Bidirectional pilus processing in the Tad pilus system motor CpaF.

The bacterial tight adherence pilus system (TadPS) assembles surface pili essential for adhesion and colonisation in many human pathogens. Pilus dynamics are powered by the ATPase CpaF (TadA), which drives extension and retraction cycles in Caulobacter crescentus through an unknown mechanism. Here we use cryogenic electron microscopy and cell-based light microscopy to characterise CpaF mechanism. We show that CpaF assembles into a hexamer with C2 symmetry in different nucleotide states. Nucleotide cycling occurs through an intra-subunit clamp-like mechanism that promotes sequential conformational changes between subunits. Moreover, a comparison of the active sites with different nucleotides bound suggests a mechanism for bidirectional motion. Conserved CpaF residues, predicted to interact with platform proteins CpaG (TadB) and CpaH (TadC), are mutated in vivo to establish their role in pilus processing. Our findings provide a model for how CpaF drives TadPS pilus dynamics and have broad implications for how other ancient type 4 filament family members power pilus assembly.

Conserved residues Glu37 and Trp229 play an essential role in protein folding of ß-lactamase.

Evolutionary robustness requires that the number of highly conserved amino acid residues in proteins is minimized. In enzymes, such conservation is observed for catalytic residues but also for some residues in the second shell or even further from the active site. ß-Lactamases evolve in response to changing antibiotic selection pressures and are thus expected to be evolutionarily robust, with a limited number of highly conserved amino acid residues. As part of the effort to understand the roles of conserved residues in class A ß-lactamases, we investigate the reasons leading to the conservation of two amino acid residues in the ß-lactamase BlaC, Glu37, and Trp229. Using site-directed mutagenesis, we have generated point mutations of these residues and observed a drastic decrease in the levels of soluble protein produced in Escherichia coli, thus abolishing completely the resistance of bacteria against ß-lactam antibiotics. However, the purified proteins are structurally and kinetically very similar to the wild-type enzyme, only differing by exhibiting a slightly lower melting temperature. We conclude that conservation of Glu37 and Trp229 is solely caused by an essential role in the folding process, and we propose that during folding Glu37 primes the formation of the central ß-sheet and Trp229 contributes to the hydrophobic collapse into a molten globule. ENZYME: EC 3.5.2.6. DATABASE: Structural data are available in PDB database under the accession number 7A5U.