Dimerisation induced formation of the active site and the identification of three metal sites in EAL-phosphodiesterases

Dom Bellini; Sam Horrell; Andrew Hutchin; Curtis W Phippen; Richard W Strange; Yuming Cai; Armin Wagner; Jeremy S Webb; Ivo Tews; Martin A Walsh

doi:10.1038/srep42166

Dimerisation induced formation of the active site and the identification of three metal sites in EAL-phosphodiesterases

Sci Rep. 2017 Feb 10:7:42166. doi: 10.1038/srep42166.

Authors

Dom Bellini^{1

2}, Sam Horrell^{1

3}, Andrew Hutchin^{1

4}, Curtis W Phippen⁴, Richard W Strange³, Yuming Cai⁴, Armin Wagner¹, Jeremy S Webb⁴, Ivo Tews⁴, Martin A Walsh^{1

2}

Affiliations

¹ Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0FA, United Kingdom.
² Research Complex at Harwell, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0FA, United Kingdom.
³ School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, Essex, United Kingdom.
⁴ Centre for Biological Sciences and Institute for Life Sciences, Life Sciences Building B85, The University of Southampton, University Rd, Southampton, Hampshire, SO17 1BJ, United Kingdom.

Abstract

The bacterial second messenger cyclic di-3',5'-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825^EAL) and PA1727 (MucR^EAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825^EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5'-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacterial Proteins / chemistry*
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Biocatalysis
Catalytic Domain
Cations, Divalent
Cloning, Molecular
Crystallography, X-Ray
Cyclic GMP / analogs & derivatives*
Cyclic GMP / chemistry
Cyclic GMP / metabolism
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression
Genetic Vectors / chemistry
Genetic Vectors / metabolism
Hydrolysis
Kinetics
Magnesium / chemistry*
Magnesium / metabolism
Manganese / chemistry*
Manganese / metabolism
Models, Molecular
Phosphoric Diester Hydrolases / chemistry*
Phosphoric Diester Hydrolases / genetics
Phosphoric Diester Hydrolases / metabolism
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Interaction Domains and Motifs
Protein Multimerization
Pseudomonas aeruginosa / chemistry*
Pseudomonas aeruginosa / enzymology
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Substrate Specificity

Substances

Bacterial Proteins
Cations, Divalent
Recombinant Proteins
Manganese
bis(3',5')-cyclic diguanylic acid
Phosphoric Diester Hydrolases
Cyclic GMP
Magnesium

Abstract

Publication types

MeSH terms

Substances

Grants and funding