Structural insights into allosteric regulation of GdpP: A conformationally dynamic phosphodiesterase

The phosphodiesterase GdpP regulates the bacterial second messenger c-di-AMP and drives antibiotic resistance in Firmicutes, yet the structural basis for its allosteric regulation has remained unknown. Here, we present cryo-EM structures of the cytosolic region of Streptococcus mutans GdpP (SmGdpP₇₄) in apo, heme-CN, and product-bound states, revealing a tetrameric architecture that enables asymmetric catalytic regulation. The non-canonical GGDEF domain functions as a scaffolding hub that positions the DHH-DHHA1 catalytic domains, representing the first example of a GGDEF domain repurposed for heterologous domain stabilization. We identify the DHHA1 dimerization interface as the primary determinant of asymmetric catalysis, and a conserved KRSR motif within the GGDEF-DHH linker as a molecular switch for heme-mediated inhibition. These findings establish a mechanistic framework for GdpP allostery and reveal structural sites for therapeutic targeting of c-di-AMP signaling in pathogenic bacteria.