title: Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti-trp RNA-binding attenuation protein.
abstract: Anti-TRAP (AT) is a small zinc-binding protein that regulates tryptophan biosynthesis in Bacillus subtilis by binding to tryptophan-bound trp RNA-binding attenuation protein (TRAP), thereby preventing it from binding RNA, and allowing transcription and translation of the trpEDCFBA operon. Crystallographic and sedimentation studies have shown that AT can homooligomerize to form a dodecamer, AT(12), composed of a tetramer of trimers, AT(3). Structural and biochemical studies suggest that only trimeric AT is active for binding to TRAP. Our chromatographic and spectroscopic data revealed that a large fraction of recombinantly overexpressed AT retains the N-formyl group (fAT), presumably due to incomplete N-formyl-methionine processing by peptide deformylase. Hydrodynamic parameters from NMR relaxation and diffusion measurements showed that fAT is exclusively trimeric (AT(3)), while (deformylated) AT exhibits slow exchange between both trimeric and dodecameric forms. We examined this equilibrium using NMR spectroscopy and found that oligomerization of active AT(3) to form inactive AT(12) is linked to protonation of the amino terminus. Global analysis of the pH dependence of the trimer-dodecamer equilibrium revealed a near physiological pK(a) for the N-terminal amine of AT and yielded a pH-dependent oligomerization equilibrium constant. Estimates of excluded volume effects due to molecular crowding suggest the oligomerization equilibrium may be physiologically important. Because deprotonation favors "active" trimeric AT and protonation favors "inactive" dodecameric AT, our findings illuminate a possible mechanism for sensing and responding to changes in cellular pH.
authors: Department of Biochemistry, Ohio State University, Columbus, OH 43210, USA.
authors: Joseph R
authors: JR
authors: Sachleben
authors: Craig A
authors: CA
authors: McElroy
authors: Paul
authors: P
authors: Gollnick
authors: Mark P
authors: MP
authors: Foster
mesh_terms: D000465:Algorithms; D001426:Bacterial Proteins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D015394:Molecular Structure; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D016601:RNA-Binding Proteins / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D016601:RNA-Binding Proteins; D014157:Transcription Factors; C435924:yczA protein, Bacillus subtilis