RESUMO
Bacterial toxin-antitoxin (TA) systems regulate key cellular processes to promote cell survival during periods of stress. During steady-state cell growth, antitoxins typically interact with their cognate toxins to inhibit activity presumably by preventing substrate recognition. We solved two x-ray crystal structures of the Proteus vulgaris tetrameric HigB-(HigA)2-HigB TA complex and found that, unlike most other TA systems, the antitoxin HigA makes minimal interactions with toxin HigB. HigB adopts a RelE family tertiary fold containing a highly conserved concave surface where we predict its active site is located. HigA does not cover the solvent-exposed HigB active site, suggesting that, in general, toxin inhibition is not solely mediated by active site hindrance by its antitoxin. Each HigA monomer contains a helix-turn-helix motif that binds to its own DNA operator to repress transcription during normal cellular growth. This is distinct from antitoxins belonging to other superfamilies that typically only form DNA-binding motifs upon dimerization. We further show that disruption of the HigB-(HigA)2-HigB tetramer to a HigBA heterodimer ablates operator binding. Taken together, our biochemical and structural studies elucidate the novel molecular details of the HigBA TA system.
Assuntos
Antitoxinas/química , Proteínas de Bactérias/química , Complexos Multiproteicos/química , Sequência de Aminoácidos , Antitoxinas/genética , Antitoxinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteus vulgaris/genética , Proteus vulgaris/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The RNA splicing and processing endonuclease from Nanoarchaeum equitans (NEQ) belongs to the recently identified (alphabeta)(2) family of splicing endonucleases that require two different subunits for splicing activity. N. equitans splicing endonuclease comprises the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Here, we report the crystal structure of the functional NEQ enzyme at 2.1 A containing both subunits, as well as that of the NEQ261 subunit alone at 2.2 A. The functional enzyme resembles previously known alpha(2) and alpha(4) endonucleases but forms a heterotetramer: a dimer of two heterodimers of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Surprisingly, NEQ261 alone forms a homodimer, similar to the previously known homodimer of the catalytic subunit. The homodimers of isolated subunits are inhibitory to heterodimerization as illustrated by a covalently linked catalytic homodimer that had no RNA cleavage activity upon mixing with the structural subunit. Detailed structural comparison reveals a more favorable hetero- than homodimerization interface, thereby suggesting a possible regulation mechanism of enzyme assembly through available subunits. Finally, the uniquely flexible active site of the NEQ endonuclease provides a possible explanation for its broader substrate specificity.