The role of glutamate 90 in drug binding by QacR, a multidrug-resistance regulator inStaphylococcus aureus

Abstract

Mercury resistance regulator (MerR) is a 32 kD metalloregulatory protein that controls mercury resistance in Gram-negative bacteria by regulating transcription of the mer-operon. It is a member in the diverse MerR-family of transcriptional activators. In the presence of Hg (II) the regulator functions as an activator of the mer-genes while in the absence of Hg(II) it is a weak repressor.The monomer folds into three functional domains: a N-terminal DNA-binding domain, which is homologous among the MerR family proteins, a C-terminal Hg-binding domain, and an intervening region, which plays a role in active repression and in transcriptional activation. The DNA-binding domain contains a helix-turn-helix motif that binds to DNA between positions -35 and -10 on the operon. The Hg-binding domain has three conserved cysteine residues at positions C82, C117, and C126, which form the trigonal high-affinity Hg-binding site. Every dimer binds one Hg atom, but the mechanism of binding is still unknown. High affinity and selectivity of MerR for Hg has been utilized in biosensors and has been engineered for purification of mercury-contaminated drinking water [1,2].In the absence of Hg the inactive form of MerR causes a distortion of DNA in the middle of the mer-operator. In this inactive form the RNA polymerase binds adjacent to MerR in a closed complex, which has access only to the -35 site of the promoter, while the -10 site remains inaccessible. The distortion is caused by a 19 bp spacing between the -35 and -10 elements, which is unusually long compared to normal 17 bp in bacteria. Incoming Hg binds to MerR and causes unwinding and unkinking of DNA. The helix backbone is straightened out and unwound which brings the -35 and -10 promoter elements onto the same face of the DNA double helix. The reorientation of the -35 and -10 sequences allows them to interact with the RNA polymerase to form an open transcriptional complex and transcription is initiated [3].Our aims are to solve the crystal structure of MerR in active and inactive form and study the specific protein-DNA interactions, binding mechanisms and structural changes occurring upon ligand binding.[1] Brown, N.L., Stoyanov,