Structural mapping of the coiled-coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins

Abstract

The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N- and C- terminal regions pack against one another to form a globular ATPase domain. This "head" domain is connected to a central, globular, "hinge" or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50-nm coiled-coil domain of MukB, the divergent SMC protein found in γ-proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled-coil domain. We find that, in contrast to the relatively complicated coiled-coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled-coil interruptions. Near the middle of the domain is a break in coiled-coil structure in which there are three more residues on the C-terminal strand than on the N-terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled-coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled-coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans.

Structure of Full-Length SMC and Rearrangements Required for Chromosome Organization

“…Several attempts with full-length Smc coiled coils only yielded poorly diffracting crystals. Taking advantage of recently gained knowledge on the register of the Bs Smc coiled coil, we thus designed shorter Bs Smc fragments (Minnen et al., 2016, Waldman et al., 2015). A structure of a Smc middle segment, designated as Bs SmcCC2, comprising residues 246–379 and 793–929 connected via a short linker (Figure 2A), was solved by crystallography at a resolution of 3.2 Å (Table S1).…”

Tuned SMC Arms Drive Chromosomal Loading of Prokaryotic Condensin

“…On the basis of available disulfide register mapping and structural information (Bürmann et al., 2013, Minnen et al., 2016, Soh et al., 2015, Waldman et al., 2015), we designed a series of 258 successively shortened Smc constructs, covering most of the coiled coil at amino-acid resolution (Figure 2A). We then used a Golden Gate assembly driven allelic replacement strategy to regenerate the endogenous locus of a smc deletion strain with the synthetic variants (Figure S2A).…”

“…Whereas the arm accommodated 70% of inserts in the input library, this fraction was reduced to 42% in the set of functional isolates (p < 0.001 by approximate permutation test). Furthermore, the distribution of coiled-coil inserts among the functional proteins was highly non-uniform, with hotspots close to the hinge and at the head- and hinge-proximal coiled-coil breaks, respectively (Minnen et al., 2016, Waldman et al., 2015). Insertions in the N-terminal helix were particularly rarely recovered.…”

Topology and structure of an engineered human cohesin complex bound to Pds5B

“…Previous work revealed that the coiled coils are formed intra-molecularly by two anti-parallel strands11 and that their length of almost 50 nm, as well as the position of several coiled coil-interrupting regions are conserved596061. Compared with other coiled coil-containing proteins, the sequence of cohesin is remarkably conserved among metazoans but not among all eukaryotes62.…”