Biology subjects

Calderone, L. A.

Publications and source records attributed to Calderone, L. A..

4 recordsLinked to original sources

bioRxiv2026

Transition metal activation reframes SAMHD1 regulation

SAMHD1 is the lone human dNTP triphosphohydrolase and is intimately linked to HIV viral restriction, dNTP pool maintenance, resistance to chemotherapy, and the autoinflammatory Aicardi-Goutieres syndrome. While its substrate promiscuity and nucleotide basis of activity have been extensively studied, the identity and mechanistic roles of its metal cofactors remain poorly defined. Here, we integrate elemental analysis, spectroscopy, protein cross-linking, and enzyme kinetics to elucidate the molecular mechanisms underlying metal-dependent activation and catalysis in SAMHD1. Our findings establish that transition metals are essential components of SAMHD1 function, highlight their overlooked role in allosteric regulation, and reveal a central role for iron in organizing the dinuclear active site. We show that iron is preferentially incorporated in one position of the bimetallic core, where it promotes recruitment of a second divalent metal ion required for activity. While manganese can substitute for iron, it alters the metal binding equilibria, highlighting the unique functional properties of iron. Notably, SAMHD1 exhibits metal cofactor promiscuity at the second metal site, accommodating diverse divalent metals with distinct effects on activity. Cumulatively, our findings establish iron as a core structural and functional determinant of SAMHD1 catalysis and reveal how transition metal selectivity and flexibility enable enzymatic activity across diverse cellular environments and metal flux conditions. Significance StatementSAMHD1 is a central regulator of cellular dNTP poolsand an essential antiviral restriction factor, yet its metal dependence remains poorly defined. Here, we show that SAMHD1 is not a magnesium-driven enzyme but a transition-metal-dependent hydrolase in which iron plays a central structural and regulatory role. We define the metal requirements of the active and allosteric sites and demonstrate that diiron and heterodinuclear iron-containing cofactors form in solution and support catalysis. Transition metals such as iron and manganese act as more effective activators than magnesium, while plasticity at the second metal-binding site enables activity across dynamic metalation and oxidation states. SAMHD1 can thus flexibly tune antiviral defense and nucleotide metabolism, bypassing constraints imposed by metal availability and the cellular redox environment.

biochemistry↗

bioRxiv2024

The conformational landscape of fold-switcher KaiB is tuned to the circadian rhythm timescale

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 KaiBs landscape. Firstly, the Ground state experiences cold denaturation: at 4{degrees}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. Significance StatementOne can conceptualize KaiB as an on-off switch to regulate circadian rhythms in bacteria, where the "On state" is the Fold-switched state that binds KaiC and other proteins, and the "Off state" is the Ground state. Our work exemplifies how evolution tuned the kinetics of interconversion to align with the hour-long timescale of its biological function. The Ground state is dramatically destabilized at cold temperatures, and the system contains an alternate "off" conformation that exchanges with the primary "off" conformation at faster timescales than the rate-limiting step. Our findings demonstrate a simple principle for evolving a protein switch: one part of a protein domain remains stably folded to serve as a scaffold for the rest of the protein to re-fold.

biochemistry↗

bioRxiv2024

A General Mechanism for the General Stress Response in Bacteria

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. This coiled-coil linker transduction mechanism additionally suggests a resolution to the mystery of how shared sensory domains control serine/threonine phosphatases, diguanylate cyclases and histidine kinases. We propose that this provides bacteria with a modularly exchangeable toolkit for the evolution of diverse signaling pathways.

microbiology↗

bioRxiv2023

Oxidative rearrangement of tryptophan to indole nitrile by a single diiron enzyme

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.

biochemistry↗