Structural Basis For The C-di-GMP Regulated Interaction Between MapZ And CheR1 In Pseudomonas Aeruginosa

Pseudomonas aeruginosfa(P.aeruginosa)is a common gram-negative bacterium that can infect and when there is a wound in the body or the immune system is compromized,and it is very difficult to remove.Chronic infection can eventually lead to death.Therefore,it is a major source of infection after surgery and some treatments in hospital.In addition,it is often present in individuals with cystic fibrosis causing lung infections.Cyclic diguanosine(c-di-GMP)is a second messenger of bacteria.It plays an important role in various cell functions.It is well establised that c-di-GMP stimulates the promotion of the synthesis of adhesins in biofilms and inhibits various forms of movement,thus controlling bacterial lifestyle changing from being suspended in motion to a static biofilm.The concentration of c-di-GMP pool in bacterial cells is mainly maintained by two pathways:c-di-GMP can be degraded by diguanylate cyclase(DGCs)and synthesised by phosphodiesterases(PDEs).Different from the planktonic bacteria,Biofilm adheres to the surface of living organisms or tissue and protects the microbial community from external damage.Bacteria is coated in exopolysaccharide matrix,which is composed by complex components,including polysaccharide,protein and DNA.While the biofilm keeps a certain level of isolation for the bacteria,water and other nessassary nurtrient are permitted to pass though.Chemotaxis is the tropism produced by the organism in response to the stimulation of chemical substances from the external environment.It refers to the movement of body cells,bacteria,and other unicellular and multicellular organisms based on certain chemical substances in the environment.It is one of the most basic cellular physiological responses.Methyltransferases(MTases)are ubiquitous in various life forms and involveed in the covalent modification of specific sites of DNAs,RNAs,and proteins.Methyltransferases are divided into five categories according to their structural characteristics.Most S-adenosyl-(L)-methionine(SAM)-dependent MTases(SAM-MTases)belong to Class I and use cofactor SAM as a methyl donor.The reactions participated by MTases play a key role in various biological pathways.Previous studies of chemosensory pathways have focused on Escherichia coli and Salmonella typhimurium,both of which have only one single CheR gene and a limited number of chemosensors.P.aeruginosa have four CheR paralogs and 26 chemoreceptors.These CheR methyltransferases(CheRl,CheR2,CheR3,and WspC)are encoded within four different chemical sensory gene clusters.Former research reveals that CheR proteins contain a chemical receptor binding pocket formed by a methyltransferase active site and a ?-subdomain.This active site interacts with the receptor methylation domain while the ?-subdomain binds to a specific sequence(pentapeptide)which is generally present in the C-terminus of a chemical receptor.However,CheRl,CheR3 and WspC do not bind to the GWEEF pentapeptide,whereas CheR2 binds to this pentapeptide with high affinity.The signal of c-di-GMP concentration is detected by many corresponding receptors or effectors.The signal transmission has high diversity and complexity,and most of its binding partners with c-di-GMP are not konwn.P.aeruginosa has a highly complex c-di-GMP signaling network,consisting of 18 GGDEF,5 EAL,16 GGDEF/EAL and 3 HD-GYP proteins and dozens of c-di-GMP-binding effector proteins.In P.aeruginosa genome,there are seven PilZ-domain-containing proteins,including the multidomain protein Alg44,the dual domain proteins FlgZ and PA2989,and four single-domain PilZ proteins,HapZ,MapZ,PA0012 and PA4324.It has been reported that HapZ interacts with the histidine kinase SagS and this interaction can be further enhanced by increasing the c-di-GMP concentration.Similar to HapZ,MapZ has also been reported to inhibit the activity of the methyltransferase CheR1,which can be enhanced in the presence of c-di-GMP.Here,we report the crystal structure of the CheRl C-domain with SAH and the complex crystal structure of the CheRl and MapZ binding with c-di-GMP.It can be observed that part of the binding area of MapZ with CheRl overlaps with the SAH/S AM-binding pocket.Therefore,the binding of MapZ can hinder the binding of SAH/SAM.This provides direct structural evidence for the mechanism of inhibition of CheRl by MapZ under the action of c-di-GMP.