RESUMO
Light-oxygen-voltage (LOV) domains are blue light-activated signaling modules integral to a wide range of photosensory proteins. Upon illumination, LOV domains form internal protein-flavin adducts that generate conformational changes which control effector function. Here we advance our understanding of LOV regulation with structural, biophysical, and biochemical studies of EL222, a light-regulated DNA-binding protein. The dark-state crystal structure reveals interactions between the EL222 LOV and helix-turn-helix domains that we show inhibit DNA binding. Solution biophysical data indicate that illumination breaks these interactions, freeing the LOV and helix-turn-helix domains of each other. This conformational change has a key functional effect, allowing EL222 to bind DNA in a light-dependent manner. Our data reveal a conserved signaling mechanism among diverse LOV-containing proteins, where light-induced conformational changes trigger activation via a conserved interaction surface.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Luz , Estrutura Terciária de Proteína , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Sequências Hélice-Volta-Hélice/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica/efeitos da radiação , Conformação Proteica/efeitos da radiação , Estrutura Secundária de Proteína , EspectrofotometriaRESUMO
The first committed step of fatty acid and polyketides biosynthesis, the biotin-dependent carboxylation of an acyl-CoA, is catalyzed by acyl-CoA carboxylases (ACCases) such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC). ACC and PCC in Streptomyces coelicolor are homologue multisubunit complexes that can carboxylate different short chain acyl-CoAs. While ACC is able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity, PCC only recognizes propionyl- and butyryl-CoA as substrates. How ACC and PCC have such different specificities toward these substrates is only partially understood. To further understand the molecular basis of how the active site residues can modulate the substrate recognition, we mutated D422, N80, R456, and R457 of PccB, the catalytic beta subunit of PCC. The crystal structures of six PccB mutants and the wild type crystal structure were compared systematically to establish the sequence-structure-function relationship that correlates the observed substrate specificity toward acetyl-, propionyl-, and butyryl-CoA with active site geometry. The experimental data confirmed that D422 is a key determinant of substrate specificity, influencing not only the active site properties but further altering protein stability and causing long-range conformational changes. Mutations of N80, R456, and R457 lead to variations in the quaternary structure of the beta subunit and to a concomitant loss of enzyme activity, indicating the importance of these residues in maintaining the active protein conformation as well as a critical role in substrate binding.