Dimer-induced signal propagation in Spo0A.
Spo0A, the response regulator protein controlling the initiation of sporulation in Bacillus, has two distinct domains, an N-terminal phosphoacceptor (or receiver) domain and a C-terminal DNA-binding (or effector) domain. The phosphoacceptor domain mediates dimerization of Spo0A on phosphorylation. A comparison of the crystal structures of phosphorylated and unphosphorylated response regulators suggests a mechanism of activation in which structural changes originating at the phosphorylatable aspartate extend to the alpha4beta5alpha5 surface of the protein. In particular, the data show an important role in downstream signalling for a conserved aromatic residue (Phe-105 in Spo0A), the conformation of which alters upon phosphorylation. In this study, we have prepared a Phe-105 to Ala mutant to probe the contribution of this residue to Spo0A function. We have also made an alanine substitution of the neighbouring residue Tyr-104 that is absolutely conserved in the Spo0As of spore-forming Bacilli. The spo0A(Y104A) and spo0A(F105A) alleles severely impair sporulation in vivo. In vitro phosphorylation of the purified proteins by phosphoramidate is unaffected, but dimerization and DNA binding are abolished by the mutations. We have identified intragenic suppressor mutations of spo0A(F105A) and shown that these second-site mutations in the purified proteins restore phosphorylation-dependent dimer formation. Our data support a model in which dimerization and signal transduction between the two domains of Spo0A are mediated principally by the alpha4beta5alpha5 signalling surface in the receiver domain.
Muchová
K
K
Institute of Molecular Biology, Slovak Academy of Sciences, 845 51 Bratislava 45, Slovakia.
Lewis
R J
RJ
Perecko
D
D
Brannigan
J A
JA
Ladds
J C
JC
Leech
A
A
Wilkinson
A J
AJ
Barák
I
I
D001412:Bacillus subtilis / Q000235:genetics / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D017931:DNA Primers; D019281:Dimerization; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013171:Spores, Bacterial / Q000502:physiology; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; C080950:Spo0A protein, Bacillus subtilis; D014157:Transcription Factors; D005020:Ethyl Methanesulfonate