Defining multiple registers within a highly segmented coiled coil of the Escherichia coli Structural Maintenance of Chromosomes (SMC) protein MukB

Many aspects of chromosomal dynamics, including DNA condensation, cohesion, and segregation, rely on the activities of the Structural Maintenance of Chromosomes (SMC) proteins. Unlike eukaryotes, which contain multiple SMC complexes for regulating these important cellular processes, prokaryotes have a single SMC protein. In gamma-proteobacteria, SMC proteins have been replaced with MukB, a protein that is structurally similar to, but divergent from SMC proteins in terms of sequence identity. These proteins share a common five domain structure consisting of globular N- and C-terminal domains connected by an antiparallel coiled coil which folds at a centralized dimerization domain. The globular N- and C-terminal domains pack against one another to form an ABC (ATP-Binding-Cassette)-like ATPase domain.;The ATPase and dimerization domains of SMC proteins have been the subject of both biochemical and structural studies. However, little is known about the antiparallel coiled coil which is clearly important for function and accounts for approximately 60% of the protein's primary sequence. This is largely due to a lack of structural information for this region of the protein. To decipher the importance of the coiled coil for MukB function, we need the ability to accurately manipulate this region which requires a detailed understanding of its structure. Coiled coil strands rely extensively on their partner strands for proper folding; therefore, deletions or insertions in one strand must be accompanied by counteracting deletions or insertions in the other strand. If this condition is not satisfied, the protein will likely be degraded. Initial studies used limited proteolysis and photoaffinity crosslinking in order to determine interacting regions within MukB's coiled coil. Subsequently, a disulfide crosslinking assay was exploited to detect interior packing interactions between paired residues within MukB's antiparallel coiled coil. This method ultimately provided not only accurate register data but was also capable of detecting deviations from canonical coiled-coil structure. These studies led to a revised model for the MukB coiled coil in which the rod-like domain is broken up into at least five different segments with a conserved intervening structural motif that is likely to be required for the observed flexibility of this coiled-coil domain.