300
heptad periodicity in their linker lengths. Whereas light-repressed
hybrid variants predominantly possessed linkers of 7n and 7n + 5
residues, those with light-induced activity mostly had linkers of 7n
+ 1 residues (n ∈ {1, …, 7}). The heptad dependence on linker
length can be accounted for by the coiled-coil nature of the linker.
Sequence analysis of a large group of related, natural sensor histidine kinases revealed their sensor and effector modules to be
mainly connected by coiled-coil linkers of length 7n and 7n + 5
residues [10].
The difference in the preferred linker lengths for light-repressed
and light-activated hybrid variants provides clues as to the mechanistic bases for signal transduction in YF1. Notably, the registers
7n and 7n + 1 differ by insertion of a single residue; within a
canonical coiled coil such an insertion induces an angular shift of
around 100° [18]. We thus propose that signal transduction in
YF1 is predicated on angular reorientation of the LOV photosensor relative to the histidine kinase effector modules [8, 9, 19, 20].
This type of angular reorientation could also be brought about by
a change in the degree of supercoiling of the linker. We recently
investigated light-dependent signal transduction in YF1 by electron paramagnetic resonance (EPR) spectroscopy and X-ray solution scattering [19, 20]. Indeed, the two independent and
complementary approaches identify light-induced left-handed
supercoiling as the mode for signal transduction and thereby provide a structural rationale for the PATCHY data.
The inversion of the response to light in YF1 cannot only be
achieved by linker variations but also by the replacement of certain
residues within the LOV photosensor units [9, 17]. To investigate
the basis for this profound effect on signal transduction, we
repeated the PATCHY analysis of the BsYtvA-BjFixL chimerae in
the background of either the D21V or H22P exchange, both of
which induce inversion of the light response in YF1.
In case of D21V, hybrid variants with the same qualitative signal response as the original YF1 D21V, i.e., with an increase in
activity upon blue-light exposure, fell into two distinct classes with
linkers of 7n and 7n + 1 residues, respectively (Fig. 5b). The preference for these classes is indicative of the coiled-coil structure of
the linker in the D21V variants. Interestingly, in the YF1 context
the clades 7n and 7n + 1 were associated with light-repressed and
light-induced activity, respectively, whereas in case of D21V they
both predominantly gave rise to light activation. However, two
D21V variants with linkers of 31 (= 7 ∙ 4 + 3) and 42 (= 7 ∙ 6) residues displayed light-repressed activity; in particular, in the former
construct, DsRed reporter fluorescence was enhanced by around
9-fold in darkness versus blue light. Intriguingly, the signal inversion introduced by the D21V exchange could thus be reverted in a
3.3.2 Linker Libraries
of Signal- Inverted YF1
Robert Stabel et al.
heptad periodicity in their linker lengths. Whereas light-repressed
hybrid variants predominantly possessed linkers of 7n and 7n + 5
residues, those with light-induced activity mostly had linkers of 7n
+ 1 residues (n ∈ {1, …, 7}). The heptad dependence on linker
length can be accounted for by the coiled-coil nature of the linker.
Sequence analysis of a large group of related, natural sensor histidine kinases revealed their sensor and effector modules to be
mainly connected by coiled-coil linkers of length 7n and 7n + 5
residues [10].
The difference in the preferred linker lengths for light-repressed
and light-activated hybrid variants provides clues as to the mechanistic bases for signal transduction in YF1. Notably, the registers
7n and 7n + 1 differ by insertion of a single residue; within a
canonical coiled coil such an insertion induces an angular shift of
around 100° [18]. We thus propose that signal transduction in
YF1 is predicated on angular reorientation of the LOV photosensor relative to the histidine kinase effector modules [8, 9, 19, 20].
This type of angular reorientation could also be brought about by
a change in the degree of supercoiling of the linker. We recently
investigated light-dependent signal transduction in YF1 by electron paramagnetic resonance (EPR) spectroscopy and X-ray solution scattering [19, 20]. Indeed, the two independent and
complementary approaches identify light-induced left-handed
supercoiling as the mode for signal transduction and thereby provide a structural rationale for the PATCHY data.
The inversion of the response to light in YF1 cannot only be
achieved by linker variations but also by the replacement of certain
residues within the LOV photosensor units [9, 17]. To investigate
the basis for this profound effect on signal transduction, we
repeated the PATCHY analysis of the BsYtvA-BjFixL chimerae in
the background of either the D21V or H22P exchange, both of
which induce inversion of the light response in YF1.
In case of D21V, hybrid variants with the same qualitative signal response as the original YF1 D21V, i.e., with an increase in
activity upon blue-light exposure, fell into two distinct classes with
linkers of 7n and 7n + 1 residues, respectively (Fig. 5b). The preference for these classes is indicative of the coiled-coil structure of
the linker in the D21V variants. Interestingly, in the YF1 context
the clades 7n and 7n + 1 were associated with light-repressed and
light-induced activity, respectively, whereas in case of D21V they
both predominantly gave rise to light activation. However, two
D21V variants with linkers of 31 (= 7 ∙ 4 + 3) and 42 (= 7 ∙ 6) residues displayed light-repressed activity; in particular, in the former
construct, DsRed reporter fluorescence was enhanced by around
9-fold in darkness versus blue light. Intriguingly, the signal inversion introduced by the D21V exchange could thus be reverted in a
3.3.2 Linker Libraries
of Signal- Inverted YF1
Robert Stabel et al.