Structural Basis of Functional Diversification of the HD-GYP Domain Revealed by the Pseudomonas aeruginosa PA4781 Protein, Which Displays an Unselective Bimetallic Binding Site

Abstract

The intracellular level of the bacterial secondary messenger cyclic di-3=,5=-GMP (c-di-GMP) is determined by a balance between its biosynthesis and degradation, the latter achieved via dedicated phosphodiesterases (PDEs) bearing a characteristic EAL or HD-GYP domain. We here report the crystal structure of PA4781, one of the three Pseudomonas aeruginosa HD-GYP proteins, which we have previously characterized in vitro. The structure shows a bimetallic active site whose metal binding mode is different from those of both HD-GYP PDEs characterized so far. Purified PA4781 does not contain iron in the active site as for other HD-GYPs, and we show that it binds to a wide range of transition metals with similar affinities. Moreover, the structural features of PA4781 indicate that this is preferentially a pGpG binding protein, as we previously suggested. Our results point out that the structural features of HD-GYPs are more complex than predicted so far and identify the HD-GYP domain as a conserved scaffold which has evolved to preferentially interact with a partner GGDEF but which harbors different functions obtained through diversification of the active site. IMPORTANCEIn bacteria, the capability to form biofilms, responsible for increased pathogenicity and antibiotic resistance, is almost universally stimulated by the second messenger cyclic di-GMP (c-di-GMP). To design successful strategies for targeting biofilm formation, a detailed characterization of the enzymes involved in c-di-GMP metabolism is crucial. We solved the structure of the HD-GYP domain of PA4781 from Pseudomonas aeruginosa, involved in c-di-GMP degradation. This is the third structure of this class of phosphodiesterases to be solved, and with respect to its homologues, it shows significant differences both in the nature and in the binding mode of the coordinated metals, indicating that HD-GYP proteins are able to fine-tune their function, thereby increasing the chances of the microorganism to adapt to different environmental needs. The dinucleotide cyclic di-GMP (c-di-GMP) serves as a core signal in the coordinated lifestyle switch of most bacteria in response to different environmental stimuli. In particular, high levels of c-di-GMP generally flag hostile conditions and result in reduced motility, attachment, and biofilm formation, while low levels of c-di-GMP indicate that it is time to go back to virulence and cell division (1-5). The intracellular levels of c-di-GMP are fine-tuned by the opposite action of dedicated enzymes: diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) (6). The first class, characterized by a GGDEF domain, synthesize c-di-GMP from two GTP molecules, while the second one hydrolyzes it. PDEs are further divided into two unrelated superfamilies of hydrolases: the more abundant EAL proteins, which degrade c-di-GMP to the linear dinucleotide pGpG, and the HD-GYP proteins, which are able to hydrolyze c-di-GMP into two molecules of GMP. Bacteria display a wide variety of genes encoding these three enzymatic domains,...

Progress in Understanding the Molecular Basis Underlying Functional Diversification of Cyclic Dinucleotide Turnover Proteins

“…In general, the HD domain superfamily of enzymes has been shown to catalyze phosphomonoesterase and phosphodiesterase reactions, depending on their catalytic metal center being mono-or binuclear, respectively. Variations in the metallic center of the HD-GYP domain were seen in the structure of the unconventional catalytically inactive Bd1817 from Bdellovibrio bacteriovorus (72), and PA4781, a two-component regulatory protein from Pseudomonas aeruginosa (73), which harbor binuclear metal centers, although of a distinct nature.…”

“…The structure of PA4781 reveals potential steric hindrance of cyclic di-GMP binding by a glutamate at position 314 (73). Accordingly, the purified enzyme has a relatively low affinity for cyclic di-GMP (K m , ϳ120 M) compared to 5=-pGpG (K m , ϳ27 M).…”

Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP

“…The crystal structure of a third HD-GYP, PA4781 (Figure 1C), showed occupancy by two nickel ions, one at the M1 position and a second at approximately the same position as M3 in the PmGH structure, denoted as M3′ in Figure 1C. 14 PA4781 was reported to bind various divalent metals with similar affinities and was proposed to be a pGpG binding protein. There is no structural or functional evidence of direct interactions of the GYP residues with the metals in any HD-GYP.…”

“…12 They typically contain N-terminal regulatory domains and C-terminal HD-GYP domains. X-ray crystal structures of three different proteins classified as HD-GYPs have been reported, 8,13,14 but only one of them, PmGH from Persephonella marina , has been reported to show appreciable c-di-GMP PDE activity. 8 When discussing the HD-GYP domain structures, we use the labels M1, M2, or M3 to discriminate the metal positions and to identify analogous positions in the three structures.…”

Mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector

“…Despite sharing 23% sequence similarity with a well-characterized cyclic di-3′,5′-GMP phosphodiesterase in Pseudomonas aeruginosa PA4781 25 , the PDE domain of SdeA uses ADPR-Ub as its substrate and catalyzes the unprecedented serine PR-ubiquitination reaction. This striking difference raised the question of how ADPR-Ub is recognized by the SdeA PDE domain.…”