Cryo-EM reveals species-specific components within the Helicobacter pylori Cag type IV secretion system core complex.

The pathogenesis of Helicobacter pylori-associated gastric cancer is dependent on delivery of CagA into host cells through a type IV secretion system (T4SS). The H. pylori Cag T4SS includes a large membrane-spanning core complex containing five proteins, organized into an outer membrane cap (OMC), a periplasmic ring (PR) and a stalk. Here, we report cryo-EM reconstructions of a core complex lacking Cag3 and an improved map of the wild-type complex. We define the structures of two unique species-specific components (Cag3 and CagM) and show that Cag3 is structurally similar to CagT. Unexpectedly, components of the OMC are organized in a 1:1:2:2:5 molar ratio (CagY:CagX:CagT:CagM:Cag3). CagX and CagY are components of both the OMC and the PR and bridge the symmetry mismatch between these regions. These results reveal that assembly of the H. pylori T4SS core complex is dependent on incorporation of interwoven species-specific components.

Hsp104 and Potentiated Variants Can Operate as Distinct Nonprocessive Translocases.

Heat shock protein (Hsp) 104 is a hexameric ATPases associated with diverse cellular activities motor protein that enables cells to survive extreme stress. Hsp104 couples the energy of ATP binding and hydrolysis to solubilize proteins trapped in aggregated structures. The mechanism by which Hsp104 disaggregates proteins is not completely understood but may require Hsp104 to partially or completely translocate polypeptides across its central channel. Here, we apply transient state, single turnover kinetics to investigate the ATP-dependent translocation of soluble polypeptides by Hsp104 and Hsp104A503S, a potentiated variant developed to resolve misfolded conformers implicated in neurodegenerative disease. We establish that Hsp104 and Hsp104A503S can operate as nonprocessive translocases for soluble substrates, indicating a "partial threading" model of translocation. Remarkably, Hsp104A503S exhibits altered coupling of ATP binding to translocation and decelerated dissociation from polypeptide substrate compared to Hsp104. This altered coupling and prolonged substrate interaction likely increases entropic pulling forces, thereby enabling more effective aggregate dissolution by Hsp104A503S.

Escherichia coli DnaK Allosterically Modulates ClpB between High- and Low-Peptide Affinity States.

ClpB and DnaKJE provide protection to Escherichia coli cells during extreme environmental stress. Together, this co-chaperone system can resolve protein aggregates, restoring misfolded proteins to their native form and function in solubilizing damaged proteins for removal by the cell's proteolytic systems. DnaK is the component of the KJE system that directly interacts with ClpB. There are many hypotheses for how DnaK affects ClpB-catalyzed disaggregation, each with some experimental support. Here, we build on our recent work characterizing the molecular mechanism of ClpB-catalyzed polypeptide translocation by developing a stopped-flow FRET assay that allows us to detect ClpB's movement on model polypeptide substrates in the absence or presence of DnaK. We find that DnaK induces ClpB to dissociate from the polypeptide substrate. We propose that DnaK acts as a peptide release factor, binding ClpB and causing the ClpB conformation to change to a low-peptide affinity state. Such a role for DnaK would allow ClpB to rebind to another portion of an aggregate and continue nonprocessive translocation to disrupt the aggregate.