Structural remodelling of the carbon-phosphorus lyase machinery by a dual ABC ATPase

Abstract

In Escherichia coli, the 14-cistron phn operon encoding carbon-phosphorus lyase allows for utilisation of phosphorus from a wide range of stable phosphonate compounds containing a C-P bond. As part of a complex, multi-step pathway, the PhnJ subunit was shown to cleave the C-P bond via a radical mechanism, however, the details of the reaction could not immediately be reconciled with the crystal structure of a 220 kDa PhnGHIJ C-P lyase core complex, leaving a significant gap in our understanding of phosphonate breakdown in bacteria. Here, we show using single-particle cryogenic electron microscopy that PhnJ mediates binding of a double dimer of the ATP-binding cassette proteins, PhnK and PhnL, to the core complex. ATP hydrolysis induces drastic structural remodelling leading to opening of the core complex and reconfiguration of a metal-binding and putative active site located at the interface between the PhnI and PhnJ subunits.

Fig. 1. The C-P lyase core complex can bind a single, flexible PhnK subunit.

a Overview of the structure of the C–P lyase core complex (PhnGHIJ, blue/green) bound to a single subunit of PhnK (red) with the names of individual subunits indicated. The ATP-binding pocket of PhnK is shown with a blue ellipse. b Surface representation of the EM density showing the extent of the tilting motion observed in PhnK. The two extreme states are shown in grey and red, respectively. c Close-up of the interaction between PhnK (red) and the PhnJ central insertion domain (CID, blue) with relevant residues shown as labelled sticks and interactions with dashed lines. d Overview of the domain structure of PhnK (top, red) and PhnJ (bottom, blue) with residue numbers indicated. For PhnK, the location of the core ABC motifs (Walker A and B, Q loop, signature motif, and H switch) are indicated in orange and the C-terminal domain in dark red. For PhnJ, the features that distinguish the protein from PhnH with which it is homologous (the Central Insertion Domain, CID, residues 129–169, and the C-terminal Domain, CTD, residues 235–281) are shown in darker blue. The four Cys residues near the C-terminus involved in Fe₄S₄ cluster binding are indicated with C. Interactions between the proteins are shown with dashed lines. e Close-up view of PhnK with catalytically important residues (Tyr15, A loop; Gln90, Q loop; Asp170/Glu171, Walker A motif; His204, H loop) shown as sticks. An extra density observed in several 3D classes at the nucleotide binding site is shown in blue. f Structure of the S. aureus Sav1866 multidrug transporter ABC domain (2ONJ) in the same orientation with the nucleotide (AMPPNP) and catalytically important residues (Tyr349, A loop; Gln422, Q loop; Glu503, Walker A motif; His534, H loop) as sticks and the Mg²⁺ ion as a purple sphere.

Fig. 2. PhnGHIJKL binds a double dimer of PhnK and PhnL.

a Top, an overview of the domain structure and sequences features of PhnL (top, yellow) and PhnK (bottom, red) with residue numbers indicated. The core ATPase motifs (Walker A and B, Q loop, signature motif, and H switch) are indicated in orange, the β hairpin extension of PhnL (residues 7–14) is shown in green and the C-terminal domain of PhnK is shown in dark red. Interactions between the proteins are shown with dashed lines. b An overview of the structure of PhnGHIJKL with the C-P lyase core complex PhnGHIJ in shades of blue/green and corresponding light colours for the other half, the PhnK dimer in red/light red, and the PhnL dimer in yellow/light yellow. Boxes indicate the approximate location of the close-up views (c–f). c Details of the PhnK ATP binding site with the AMPPNP (white/orange) shown alongside relevant, interacting side chains (labelled sticks), and the Mg²⁺ ion as a blue sphere. d Stacking interaction observed between PhnK Trp29 and PhnL Arg82. e Interaction between the PhnK C-terminus (helices α8 and α9, residues 236–247) and the PhnL β hairpin extension (residues 7–14). f Charged interactions observed between the PhnK C-terminal region (residues 210–236) and PhnL.

Fig. 3. ATP hydrolysis by PhnK and PhnL is required for phosphonate utilisation in vivo.

a In vivo functional assay using the E. coli HO1488 strain (ΔphnHIJKLMNOP) grown either without plasmid (−) or complemented with pSKA03 containing the entire phn operon including the pho box and including the following specific mutations: wt (none), PhnJ Y158A, PhnJ E149A, PhnK R78A/R82A, PhnK E171Q, and PhnL E175Q. Cells were plated on MOPS minimal agar plates containing either K₂HPO₄, 2-AmEtPn, MePn, or no added phosphorus source (no P) as indicated. The plates are representative of two repetitions. Source data are provided as a Source Data file. b ATPase activity using purified Phn(GHIJKL)₂ wildtype, Phn(GHIJKL)₂-PhnK E171Q (PhnK E171Q), and Phn(GHIJKL)₂-PhnL E175Q (PhnL E175Q) measured by separation of nucleotide species by ion exchange chromatography after overnight incubation with ATP. c Coupled assay ATPase activity (μmol/min/mg) using Phn(GHIJKL)₂ wildtype (wt), Phn(GHIJKL)₂-PhnK E171Q (PhnK E171Q), Phn(GHIJKL)₂-PhnL E175Q (PhnL E175Q), and the double mutant Phn(GHIJKL)₂-PhnK E171Q-PhnL E175Q (E171Q/E175Q). Bars show the mean from three independent reactions and error bars show standard deviation from the mean. One measurement with negative value was excluded from E171Q/E175Q. Source data are provided as a Source Data file.

Fig. 4. ATP hydrolysis by PhnK leads to opening of the C–P lyase core complex.

Overview of the structural changes taking place in the Phn(GHIJK)₂ complex between the closed (a) and open (b) states. The Phn(GHIJK)₂ complex is shown as a surface representation with the C–P lyase core complex in shades of blue/green and PhnK in red/light red. The arrow indicates the extent of opening at the Zn²⁺ site. c Details of the presumed active site at the interface between PhnI and PhnJ in the closed (top) and open (bottom) states. The Zn²⁺ ion is shown as a grey sphere with coordination geometry indicated and relevant, interacting residues with labelled sticks. d Details of the active site pocket with a bound molecule of 5-phospho-α-D-ribose-1,2-cyclic-phosphate (PRcP) as modelled in the closed (top) and open (bottom) states. e Coordinated movement of the PhnI N terminus and PhnG C terminus from the surface of the PhnGHIJK complex in the closed state (top) to the active site in the open state (bottom).

Fig. 5. Comparison to the ABC transporters.

a Overview of the structure of the S. aureus Sav1866 ABC transporter in the ADP-bound conformation with the ABC module in green and transmembrane domain in grey, except for the coupling helix, responsible for the interaction between the two, which is shown in cyan (2HYD) b Overview of the structure of the C-P lyase core complex with a dimer of PhnK E171Q in the ATP bound conformation with the PhnK dimer in red and the C-P lyase core complex in grey except for the PhnJ Central Insertion Domain (CID), responsible for PhnK binding, which is shown in blue. c Overview of the structure of the PhnK ABC dimer bound to PhnL in the open conformation with the PhnL dimer in yellow and PhnK in grey except for the C-terminal region, responsible for the interaction, which is shown in red. d Details of the S. aureus Sav1866 ABC transporter ATP binding site (bound to ADP). e Details of the PhnK ATP binding site (bound to AMPPNP). f Details of ATP bound to the PhnL active site.

Fig. 6. Model for the role of a double ABC module in catalysis by C–P lyase.

A schematic model depicting a possible functional cycle for C–P lyase with known (observed) states in colours and putative states in grey. At the top left, the C–P lyase core complex (PhnGHIJ, blue) binds two dimers of PhnK (red, in the ATP-bound state) and PhnL (partly open) as well as substrate. This complex can bind ATP in PhnL before possibly triggering a reaction in the core complex and ATP hydrolysis and P_i release in the PhnK dimer. PhnK ATP hydrolysis causes opening of the core to allow product release while the PhnL subunits are allowed to form a closed dimer and substrate is exchanged. Following renewed substrate binding, the core closes while bringing the two PhnK subunits in close proximity in an ATP bound state. Finally, PhnL hydrolyses ATP, releases ADP + P_i, and separates leading back to the starting position.