The symmetrical structure of structural maintenance of chromosomes (SMC) and MukB proteins: long, antiparallel coiled coils, folded at a flexible hinge

Abstract

Structural maintenance of chromosomes (SMC) proteins function in chromosome condensation and several other aspects of DNA processing. They are large proteins characterized by an NH2-terminal nucleotide triphosphate (NTP)-binding domain, two long segments of coiled coil separated by a hinge, and a COOH-terminal domain. Here, we have visualized by EM the SMC protein from Bacillus subtilis (BsSMC) and MukB from Escherichia coli, which we argue is a divergent SMC protein. Both BsSMC and MukB show two thin rods with globular domains at the ends emerging from the hinge. The hinge appears to be quite flexible: the arms can open up to 180 degrees, separating the terminal domains by 100 nm, or close to near 0 degrees, bringing the terminal globular domains together. A surprising observation is that the approximately 300-amino acid-long coiled coils are in an antiparallel arrangement. Known coiled coils are almost all parallel, and the longest antiparallel coiled coils known previously are 35-45 amino acids long. This antiparallel arrangement produces a symmetrical molecule with both an NH2- and a COOH-terminal domain at each end. The SMC molecule therefore has two complete and identical functional domains at the ends of the long arms. The bifunctional symmetry and a possible scissoring action at the hinge should provide unique biomechanical properties to the SMC proteins.

Figure 1

Coiled-coil segments predicted by the program Protean (DNAstar) are shown as black rectangles. Numbers above the vertical lines indicate the aa number, and numbers in parentheses between arrows indicates the total number of aa between the lines. The 275–300-aa segment for BsSMC and the 330–335-aa segment of EcMukB were initially selected as the coiled coil because they matched the 41- and 51-nm lengths measured by EM. However, measurements of the truncated construct MukBcoil (see Discussion) indicate that the coiled coil of MukB probably includes the short segment 1205–1243. The boundaries and alignment of the coiled-coil segments are therefore still ambiguous. Heterodimeric SMCs may also pair by the antiparallel coiled-coil arrangement, as illustrated for Cut3/Cut14 and XCAP-E/XCAP-C.

Figure 2

Expression and purification of BsSMC. The first three lanes show bacterial lysate, supernatant, and pellet. About one-third of the BsSMC is in the supernatant in these expressing bacteria grown at 22°C. The next seven lanes show fractions from the Sephacryl column, and the final three lanes show fractions from the glycerol gradient (of the peak Sephacryl fraction). The molecular mass markers at 200, 160, and 120 kD are indicated.

Figure 3

Electron micrographs of BsSMC and MukB. Selected fields are presented in A and B. (C) The most common conformation, “folded-rod.” (D) The “coils-spread” conformation. (E) Molecules with the coils together but with the terminal domains split, which was seen reproducibly for BsSMC. (F) The most informative “open-V” conformation. Note the symmetry of the molecules in the open-V conformation: whenever one arm shows two small globular domains, the other arm does also. Bars, 100 nm.

Figure 4

Electron micrographs of truncated and chimeric constructs. (A) MukBcoil, in which both the NH₂- and COOH-terminal domains were deleted. The globular domain is the hinge. The ends seem to be sticky and are frequently together (right-hand images), although some open-V molecules could be found. (B) FN-MukBcoil, in which a thick segment of fibronectin was attached to the NH₂ terminus of the coiled coil. This segment completely eliminated the stickiness of the ends, and all molecules are in the open-V configuration. The thick FN segment is seen projecting from each end, confirming the antiparallel coiled-coil arrangement.

Figure 5

Molecules of FN-MukBcoil in the open-V conformation were measured for the angle of the two arms. The number of molecules found in 20° increments is shown as a histogram. Note that angles larger than 180° are possible but could not be distinguished, so they would be grouped with the smaller angle.

Figure 6

Phylogenetic trees of the SMC proteins and some outliers more distantly related. Separate trees are shown for the ∼220–240-aa NH₂-terminal domain (FLKRL...LEHVE in BsSMC), and the ∼80-aa COOH-terminal domain (LSGGE...YSSDT in BsSMC). The circles indicate the six groups of SMCs: 1–4, eukaryal SMCs; B, bacterial; A, archaeal. The group under the circle “out” are outliers, distantly related to SMCs. Each of the outlier sequences has an NH₂-terminal domain with a related ATP-binding motif, some limited sequence identity in the COOH-terminal domain, and long coiled coils separating the two, but usually no hinge. Accession numbers for SMC sequences (mostly Swiss Prot). Group 1 SMC: SMC1 (S. cer), P41003; H. sapiens, S78271; XSMC1 (X. la), AF051784. Group 2 SMC: SMC2 (S. cer), P38989; Cut14 (S. pom), P41003; MIX-1 (C. el), U96387; XCAP-E (X. la), P50533; ScII (chick), Q90988. Group 3 SMC: SMC3 (S. cer), P47037; SMC3 (S. pom), AL009197; A.(E.) nidulans, S65799; D. melanog., U30492; Bamacan (rat), U82626; XSMC3 (X. la), AF051785. Group 4 SMC: SMC4 (S. cer), U53880; Cut3 (S. pom), P41004; DPY-27 (C. el), P48996; SMC4a (C. el), Z46242; XCAP-C (X. la), P50532. Group B Bacterial SMCs: Bacillus subtilis, P51834; Mycobacterium tuberculosis, Q10970; Treponema palidum, ORF00437; Mycoplasma hyorhinis, P41508; Mycoplasma pneumoniae, P75361. Group A (mostly) archaeal SMCs: Methanococcus jannaschii, U67604; Aquifex aeolicus, AE000699; Archeoglobus fulgidus, AE000995; Synechocystis, D90905; Pyrococcus horikoshii, D90905. Outliers—proteins distantly related to SMC: MukB (E. coli), P22523; M. jannaschii 1322, A64465; Methanobacterium thermoautotrophican, AE000837; Sulfolobus acidophilum, Y10687; Rad50 (mouse), U66887; Rad50 (S. cer), P12753; Rad18 (S. pom), P53692; RHC18 (S. cer), Q12749; SbcC (E. coli), P13458.

Figure 6

Phylogenetic trees of the SMC proteins and some outliers more distantly related. Separate trees are shown for the ∼220–240-aa NH₂-terminal domain (FLKRL...LEHVE in BsSMC), and the ∼80-aa COOH-terminal domain (LSGGE...YSSDT in BsSMC). The circles indicate the six groups of SMCs: 1–4, eukaryal SMCs; B, bacterial; A, archaeal. The group under the circle “out” are outliers, distantly related to SMCs. Each of the outlier sequences has an NH₂-terminal domain with a related ATP-binding motif, some limited sequence identity in the COOH-terminal domain, and long coiled coils separating the two, but usually no hinge. Accession numbers for SMC sequences (mostly Swiss Prot). Group 1 SMC: SMC1 (S. cer), P41003; H. sapiens, S78271; XSMC1 (X. la), AF051784. Group 2 SMC: SMC2 (S. cer), P38989; Cut14 (S. pom), P41003; MIX-1 (C. el), U96387; XCAP-E (X. la), P50533; ScII (chick), Q90988. Group 3 SMC: SMC3 (S. cer), P47037; SMC3 (S. pom), AL009197; A.(E.) nidulans, S65799; D. melanog., U30492; Bamacan (rat), U82626; XSMC3 (X. la), AF051785. Group 4 SMC: SMC4 (S. cer), U53880; Cut3 (S. pom), P41004; DPY-27 (C. el), P48996; SMC4a (C. el), Z46242; XCAP-C (X. la), P50532. Group B Bacterial SMCs: Bacillus subtilis, P51834; Mycobacterium tuberculosis, Q10970; Treponema palidum, ORF00437; Mycoplasma hyorhinis, P41508; Mycoplasma pneumoniae, P75361. Group A (mostly) archaeal SMCs: Methanococcus jannaschii, U67604; Aquifex aeolicus, AE000699; Archeoglobus fulgidus, AE000995; Synechocystis, D90905; Pyrococcus horikoshii, D90905. Outliers—proteins distantly related to SMC: MukB (E. coli), P22523; M. jannaschii 1322, A64465; Methanobacterium thermoautotrophican, AE000837; Sulfolobus acidophilum, Y10687; Rad50 (mouse), U66887; Rad50 (S. cer), P12753; Rad18 (S. pom), P53692; RHC18 (S. cer), Q12749; SbcC (E. coli), P13458.

Figure 7

The model of the SMC protein structure. Arrows indicate the N→ C direction of the polypeptide. The NH₂- and COOH-terminal domains are shown schematically, without attempting to identify them with the two globular domains seen by EM in some open molecules. The coiled-coil rods are rather rigid, but the hinge is quite flexible, permitting a scissoring movement with the coils separated at angles from 0 to 180° or more. The terminal domains associate with each other to lock the molecule into the “folded-rod” conformation, but this association is reversible, permitting transition to the open-V conformation.