) Deletion analysis of ULP1 and ulpΔ complementation indicates an essential element N-terminal to the conserved Ulp1 protease domain that is required for yeast cell growth.
Molecular Cell 2000 5, DOI: ( /S (00) )