The Intrinsically Disordered Region of ExbD Is Required for Signal Transduction

Abstract

The TonB system actively transports vital nutrients across the unenergized outer membranes of the majority of Gram-negative bacteria. In this system, integral membrane proteins ExbB, ExbD, and TonB work together to transduce the proton motive force (PMF) of the inner membrane to customized active transporters in the outer membrane by direct and cyclic binding of TonB to the transporters. A PMF-dependent TonB-ExbD interaction is prevented by 10-residue deletions within a periplasmic disordered domain of ExbD adjacent to the cytoplasmic membrane. Here, we explored the function of the ExbD disordered domain in more detail. In vivo photo-cross-linking through sequential pBpa substitutions in the ExbD disordered domain captured five different ExbD complexes, some of which had been previously detected using in vivo formaldehyde cross-linking, a technique that lacks the residue-specific information that can be achieved through photo-cross-linking: two ExbB-ExbD heterodimers (one of which had not been detected previously), previously detected ExbD homodimers, previously detected PMF-dependent ExbD-TonB heterodimers, and for the first time, a predicted, ExbD-TonB PMF-independent interaction. The fact that multiple complexes were captured by the same pBpa substitution indicated the dynamic nature of ExbD interactions as the energy transduction cycle proceeded in vivo. In this study, we also discovered that a conserved motif—V45, V47, L49, and P50—within the disordered domain was required for signal transduction to TonB and to the C-terminal domain of ExbD and was the source of motif essentiality. IMPORTANCE The TonB system is a virulence factor for Gram-negative pathogens. The mechanism by which cytoplasmic membrane proteins of the TonB system transduce an electrochemical gradient into mechanical energy is a long-standing mystery. TonB, ExbB, and ExbD primary amino acid sequences are characterized by regions of predicted intrinsic disorder, consistent with a proposed multiplicity of protein-protein contacts as TonB proceeds through an energy transduction cycle, a complex process that has yet to be recapitulated in vitro. This study validates a region of intrinsic disorder near the ExbD transmembrane domain and identifies an essential conserved motif embedded within it that transduces signals to distal regions of ExbD suggested to configure TonB for productive interaction with outer membrane transporters.

Antibiotic Conjugates with an Artificial MECAM-Based Siderophore Are Potent Agents against Gram-Positive and Gram-Negative Bacterial Pathogens

“…The causal role of this mutation was proven by the observation that a complementation of ExbB Q163*, but not wild type of ExbB, into a Δ exbB strain conferred resistance to 27 . Previous studies showed that ExbB is an integral cytoplasmic membrane (CM) protein with three transmembrane domains. , It is believed that ExbB has three functions: as a scaffold on stabilizing the structure of Ton machinery, supplier of proton motive force (PMF) for conformational changes of TonB and the associated outer membrane receptor, and signal transduction. , ExbB Q163* is a truncated form of ExbB lacking the third transmembrane domain (TMD3) and the cytoplasmic carboxy terminus. By site-directed mutagenesis, Baker et al found key residues located in the three TMDs of ExbB and proposed that TMD 1 mainly interacts with the TMD of TonB, TMD 2 interacts with ExbD, whereas TMD 3 is involved in signal transduction.…”

“…Previous studies showed that ExbB is an integral cytoplasmic membrane (CM) protein with three transmembrane domains. 46,51 It is believed that ExbB has three functions: as a scaffold on stabilizing the structure of Ton machinery, supplier of proton motive force (PMF) for conformational changes of TonB and the associated outer membrane receptor, and signal transduction. 46,47 ExbB Q163* is a truncated form of ExbB lacking the third transmembrane domain (TMD3) and the cytoplasmic carboxy terminus.…”

The Ton Motor

“…Recent studies have highlighted the importance of the disordered, periplasmic linker domain of Ec ExbD. A conserved motif just upstream of the TM domain of E cExbD, V45, V47, L49, and P50 was found to be required for Ton function ( Kopp and Postle, 2020 ). The TonB periplasmic linker is long enough to allow the C-terminal folded domain of TonB to reach the TBDTs in the OM.…”

Primary role of the Tol-Pal complex in bacterial outer membrane lipid homeostasis

“…Both systems are also hijacked by bacteriocins for cell entry. Even though ExbBD-TonB does not display preference for the septal region (45), some degree of cross complementation has been reported between ExbBD and TolQR (46)(47)(48)(49)(50), indicative of possible interchangeability across the IM subcomplexes. In this work, we engineer a chimeric version of TonB-TolA that can work with the ExbBD subcomplex in Escherichia coli, giving rise to an assembled IM complex that no longer localizes specifically to the cell division site.…”