title: The length of glycine-rich linker in DNA-binding domain is critical for optimal functioning of quorum-sensing master regulatory protein HapR.
abstract: HapR is a quorum-sensing master regulatory protein in Vibrio cholerae. Though many facts are known regarding its structural and functional aspects, much still can be learnt from natural variants of this wild-type protein. While unraveling the underlying cause of functional inertness of a natural variant (HapRV2), the significance of a conserved glycine residue at position 39 in a glycine-rich linker in DNA-binding domain comes into light. This work aims at investigating how the length of glycine-rich linker (R(33)GIGRGG(39)) bridging helices α1 and α2 modulates the functionality of HapR. In pursuit of our interest, glycine residues were inserted after terminal glycine (G39) of the linker in a sequential manner. To evaluate functionality, all the glycine linker variants were subjected to a battery of performance tests under various conditions. Combined in vitro and in vivo results clearly demonstrated a gradual functional impairment of HapR linker variants coupled with increasing length of glycine-rich linker and finally, linker variant harboring four glycine residues resulted in a functionally compromised protein with significant loss of communication with cognate DNAs. Molecular dynamics studies of modeled HapR linker variants in complex with cognate promoter region show that residues namely Ser50, Thr53 and Asn56 are involved in varying degree of interactions with different nucleotides of HapR-DNA complex. The diminished functionality between variants and DNA appears to result from reduced or no interactions between Phe55 and nucleotides of cognate DNA as observed during simulations.
authors.affiliation: Molecular Biology and Microbial Physiology Division, Institute of Microbial Technology (Council of Scientific and Industrial Research), Sector 39A, Chandigarh, 160036, India.
authors.forename: Naorem Santa
authors.initials: NS
authors.lastname: Singh
authors.forename: Sangita
authors.initials: S
authors.lastname: Kachhap
authors.forename: Richa
authors.initials: R
authors.lastname: Singh
authors.forename: Rahul Chandra
authors.initials: RC
authors.lastname: Mishra
authors.forename: Balvinder
authors.initials: B
authors.lastname: Singh
authors.forename: Saumya
authors.initials: S
authors.lastname: Raychaudhuri
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D008970:Molecular Weight; D010649:Phenylalanine / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053038:Quorum Sensing; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D014734:Vibrio cholerae / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D010649:Phenylalanine; D005998:Glycine