A Q63E <i>Rhodobacter sphaeroides</i> AppA BLUF Domain Mutant Is Locked in a Pseudo-Light-Excited Signaling State

The AppA BLUF photoreceptor from Rhodobacter sphaeroides contains a conserved key residue, Gln63 that is thought to undergo a shift in hydrogen bonding interactions when a bound flavin is light excited. In this study we have characterized two substitution mutants of Gln63 (Q63E, Q63L) in the context of two constructs of the BLUF domain that have differing lengths, AppA1–126 and AppA17–133. Q63L mutations in both constructs exhibit a blue-shifted flavin absorption spectrum as well as a loss of the photocycle. Altered fluorescence emission and fluorescence quenching of the Q63L mutant indicates significant perturbations of hydrogen bonding to the flavin and surrounding amino acids which is confirmed by (1)H-(15)N HSQC NMR spectroscopy. The Q63E substitution mutant is constitutively locked in a lit signaling state as evidenced by a permanent 3 nm red-shift of the flavin absorption, quenching of flavin fluorescence emission, analysis of (1)H-(15)N HSQC spectra and the inability of full length AppA Q63E to bind to the PpsR repressor. The significance of these findings on the mechanism of light-induced output signaling is discussed

Structural Insights into Homo- and Heterotropic Allosteric Coupling in the Zinc Sensor <i>S. aureus</i> CzrA from Covalently Fused Dimers

The Zn(II)/Co(II)-sensing transcriptional repressor, Staphylococcus aureus CzrA, is a homodimer containing a symmetry-related pair of subunit-bridging tetrahedral N3O metal sensor coordination sites. A metal-induced quaternary structural change within the homodimer is thought to govern the biological activity of this and other metal sensor proteins. Here, we exploit covalent (Gly4Ser)n linkers of variable length in “fused” CzrAs, where n = 1 (designated 5L-fCzrA), 2 (10L-fCzrA), or 3 (15L-fCzrA), as molecular rulers designed to restrict any quaternary structural changes that are associated with metal binding and metal-mediated allosteric regulation of DNA binding to varying degrees. While 15L-fCzrA exhibits properties most like homodimeric CzrA, shortening the linker in 10L-fCzrA abolishes negative homotropic cooperativity of Zn(II) binding and reduces DNA binding affinity of the apoprotein significantly. Decreasing the linker length further in 5L-fCzrA effectively destroys one metal site altogether and further reduces DNA binding affinity. However, Zn(II) negatively regulates DNA binding of all fCzrAs, with allosteric coupling free energies (ΔG1c) of 4.6, 3.1, and 2.7 kcal mol-1 for 15L-, 10L-, and 5L-fCzrAs, respectively. Introduction of a single nonliganding H97N substitution into either the N-terminal or C-terminal protomer domain in 10L-fCzrA results in ΔG1c = 2.6 kcal mol-1 or ≈83% that of 10L-fCzrA; in contrast, homodimeric H97N CzrA gives ΔG1c = 0. 1H−15N HSQC spectra acquired for wt-, 10L-fCzrA and H97N 10L-fCzrA in various Zn(II) ligation states reveal that the allosteric change of the protomer domains within the fused dimer is independent and not concerted. Thus, occupancy of a single metal site by Zn(II) introduces asymmetry into the CzrA homodimer that leads to significant allosteric regulation of DNA binding

Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex

We report that during activation of the simian virus 40 (SV40) pre-replication complex, SV40 T antigen (Tag) helicase actively loads replication protein A (RPA) on emerging single-stranded DNA (ssDNA). This novel loading process requires physical interaction of Tag origin DNA-binding domain (OBD) with the RPA high-affinity ssDNA-binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag-OBD binds to RPA70AB at a site distal from the ssDNA-binding sites and that RPA70AB, Tag-OBD, and an 8-nucleotide ssDNA form a stable ternary complex. Intact RPA and Tag also interact stably in the presence of an 8-mer, but Tag dissociates from the complex when RPA binds to longer oligonucleotides. Together, our results imply that an allosteric change in RPA quaternary structure completes the loading reaction. A mechanistic model is proposed in which the ternary complex is a key intermediate that directly couples origin DNA unwinding to RPA loading on emerging ssDNA