Structure and assembly of the pseudopilin PulG.

The pseudopilin PulG is one of several essential components of the type II pullulanase secretion machinery (the Pul secreton) of the Gram-negative bacterium Klebsiella oxytoca. The sequence of the N-terminal 25 amino acids of the PulG precursor is hydrophobic and very similar to the corresponding region of type IV pilins. The structure of a truncated PulG (lacking the homologous region), as determined by X-ray crystallography, was found to include part of the long N-terminal alpha-helix and the four internal anti-parallel beta-strands that characterize type IV pilins, but PulG lacks the highly variable loop region with a disulphide bond that is found in the latter. When overproduced, PulG forms flexible pili whose structural features, as visualized by electron microscopy, are similar to those of bacterial type IV pili. The average helical repeat comprises 17 PulG subunits and four helical turns. Electron microscopy and molecular modelling show that PulG probably assembles into left-handed helical pili with the long N-terminal alpha-helix tightly packed in the centre of the pilus. As in the type IV pilins, the hydrophobic N-terminal part of the PulG alpha-helix is necessary for its assembly. Subtle sequence variations within this highly conserved segment seem to determine whether or not a type IV pilin can be assembled into pili by the Pul secreton.

X-ray structures of the maltose-maltodextrin-binding protein of the thermoacidophilic bacterium Alicyclobacillus acidocaldarius provide insight into acid stability of proteins.

Maltose-binding proteins act as primary receptors in bacterial transport and chemotaxis systems. We report here crystal structures of the thermoacidostable maltose-binding protein from Alicyclobacillus acidocaldarius, and explore its modes of binding to maltose and maltotriose. Further, comparison with the structures of related proteins from Escherichia coli (a mesophile), and two hyperthermophiles (Pyrococcus furiosus and Thermococcus litoralis) allows an investigation of the basis of thermo- and acidostability in this family of proteins.The thermoacidophilic protein has fewer charged residues than the other three structures, which is compensated by an increase in the number of polar residues. Although the content of acidic and basic residues is approximately equal, more basic residues are exposed on its surface whereas most acidic residues are buried in the interior. As a consequence, this protein has a highly positive surface charge. Fewer salt bridges are buried than in the other MBP structures, but the number exposed on its surface does not appear to be unusual. These features appear to be correlated with the acidostability of the A. acidocaldarius protein rather than its thermostability. An analysis of cavities within the proteins shows that the extremophile proteins are more closely packed than the mesophilic one. Proline content is slightly higher in the hyperthermophiles and thermoacidophiles than in mesophiles, and this amino acid is more common at the second position of beta-turns, properties that are also probably related to thermostability. Secondary structural content does not vary greatly in the different structures, and so is not a contributing factor.