A Novel Ligand-binding Domain Involved in Regulation of Amino Acid Metabolism in Prokaryotes

Thijs J.G Ettema,Travis H Tani,Arie B Brinkman,John Van Der Oost,John B Rafferty

doi:10.1074/jbc.m206063200

Abstract

A combination of sequence profile searching and structural protein analysis has revealed a novel type of small molecule binding domain that is involved in the allosteric regulation of prokaryotic amino acid metabolism. This domain, designated RAM, has been found to be fused to the DNA-binding domain of Lrp-like transcription regulators and to the catalytic domain of some metabolic enzymes, and has been found as a stand-alone module. Structural analysis of the RAM domain of Lrp reveals a betaalphabetabetaalphabeta-fold that is strikingly similar to that of the recently described ACT domain, a ubiquitous allosteric regulatory domain of many metabolic enzymes. However, structural alignment and re-evaluation of previous mutagenesis data suggest that the effector-binding sites of both modules are significantly different. By assuming that the RAM and ACT domains originated from a common ancestor, these observations suggest that their ligand-binding sites have evolved independently. Both domains appear to play analogous roles in controlling key steps in amino acid metabolism at the level of gene expression as well as enzyme activity.

Highlights

A combination of sequence profile searching and structural protein analysis has revealed a novel type of small molecule binding domain that is involved in the allosteric regulation of prokaryotic amino acid metabolism
In the present study we describe a novel ligand-binding module that we named the RAM domain because of its general involvement in the allosteric Regulation of Amino acid Metabolism
The ␤␣␤␤␣␤-motif appears to be a common regulatory structure in amino acid metabolic enzymes and transcriptional regulators; both the RAM and the ACT domains share this fold and are associated with proteins that are involved with amino acid metabolism either as part of enzymes, as part of transcriptional regulators, or as stand-alone SMBD

Summary

EXPERIMENTAL PROCEDURES

PSI-BLAST Analysis and Multiple Sequence Alignments, Domain Analysis of Proteins—In order to verify and characterize the relationship between distant RAM domains at the sequence level, we performed several PSI-BLAST searches [8] at the National Center of Biotechnology Information. When a PSI-BLAST search was seeded with the Cterminal domain of the Pyrococcus furiosus LrpA (residues 62–141), using a BLOSUM80 matrix and an expect value threshold of 0.001, the first stand-alone versions of RAM (lacking the HTH domain) were retrieved within the first iteration (10580664; E ϭ 6 ϫ 10Ϫ12); the RAM domains within the Sulfolobus solfataricus and Sulfolobus tokodaii 2-isopropylmalate synthase (13814162, respectively, 15623321) were recovered in iteration 3 (E ϭ 7 ϫ 10Ϫ6). A reverse PSI-BLAST using the S. solfataricus 2-isopropylmalate synthase sequence (residues 342– 461) recovered HTH-RAM proteins (e.g. 13813287 at iteration 1, E ϭ 3 ϫ 10Ϫ5) and stand-alone versions of the RAM domain (e.g. 13813398 at iteration 2, E ϭ 5 ϫ 10Ϫ5), thereby connecting the most distant RAM domain containing proteins with each other on a statistical basis. A structural alignment was deduced using the Structural alignment tool of the Swiss PDB viewer [12]

RESULTS

A Novel Regulatory Domain in Amino Acid Metabolism

DISCUSSION