Docking of components in a bacterial complex

Abstract

The central issue raised by Ishikawa et al. is that of the configuration of the productive HslVU complex. We note that complexes of HslV and HslU in E. coli are labile and unstable under many conditions¹. The original electron microscopy (EM) images of Rohrwild et al.² appear to show free HslV, free HslU and HslV–HslU complex particles. To explain the discrepancy between the HslV–HslU arrangement in our co-crystals and their negatively stained EM data, we suggested that there might have been a collapse of the fragile I-domain structure in the EM preparations, or a reversal in the orientation of the HslU rings¹. Ishikawa et al. interpret their results from cryo-EM at 30 Å resolution in this latter way, using our crystal data of the components. Although these preparations preserve the native structure better than the negatively stained ones, our HslV– HslU samples are also active under crystallization conditions³.

Bochtler et al. reply

We have attempted to distinguish between the two docking modes (I-domains distal or proximal to HslV) in mutagenesis experiments involving more than two dozen mutants³. We disrupted putative contact sites to HslV in the I-domain of HslU (Fig. 3b in ref. 1) and on its opposite face and find none of these mutations has any effect on peptide hydrolysis or on casein degradation. This suggests either that no precise complex is required, or that both modes of docking are feasible. In contrast, degradation of the physiological substrate fusion protein MBP–SulA is affected by mutations both in the I-domain as well as those involving the opposite side of HslU. Small-angle X-ray scattering data and a crystal structure of the Haemophilus influenzae HslVU complex are also consistent with the EM docking mode⁴.