(A) The secondary structure of budding yeast Mms21 and the alignment of Mms21 orthologs from different species. Structure-based sequence alignment of Mms21 orthologs from S. cerevisiae (sc), S. pombe (sp), Zebrafish, Xenopus, mouse, rat, and human are shown. Conserved residues are indicated in red and identical residues are highlighted. The amino-acid numbers and secondary structures of the budding yeast Mms21 are displayed above the sequence.
(B)–(E) Electrostatic surface representations of Smc5 and Mms21 illustrate charge and shape complementarities. The electrostatic surface representations of the arm region of Smc5 (B) and Mms21 (D) are shown. In addition, each representation is shown with the ribbon image of its binding partner (C and E). (D) and (E) are viewed from the sides opposite those in (B) and (C).

(A–C) Deletions and point mutations of Mms21 diminishing their interaction with Smc5 result in cell death or growth defects. Representative tetrads from diploid strains heterozygous for the indicated mms21 deletions or mutations are shown. The spore clones containing mms21 deletions or mutations are indicated by diamonds and squares, respectively.
(D) Sensitivities of mms21 mutants toward replication blocking agents MMS, UV, and HU. Wild-type and mutant mms21 strains as indicated were examined by comparing their growth on YPD plates with or without the toxins at the indicated concentrations.
(E) The mms21 point mutations do not decrease Mms21 protein levels. Cell lysates were made from cultures growing at 23°C (left panel) or 30°C (right panel). Wild-type and mutant Mms21 proteins were tagged with HA and their levels examined by protein blotting using anti-HA antibody (top). Equal loading was shown by examining the same blot using anti-PGK antibody (bottom).
(F) Mms21-M1, -M3 and -M5 proteins affect Smc5 binding in vivo. Cells contain Smc5-TAF and HA-tagged wild-type (WT) or mutant Mms21 proteins as indicated. Smc5 and associated proteins were isolated using anti-ProA antibody and examined by Western blot (left panel). The relative amounts of Mms21 proteins that co-purified with Smc5 were quantified from several blots; the average ratio and standard deviation are presented on the right panel.

(A–B) The superimposition of Ubc9 with UbcH7 (A) and with Ubc13 (B). Shown are the region of UbcH7 (cyan) that contacts c-Cbl (gray), the region of Ubc13 (cyan) that contacts CHIP (gray), and the corresponding region of Ubc9 (green). The Phe63 residue in UbcH7 and Met68 in Ubc13, which protrude into a groove in the RING domain of c-Cbl or in the U-box domain of CHIP, respectively, are indicated. The corresponding residue Ser70 in Ubc9 is labeled.
(C) Removal of the five conserved N-terminal amino acids or mutating Ser70 greatly diminishes Ubc9 function. In vitro sumoylation assays using purified full-length Ubc9 (FL), a mutation lacking the five conserved N-terminal amino acids (ΔN), and a serine 70 to alanine mutation (S70A) were examined by anti-SUMO blot. Note that all reactions contain the same amount of SUMO, SUMO E1, Mms21 and full-length or mutant Ubc9 proteins.
(D) The superimposition of Mms21 SPL-RING domain with c-Cbl and CHIP in complex with the N-terminal regions of corresponding E2s. The SPL-RING domain of Mms21 (gold) and the RING of c-Cbl (gray) are shown with the N-terminal region of Ubc9 (green) in the left panel and with that of UbcH7 (cyan) in the middle panel. The SPL-RING domain of Mms21 (gold) and the U-box of CHIP (gray) are shown with the N-terminal region of Ubc13 (blue) in the right panel. Note that the N-terminal region of Ubc9, but not that of UbcH7 or Ubc13, forms helix turns which interact with four residues on Mms21 Loop 1.
(E) I186A and T187A mutations of Mms21 greatly reduced sumoylation function. Experiments were carried out as in (C).

(A) Schematic representation of SMC structure using Smc5 as an example. The five domains of Smc5 are depicted on the top with the coiled-coil domain present in the crystal structure marked gray. The hypothetical structure of Smc5 is illustrated at the bottom.
(B) Overall view of the Mms21-Smc5 structure. The coiled-coil regions of Smc5 are shown in blue and are composed of two helixes, H1 and H2. The corresponding amino acids at the ends of each helix are indicated. Mms21 is shown in gold and the zinc ion as a green sphere. The N- and C-termini of Mms21 are indicated.
(C) The arm region of Smc5 exhibits a moderate curvature. The Cα structural representation of the arm region of Smc5 that binds to Mms21 is shown with annotation of its length and bending.

(A) Illustration of Mms21 residues important for Smc5 interaction. Each residue is indicated by its side chain in the ribbon/electrostatic surface representations of the Mms21-Smc5 structure. Residues mutated in M1 are marked red; those mutated in M2–M5 are marked as green, magenta, cyan and blue, respectively.
(B) Deletion and mutation constructs of Mms21. The schematic map of Mms21 is shown with the annotation of Smc5-interacting segments (T1, T2, α2, and α3), along with the mutations made in these segments (M1–M5) and with the RING domain. The representations of the four deletion constructs are drawn at the bottom.
(C) Deletion mutants of Mms21 decreased the interaction with the arm region of Smc5. Wild-type as well as each of the deletion mutant Mms21 proteins were expressed as His6-tagged proteins in E. coli and purified by Ni-NTA resins. These proteins were tested for their ability to pull-down the purified arm region of Smc5 (Smc5-arm-GST) as described in the text and methods. The eluate was examined by SDS-PAGE, and Coomassie blue-stained gel images are shown with the labels of the corresponding Mms21 constructs on top. The Smc5-arm-GST protein was not pulled down by the Ni-NTA resins (E). Smc5-arm-GST protein and various Mms21 proteins are denoted by arrows. Quantification of several experiments yielded the relative ratios between Smc5 and Mms21 proteins in the eluate; the average ratio and standard deviation are graphed in the lower panel.
(E) Point mutations of Mms21 decreased the interaction with the arm region of Smc5. Same as (C), except point mutant Mms21 proteins were used.

(A) The structure of the SPL-RING domain in Mms21. This structure contains two loops (Loops 1 and 2), one short helix (α7), and three short β sheets (β1-3). The zinc ion close to Loop 2 is indicated by a green sphere.
(B) A close-up view of the zinc ion interacting with His202 and three cysteines (Cys200, Cys221, and Cys226) to stabilize Loop 2.
(C) A close-up view of the residues critical for the maintenance of Loop 1 structure. Residues critical for Loop 1 structure are indicated.
(D) The alignment of SPL-RING sequences of Mms21 proteins from budding yeast (sc), fission yeast (sp) and humans, with the RING sequences of c-Cbl and Ring1b, and with the U-box sequence of CHIP. The blue boxes indicate the three cysteines and one histidine surrounding the zinc ion near Loop 2s in Mms21, c-Cbl and Ring1b. Magenta boxes indicate the five residues required for the Loop 1 structure of Mms21. Note that these residues are conserved among Mms21 homologs, but not in c-Cbl Ring1B or CHIP. Red boxes mark the four cysteines surrounding the zinc ion in Loop 1 of c-Cbl and Ring1b.
(E) The alignment of the SPL-RING sequence of Mms21 and the SP-RING sequence of the PIAS proteins. The blue and magenta boxes are as described in (D).
(F) The superimposition of SPL-RING with RING and U-box structures. The SPL-RING of Mms21 (gold) is superimposed with the RING structure of c-Cbl (silver, left), the RING structure of Ring1b (blue, middle) and the U-box structure of CHIP (salmon, right).