Coupling of zinc and GTP binding drives G-domain folding in Acinetobacter baumannii ZigA
Biophysical Journal(2024)
Abstract
COG0523 proteins, also known as nucleotide-dependent metallochaperones, are a poorly understood class of small P-loop G3E GTPases. Multiple family members play critical roles in bacterial pathogen survival during an infection as part of the adaptive response to host-mediated “nutritional immunity.” Our understanding of the structure, dynamics, and molecular-level function of COG0523 proteins, apart from the eukaryotic homologue, Zng1, remains in its infancy. Here, we use X-ray absorption spectroscopy to establish that Acinetobacter baumannii (Ab) ZigA coordinates ZnII using all three cysteines derived from the invariant CXCC motif to form an S3(N/O) coordination complex, a feature inconsistent with an available ZnII-bound crystal structure of a distantly related COG0523 protein of unknown function from E. coli, EcYjiA. The binding of ZnII and guanine nucleotides is thermodynamically linked in AbZigA, and this linkage is more favorable for the substrate GTP relative to the product GDP. Part of this coupling originates with nucleotide-induced stabilization of the G-domain tertiary structure as revealed by global thermodynamics measurements and hydrogen-deuterium exchange-mass spectrometry (HDX-MS). HDX-MS also reveals that the HDX behavior of the G2 (switch 1) loop is highly sensitive to nucleotide status and becomes more exchange labile in the GDP (product)-bound state. Significant long-range perturbation of local stability in both the G-domain and the C-terminal domain define a candidate binding pocket for a client protein that appears sensitive to nucleotide status (GDP vs. GTP). We place these new insights into the structure, dynamics, and energetics of intermolecular metal transfer into the context of a model for AbZigA metallochaperone function.
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Key words
zinc homeostasis,allostery,x-ray absorption spectroscopy,GTPase,zinc metallochaperone,nucleotide-dependent metallochaperone,CobW
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