Structural Insights Into Hexose Phosphate Recognition By HptA In Staphylococcus Aureus And TRNA Methyltransferase TRM6-TRM61 In Saccharomyces Cerevisiae

Staphylococcus aureus is a major human pathogen that causes a diverse array of diseases in nosocomial and community-acquired settings,ranging from superficial skin infections to severe diseases.Staphylococcus aureus that invades,survives,and multiply within various host epithelial cells exploit hexose phosphates from the host cytoplasm through the hexose phosphate transport(HPT)system to gain energy and synthesize cellular components.Recently,the HPT system HptRSA in Staphylococcus aureus that could facilitate the uptake of hexose phosphate was identified and characterized.This system consists of three components which are called HptA,HptS and HptR.Therefore,this HPT system is also called three components signal transduction system.Briefly,the transcription of a hexose phosphate transporter UhpT is regulated by a novel two-component system(TCS)consisting of a membrane-bound histidine kinase HptS and a cytoplasmic response regulator HptR and by a hexose phosphate-sensing protein HptA.This HPT system is important to support intracellular survival/multiplication of Staphylococcus aureus especially when the conditions are nutritionally limited.However,the protein component interaction relationship and the detailed mechanism of signal transduction of HPT system in Staphylococcus aureus are not well known.Among the HPT system components in Staphylococcus aureus.HptA is a hexose phosphate sensor protein,which is located upstream of the whole system,however,the structural basis and mechanism by which Staphylococcus aureus senses the extracellular hexose phosphate signal and activates the downstream HptS are not well understood.The solution to above question has important significance on the relationship between structure and function of the whole HPT system.In this study,we report the crystal structure of HptA from Staphylococcus aureus in unliganded form and its complexes with glucose-6-phosphate(G6P)and galactose-6-phosphate(BGP),which was the first structure of substrate binding protein that could bind hexose phosphate in the Gram-positive bacteria.Similar to most other periplasmic substrate binding protein structures,HptA is composed of two domains termed the N-and C-domains together with an inter-domain hinge region.In both complex structures,11 residues are involved in interaction,of which Tyr37,Gly86,Thr167,Tyr216,Asn242 and Asp244 interact with the glucose or galactose ring while Pro36,Ser63,Thr64,Thr166,Thr167 and Thr198 interact with phosphate part,revealing that BGP binds basically in the same way into the binding pocket of HptA as G6P does.The hexose phosphate free and complexed HptA structures indicate that the two domains move closer to each other upon hexose phosphate binding,which reveals that HptA-apo structure may correspond to a not fully open or partially closed form while HptA-G6P or HptA-BGP present the fully closed form.Previous studies show that HptA interacts with HptS in gene level,indicating an interaction between these two proteins.Subsequently,the SPR assay was conducted to probe HptA and HptSp extracellular region(HptSp)interaction in vitro.The SPR result shows that both HptA-apo and HptA-hexose phosphate form a complex with HptSp.Although the presence of hexose phosphate does not affect the affinity between HptA and HptS,it has an influence on the dissociation rate between HptA and HptSp.Collectively,we speculate that HptA forms a complex with HptS when the signal is absent and HptS kinase activity is meanwhile inhibited.However,when the hexose phosphate signal is present,the structure of hexose phosphate bound HptA is changed to closed conformation,resulting in the change of interaction state between HptA and HptS,thus activating HptS kinase and downstream signal transduction.The N1 methylation of adenine at position 58(m1A5 8)of tRNA is an important post-transcriptional modification,which is vital for maintaining the stability of the initiator methionine tRNAiMmet.In eukaryotes,this modification is performed by the TRM6-TRM61 holoenzyme.However,the mechanism by which TRM6-TRM61 perform methyltransferase activity is not well known.To understand the molecular mechanism that underlies the cooperation of TRM6 and TRM61 in the methyl transfer reaction,we determined the crystal structure of TRM6-TRM61 holoenzyme from Saccharomyces cerevisiae in the presence and absence of its methyl donor S-Adenosyl-L-methionine(SAM).In the complex structure,two TRM6-TRM61 heterodimers assemble as a heterotetramer.Based on our structural analysis and biochemical evidence,we propose a mechanism model of m1A58 methyltransferase TRM6-TRM61 recognizes tRNA substrate occurred in the eukaryote.