RESUMO
PilT is a hexameric ATPase required for bacterial type IV pilus retraction and surface motility. Crystal structures of ADP- and ATP-bound Aquifex aeolicus PilT at 2.8 and 3.2 A resolution show N-terminal PAS-like and C-terminal RecA-like ATPase domains followed by a set of short C-terminal helices. The hexamer is formed by extensive polar subunit interactions between the ATPase core of one monomer and the N-terminal domain of the next. An additional structure captures a nonsymmetric PilT hexamer in which approach of invariant arginines from two subunits to the bound nucleotide forms an enzymatically competent active site. A panel of pilT mutations highlights the importance of the arginines, the PAS-like domain, the polar subunit interface, and the C-terminal helices for retraction. We present a model for ATP binding leading to dramatic PilT domain motions, engagement of the arginine wire, and subunit communication in this hexameric motor. Our conclusions apply to the entire type II/IV secretion ATPase family.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Fímbrias Bacterianas/fisiologia , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/fisiologia , Movimento/fisiologia , Subunidades Proteicas/química , Subunidades Proteicas/fisiologia , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Arginina/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Fímbrias Bacterianas/genética , Proteínas Motores Moleculares/genética , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , Subunidades Proteicas/genéticaRESUMO
PilT is a biological motor required for the retraction of bacterial type IV pili. Nesseria gonorrhoeae PilT has been purified and its ultrastructure has been examined by freeze-etch electron microscopy, revealing a 115 A outer diameter, 15-35 A inner diameter ring. Aquifex aeolicus PilT crystals were obtained in a primitive hexagonal space group (unit-cell parameters a = b = 107.3, c = 68.5 A) and diffract to a minimum Bragg spacing of 2.8 A when PilT is co-crystallized with adenine nucleotides. Initial phases to 3.5 A resolution have been determined by multiwavelength anomalous dispersion and density modification. Resulting electron-density maps show a hexameric A. aeolicus PilT ring 105 A wide by 55 A high, with an inner cavity that varies in shape and width from 20 to 40 A over the height of the complex. Both PilT ultrastructures are very similar to type II and type IV secretion ATPases in overall shape, size and assembly.