RESUMEN
How can a single protein domain encode a conformational landscape with multiple stably-folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB from Rhodobacter sphaeroides. Unique among known natural metamorphic proteins, this KaiB variant spontaneously interconverts between two monomeric states: the "Ground" and "Fold-switched" (FS) state. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and FS state, underscoring the ability of a single sequence to encode the slow process needed for function. We reveal the rate-limiting step between the Ground and FS state is the cis-trans isomerization of three prolines in the fold-switching region by demonstrating interconversion acceleration by the prolyl isomerase CypA. The interconversion proceeds through a "partially disordered" (PD) state, where the C-terminal half becomes disordered while the N-terminal half remains stably folded. We discovered two additional properties of KaiB's landscape. Firstly, the Ground state experiences cold denaturation: at 4°C, the PD state becomes the majorly populated state. Secondly, the Ground state exchanges with a fourth state, the "Enigma" state, on the millisecond timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to predict this "Enigma" state is a beta-strand register shift that eases buried charged residues, and support this structure experimentally. These results provide mechanistic insight in how evolution can design a single sequence that achieves specific timing needed for its function.
RESUMEN
The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates the General Stress Response in B. subtilis upon binding a partner protein (RsbT) that is released from sequestration by environmental stresses. We report that RsbT activates RsbU by inducing otherwise flexible linkers of RsbU to form a short coiled-coil that dimerizes and activates the phosphatase domains. Importantly, we present evidence that related coiled-coil linkers and phosphatase dimers transduce signals from diverse sensor domains to control the General Stress Response and other signaling across bacterial phyla. These results additionally resolve the mystery of how shared sensory domains control serine/threonine phosphatases, diguanylate cyclases and histidine kinases, revealing a common coiled-coil linker transduction mechanism. We propose that this provides bacteria with a modularly exchangeable toolkit for the evolution of diverse signaling pathways.
RESUMEN
Nitriles are uncommon in nature and are typically constructed from oximes via the oxidative decarboxylation of amino acid substrates or from the derivatization of carboxylic acids. Here we report a third strategy of nitrile biosynthesis featuring the cyanobacterial nitrile synthase AetD. During the biosynthesis of the 'eagle-killing' neurotoxin, aetokthonotoxin, AetD converts the alanyl side chain of 5,7-dibromo-L-tryptophan to a nitrile. Employing a combination of structural, biochemical, and biophysical techniques, we characterized AetD as a non-heme diiron enzyme that belongs to the emerging Heme Oxygenase-like Diiron Oxidase and Oxygenase (HDO) superfamily. High-resolution crystal structures of AetD together with the identification of catalytically relevant products provide mechanistic insights into how AetD affords this unique transformation that we propose proceeds via an aziridine intermediate. Our work presents a new paradigm for nitrile biogenesis and portrays a substrate binding and metallocofactor assembly mechanism that may be shared among other HDO enzymes.
RESUMEN
Fe and Zn ions are essential enzymatic cofactors across all domains of life. Fe is an electron donor/acceptor in redox enzymes, while Zn is typically a structural element or catalytic component in hydrolases. Interestingly, the presence of Zn in oxidoreductases and Fe in hydrolases challenge this apparent functional dichotomy. In hydrolases, Fe either substitutes for Zn or specifically catalyzes certain reactions. On the other hand, Zn can replace divalent Fe and substitute for more complex Fe assemblies, known as Fe-S clusters. Although many zinc-binding proteins interchangeably harbor Zn and Fe-S clusters, these cofactors are only sometimes functional proxies.
