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Abstract

Nature has evolved photosensory proteins that enable organisms to adapt their behavior to the prevailing light conditions. BLUF (sensor of blue light using FAD) and LOV (light, oxygen, voltage) photoreceptor domains are small globular domains that sense blue light using a flavin chromophore. These domains regulate diverse biological functions in a modular fashion, either as part of multidomain proteins or through protein–protein interactions. Natural and synthetic BLUF- and LOV-regulated proteins are popular tools for the targeted and non-invasive manipulation of biological systems by light and there is high demand for synthetic photoreceptor proteins to subject novel systems to optical control. The challenges encountered with the design of synthetic BLUF and LOV-regulated proteins illustrate the shortcomings of our current understanding of BLUF and LOV signaling mechanisms and allosteric regulation of multidomain proteins in general. This work uses a wide array of biophysical methods, including X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, small angle X-ray scattering and enzymology to characterize the structure and the photoactivation mechanism of two blue-light activated adenylyl cyclases (ACs): the BLUF-domain regulated bPAC from Beggiatoa sp. and the LOV-domain regulated mPAC from Microcoleus chtonoplastes. These two proteins contain a photoreceptor (BLUF or LOV) domain followed by an AC effector domain and serve as model systems for allosteric regulation in multidomain proteins. As experimental determination of multidomain protein structure is a challenging task, this work also explores computational routes to predict multidomain protein structure from known domain structures and unknown domain linkers. A Rosetta modeling protocol was developed and thoroughly benchmarked to investigate if multidomain protein structures can be predicted or even rationally designed. Crystal structures and high-resolution models of bPAC and mPAC revealed that the cores of the photo-receptor domains do not share an interface with the AC effector domains. Instead, this work demon-strates for the first time that the domains are linked by a coiled coil and a handle-like helical structure that constitute a conserved regulatory extension of the AC domain. Signals from the photoreceptors propagate through the coiled coil and the handles that interact with a prominent extrusion of the AC domain (the tongue). The tongue is established as a master regulator of the AC that controls both the opening angle of the AC active site cleft and the position of catalytic residues. A model is derived, explaining how the fundamentally different signal generation mechanisms of BLUF- and LOV domains are transmitted to the coiled coil where they eventually lead to similar small-scale transient motions. Analysis of the interactions between the coiled coil, the handles and the tongues demonstrates that small and transient motions are sufficient to increase AC activity several hundred-fold through slight shifts of the AC conformational equilibrium. Together, this results in a new model of allosteric AC activation that unifies many previous, seemingly contrasting observations. It is further shown that the Rosetta modeling protocol performs very well on static assemblies with large interdomain interfaces, and can be used to predict and design stable multidomain structures. However, in the light of the subtleties that nature came up with for allosteric multidomain switches such as bPAC or mPAC, structural modeling is still far away from rational design of functional linkers.