The region of Smc5 associated with Mms21 forms a moderately bent coiled-coil structure
To gain mechanistic insights into the functions of Mms21, we determined its crystal structure in conjunction with the associated Smc5 regions. Using limited protease treatment, we defined the Mms21-interacting region to a portion of the arm region of Smc5 that is comprised of the following two sequences: Asp302 to Leu369 and Lys733 to Gln813 () and (X.D., X.L., X.Z., H.Y., unpublished data). These two regions of Smc5 were linked by a four-amino-acid linker peptide (Gly-Ser-Gly-Ser) and the resulting chimeric protein formed a stable complex with the full-length Mms21. The phase of the structure of this complex was solved at 3.9Å by the Se-Met single-wavelength anomalous dispersion (SAD) method. Refinement against the native data was achieved at a resolution of 2.3Å ().
Crystallographic data statistics
In the structure of the Mms21-Smc5 complex, the two segments of Smc5 are present as two long anti-parallel helixes (H1 and H2) that wrap together for half a round forming a coiled-coil structure (). The well-refined region of the H1 helix contains 60 residues (Lys304 to Arg363) while that of H2 contains 73 residues (Asp739 to Lys811). A few residues at each end of the helixes as well as the linker between the two are not visible in the electron density map.
The core interface between the two helixes is predominantly hydrophobic. The well-complemented region of these two helixes extends for about 80Å. As the entire arm region of Smc5 is predicted to contain residues 129 to 495 and residues 660 to 944 (see methods), which is about six times as long as the coiled-coil region determined in the structure, the length of the Smc5 arm is estimated to be approximately 480Å (48 nm). This prediction is consistent with the 50 nm cohesin and condensin fibers observed in electron microscope studies (Haering et al., 2002
; Anderson et al., 2002
). Note that the lengths of the SMC proteins should be largely determined by their coiled-coil regions, as the head and hinge regions are globular structures with diameters of no more than a few nanometers (Haering et al., 2002
; Haering et al., 2004
). Another interesting feature of the Smc5 coiled-coil region is that it exhibits a moderate curvature of 165° (). In summary, this Smc5 structure provides the first glimpse into the arm regions of SMC proteins, and the bending may be an important determinant in shaping the overall structure of the Smc5/6 complex.
The N-terminal region of Mms21 forms extensive interactions with Smc5
Mms21 in this complex forms a bipartite structure. Its N-terminal domain (NTD) contributes to Smc5 binding, whereas its C-terminal domain (CTD) contains a variant RING structure and has no contact with Smc5 (). The detailed structure of the Mms21 CTD will be discussed later in the text. The core of the Mms21 NTD is composed of two elongated helixes (Pro16 to Ser52 and Glu60 to Ala100) wrapped around the coiled-coil regions of Smc5 ( and ). The first helix is divided into two short alpha helixes (α1 and α2) and a six-amino acid non-typical helix stretch (T2; ); the second helix (α3) displays a standard helical structure.
The structure of Mms21 and the interface between Mms21 and Smc5
The interface between the Mms21 NTD and Smc5 is extensive, resulting in the burial of 2360Å2 of surface area from Mms21 and 2426Å2 from Smc5 (). As a result, the NTD of Mms21 and the coiled-coil region of Smc5 form a well-structured, stable helix bundle. Nineteen hydrogen bonds and four salt bridges contribute greatly to the interaction between these two proteins (). Moreover, most of the hydrophobic residues are located in the interior of the four-helix bundle, thereby providing additional van der Waals interactions ().
We also examined the thermodynamic properties of the Mms21-Smc5 interaction. Isothermal titration calorimetry (ITC) measurements revealed relatively large negative enthalpies (between −18.21 and −22.0 kcal/mol) and positive entropies (between 10.81 and 15.20 kcal/mol) for this interaction at three temperatures (10°C, 15°C and 20°C, Table S1
). The favorable enthalpies indicate that this interaction is energetically driven by exothermic enthalpy and that the hydrophilic interactions, including hydrogen bonds and salt bridges, contribute to complex formation. The unfavorable entropies suggest a potential conformational change during complex formation. In addition, the enthalpy of the interaction displayed linear temperature dependence with a heat capacity change of −307 cal/mol·K (Table S1
and Supp Fig. 1
), suggesting significant hydrophobic interactions within the complex. Taken together, the thermodynamic properties of the Mms21-Smc5 interaction are consistent with the structural results and confirm the contributions from hydrogen bonds, salt bridges, and hydrophobic interactions.
Four segments of the N-terminal region of Mms21 are required for interaction with Smc5
To understand the functions of the Mms21 NTD, we identified four segments in the region that make the largest contributions to Smc5 binding based on the aforementioned structural information. These include: the T1 region, which is the most N-terminal region of Mms21 wrapped around Smc5; the α2 and α3 helixes, which contact Smc5 in a parallel fashion; and the T2 region, which is composed of six residues located between α1 and α2, and closely interacts with Smc5 (). We constructed deletion mutants that lack one or more of these regions and examined their interaction with the coiled-coil region of Smc5 in vitro. We found that Mms21 mutant proteins exhibited progressively weakened Smc5 binding as more of these regions were deleted (), suggesting that all of these regions contribute to the Mms21-Smc5 interaction.
The regions and residues important for Mms21-Smc5 interaction
Using the structural information, we further identified residues within these regions that are in closest proximity to Smc5 and examined their importance in Smc5 binding by testing the effects of alanine replacement at these residues. In the T1 region, four amino acids (Pro9, Val12, Leu14, and His15) were found to contact Smc5 (). His15 forms a hydrogen bond and a salt bridge with Asn791 of Smc5, whereas the three hydrophobic amino acids contribute to multiple van der Waals contacts with Smc5. A mutant protein, Mms21-M1, was made by replacing these four residues with alanines.
Similarly, two to four residues in each of the other Smc5-contacting regions were identified and mutated to alanines, resulting in four additional Mms21 mutants (Mms21-M2 through -M5). Mms21-M2 contains mutations at two hydrophobic amino acids (Leu25 and Leu30) in the T2 region (). Both leucine residues provide van der Waals contacts with Smc5. Mms21-M3 contains three mutations (Ile33Ala, Tyr34Ala and Cys37Ala) at the N-terminus of the α2 helix, resulting in the loss of multiple van der Waals contacts with Smc5 (). Mms21-M4 contains three mutations (Gln40Ala, Thr44Ala, and Gln47Ala) at the C-terminus of the α2 helix, resulting in the loss of seven hydrogen bonds and multiple van der Waals contacts with Smc5 (). Lastly, Mms21-M5 contains four mutations in the α3 helix (Tyr76Ala, Glu79Ala, Ser80Ala, and Phe83Ala), resulting in the loss of one salt bridge, one hydrogen bond, and multiple van der Waals contacts ().
The five mutated Mms21 proteins (-M1 through -M5) as well as the wild-type protein were expressed as His6-tagged proteins from E. coli and purified on nickel-nitrilotriacetic acid resins. These proteins were tested for their ability to pull down the arm region of Smc5. As shown in , mutations at these residues led to different degrees of reduction in Smc5 binding. Quantification of several trials of the pull-down experiments showed that the Mms21-M2 protein resulted in the most severe defect in Smc5 binding, followed by -M3, and then -M5. Mms21-M1 and -M4 led to moderate yet significant defects in Smc5 binding. Thus, we conclude that all the aforementioned residues contribute to Smc5 binding, though their relative contributions are different. The residues mutated in M2 are the most important, followed by those mutated in M3 and M5 and finally by those mutated in M1 and M4.
Mutations in Mms21 that affect Smc5 interaction lead to growth defects and DNA damage sensitivity
Next we investigated the biological significance of the Mms21-Smc5 interaction by testing whether Mms21 mutations that weaken the interaction with Smc5 can result in cell growth defects. First we replaced the wild-type MMS21 with each of the four aforementioned deletion constructs. This was performed in diploid yeast cells to provide one copy of wild-type MMS21 in order to avoid potential lethality during strain construction. The resulting strains were sporulated to generate haploid progeny containing only the mms21 deletions. We observed that these haploid mutants either grew slowly (ΔP16 and ΔL30) or were inviable (ΔS53 and ΔQ103); with larger deletions resulting in increased phenotypic severity (). This result is consistent with the notion that interaction with Smc5 is necessary for the essential function(s) of Mms21. Since deletion constructs can perturb protein folding, we also performed similar tests using point mutants defective in Smc5 binding.
mms21 mutations which decreased Smc5 interaction result in cell death and DNA damage sensitivity
Using an approach similar to the one described above, mms21-M1
were integrated at the MMS21
chromosomal locus. We found that mms21-M1
both led to cell death at 30°C, the normal growth temperature for yeast, but could give rise to medium and tiny sized spore clones, respectively, at 23°C (). In addition, mms21-M3
resulted in slow growth, while mms21-M4
appeared to grow as wild-type (). We further examined mms21-M4
for their sensitivity to DNA damaging agents, as the Smc5/6 complex is required to overcome replication blocks caused by hydroxyurea (HU), methyl methanesulfonate (MMS) and ultraviolet radiation (UV) (Murray and Carr, 2008
). As shown in , mms21-M5
, but not -M4,
cells were sensitive to these replication blocking agents, suggesting that mms21-M5,
but not -M4,
affects the complex’s repair function. The growth or repair defects exhibited by mms21
mutants were not due to insufficient protein levels, as the mutant proteins were expressed at or above wild-type levels (). Taken together, our results suggest that mms21
mutations affecting Smc5 binding impair the essential and repair functions of the Smc5/6 complex.
The severity of the phenotype of mms21-M2 to M5 cells correlates well with the degree of impairment in Smc5 binding in vitro. The moderate defect in Smc5 binding exhibited by the M4 mutation (85% of wild-type level, ) appears insufficient to cause an obvious phenotype when other interacting interfaces remain intact. The more pronounced impairment in Smc5 binding exhibited by the other three mutations (M2, 19% of wild-type; M3, 48% of wild-type; and M5, 61% of wild-type; ) resulted in cellular defects ranging from cell death (M2), poor growth (M3), to normal growth but with DNA damage sensitivity (M5). Thus, for mms21-M2 to M5, weaker Smc5 binding correlates with a more severe phenotype. The mms21-M1 mutant is an exception to this correlation, as it exhibited a moderate defect in Smc5 binding in vitro (84% of wild-type, ), yet led to cell death at 30°C (). To understand this discrepancy, we examined the interaction between mms21-M1 and Smc5 in vivo. As shown in , mms21-M1 led to a large reduction in Smc5 binding (5% of wild-type). This reduction was more pronounced than mms21-M3 and -M5, which exhibit 30% and 66% of wild-type level binding, respectively. This observation provides an explanation for the strong phenotype exhibited by mms21-M1. The different in vitro and in vivo binding of mms21-M1 suggests that the residues affected by M1 (the T1 region) have additional roles in Smc5 binding in vivo.
The SPL-RING structure has both similarities and differences with the RING and U-box domains of ubiquitin E3 ligases
The core of Mms21 CTD contains the SPL-RING domain, which forms a distinct type of RING structure. Similar to the RING structure of ubiquitin E3s, this region of Mms21 contains three short β strands (β1– β3), an alpha-helical domain (α7), and two loops (T7 or Loop 1, and T8 or Loop2; , , and ). Strikingly, unlike the RING structure of ubiquitin E3s, in which each loop features a zinc ion stabilized by four cysteines and/or histidines, Mms21 contains only one such loop (Loop2) while its other loop (Loop 1) does not contain any zinc ion ( and ). The zinc ion in Mms21 is located between Loop2 and beta sheets β1 and β2. It is coordinated by three cysteines (Cys200, Cys221, and Cys226) and His202 (). The Loop 1 (Leu182 to Tyr191) stays close to the β2 and α7 regions (). It does not contain a zinc ion and is stabilized by the hydrogen bonds formed between Cys184 on Loop 1 and Asp205 on β2, as well as multiple van der Waals contacts among Cys184, Pro185 and Ile186 from Loop 1 and Phe204 and Asp205 from β2 (). The three cysteines and one histidine required to incorporate the zinc ion in Loop2, as well as the five residues important for stabilizing Loop 1, are highly conserved among Mms21 homologs and PIAS family proteins (), suggesting that SPL-RING is a common structure for Mms21 proteins and is likely shared by the SP-RING type SUMO ligases.
Mms21 SPL-RING structure is different from RING and U-box structures
Comparison of the SPL-RING domain with structures in PDB using the DALI server (Holm and Sander, 1993
) revealed that it has similarities with RING structures of several ubiquitin E3s, such as the Ring1b/Bmi1 dimer of the Polycomb complex and c-Cbl (Zheng et al., 2000
; Buchwald et al., 2006
). The RING domains of Ring1b and c-Cbl can be superimposed nicely on SPL-RING with a Root Mean Square Deviation (RMSD) of 1.3 and 1.9 Å, respectively (). The Loop2 structures of the three proteins share the greatest similarities; the histidine and three cysteines that interact directly with the zinc ion are conserved (). The Loop 1 regions are different in SPL-RING and the ubiquitin E3 RINGs. In Ring1b and c-Cbl, Loop 1 contains a zinc ion that is stabilized by four cysteines (). In Mms21, this loop does not contain zinc and stays closer to the short helix (α7) and β2, such that they form one hydrogen bond and multiple van der Waals contacts. In addition to RING structures, SPL-RING also exhibits similarities with the U-box domain found in ubiquitin E3s such as CHIP (Zhang et al., 2005
; Xu et al., 2006
; Xu et al., 2008
). SPL-RING and the U-box domain of CHIP can be superimposed with an RMSD of 1.5 Å (). Differences between these two structures are found in both Loop 1 and Loop 2. In contrast to the SPL-RING or RING domains, neither Loop 1 nor Loop 2 of the U-box domain contains zinc; instead, each loop is stabilized by one set of hydrogen bonds (). In summary, SPL-RING is similar to the RING and U-box domains, but displays unique properties that can provide a structural basis for the functional specificities exhibited by SUMO and ubiquitin E3s.
Mms21 can recognize SUMO E2, but not ubiquitin E2s
The RING domains of both ubiquitin and SUMO E3s are responsible for interacting with their corresponding E2s. However, the mechanisms by which these two types of E3 specifically recognize the correct E2 are not clear. The structures of the SUMO E2, Ubc9, and those of the ubiquitin E2s, such as UbcH7 and Ubc13, are similar, with an RMSD of 1.8Å and 1.6Å, respectively. The structural similarities between SUMO and ubiquitin E2s and E3s allow us to use the determined structure of the c-Cbl-UbcH7 and CHIP-Ubc13 complexes to generate a superimposed model for the Mms21 RING-Ubc9 complex.
Comparing the superimposed structure of Mms21-Ubc9 with those of the c-Cbl-UbcH7 and CHIP-Ubc13 complexes, two differences are observed. First, the most important residues for ubiquitin E2 and E3 recognition are Phe63 in UbcH7 and Met68 in Ubc13, two relatively large hydrophobic amino acids (Zheng et al., 2000
; Zhang et al., 2005
); ). These residues protrude into a groove of the RING in c-Cbl or U-box in CHIP to make multiple van der Waals contacts. However, the residue occupying this position in Ubc9 is Ser70 (). This change ablates the van der Waals interactions with c-Cbl or CHIP, thus providing one explanation for the inability of Ubc9 to recognize ubiquitin E3s. Moreover, Ser70 is conserved in all Ubc9 homologs (Supp. Fig. 2
) and makes several new contacts with Mms21 SPL-RING based on modeling. We mutated Ser70 to alanine and found that this mutation greatly reduced sumoylation activity in vitro
(). This is consistent with the notion that Ser70 of Ubc9 is important for its function. Second, the MSΦI/LΦ (with Φ indicating hydrophobic residues) sequence at the extreme N-terminal region of Ubc9 and residues in the Mms21 Loop 1 are in close proximity and can contact each other ( and Supp. Fig. 2
). This feature is not observed for the Mms21-UbcH7 or the Mms21-Ubc13 pair in the superimposed models (). Compared to Ubc9, the N-terminus of UbcH7 lacks two residues and its first three residues are disordered in the crystal structure, while the N-terminus of Ubc13 projects away from the RING structure (Supp. Fig. 2
and ). Thus, while the N-terminal helix of Ubc9 can interact with Mms21 Loop 1, a similar interaction is unlikely to form between UbcH7 or Ubc13 and Mms21. Moreover, the N-terminal five amino acids are conserved among Ubc9 homologs (Supp. Fig. 2
), further indicating their importance. To test this idea, we examined a Ubc9 mutant that lacks these residues in the in vitro
sumoylation assay and found that it exhibited a greatly reduced activity (). Thus, this region is required for Ubc9 function. We also examined two residues, I186 and T187, on Mms21 Loop 1, which are predicted to contact Ubc9 (). We found that both I186A and T187A mutations greatly decreased Mms21 activity in vitro
(), indicating their importance for Mms21 function.
Comparison of the E2–E3 interactions for sumoylation and ubiquitination