The trans‐activation domain of the sporulation response regulator Spo0A revealed by X‐ray crystallography

Abstract

SummarySporulation in Bacillus involves the induction of scores of genes in a temporally and spatially co-ordinated programme of cell development. Its initiation is under the control of an expanded twocomponent signal transduction system termed a phosphorelay. The master control element in the decision to sporulate is the response regulator, Spo0A, which comprises a receiver or phosphoacceptor domain and an effector or transcription activation domain. The receiver domain of Spo0A shares sequence similarity with numerous response regulators, and its structure has been determined in phosphorylated and unphosphorylated forms. However, the effector domain (C-Spo0A) has no detectable sequence similarity to any other protein, and this lack of structural information is an obstacle to understanding how DNA binding and transcription activation are controlled by phosphorylation in Spo0A. Here, we report the crystal structure of C-Spo0A from Bacillus stearothermophilus revealing a single a-helical domain comprising six a-helices in an unprecedented fold. The structure contains a helix± turn±helix as part of a three a-helical bundle reminiscent of the catabolite gene activator protein (CAP), suggesting a mechanism for DNA binding. The residues implicated in forming the s A -activating region clearly cluster in a flexible segment of the polypeptide on the opposite side of the structure from that predicted to interact with DNA. The structural results are discussed in the context of the rich array of existing mutational data.

Surfaces of Spo0A and RNA Polymerase Sigma Factor A That Interact at the spoIIG Promoter in Bacillus subtilis

“…The structure of the Spo0A-DNA complex produced by Zhao et al (31) does not include all amino acyl residues of ␣-helix E, presumably because of disorder in the crystal. However, the structure of Bacillus stearothermophilus C-Spo0A produced without DNA by Lewis et al (15) includes all of ␣-helix E. Superimposition of the two Spo0A structures revealed that the two structures are nearly identical, with a root mean square deviation of 0.43 Å (31). Therefore, we superimposed the structure of Spo0A from the work of Lewis et al onto our model to estimate the position of all residues in ␣-helix E.…”

“…E221 is located near but outside of ␣-helix E in the C terminus of the DNA recognition helix. The recognition helix of the helix-turn-helix region in Spo0A is unusually long (3,15). The N-terminal amino acids of the recognition helix bind in the major groove of the DNA (31).…”

“…The structure of the Spo0A DNA binding domain has been determined by X-ray crystallography for both free Spo0A and Spo0A in complex with DNA (15,31). Interestingly, all mutations in Spo0A specifically affecting the ability of Adependent RNA polymerase to activate transcription cluster in ␣-helix E, a flexible helix in the C terminus of the protein that is positioned away from the core structure of the protein.…”

A Genomic Signature and the Identification of New Sporulation Genes

“…We first calculated a phylogenetic tree of the putative homologues of Spo0A, defined as all proteins with the same domain architecture as spo0A, which has a distinctive DNA-binding domain (68). These included, besides known Spo0A proteins, other transcription factors, such as CreB, CssR, DevR, and VraR, to name but a few, ranging from 0 to 94 sequences per species.…”

Structural Classification of Bacterial Response Regulators: Diversity of Output Domains and Domain Combinations

“…Structural characterization of the DNA-binding domains of various response regulators revealed several variations on the common helix-turn-helix (HTH) theme, exemplified by the NarL-type, OmpR-type "winged helix," Spo0A-type, and Fis-like structures (12,78,79,87,88,100). Sequence comparisons revealed additional types of DNA-binding domains in certain response regulators, some of which have been experimentally verified (94,113).…”