Assessing the Conformational Changes of pb5, the Receptor-binding Protein of Phage T5, upon Binding to Its Escherichia coli Receptor FhuA

Within tailed bacteriophages, interaction of the receptor-binding protein (RBP) with the target cell triggers viral DNA ejection into the host cytoplasm. In the case of phage T5, the RBP pb5 and the receptor FhuA, an outer membrane protein of Escherichia coli, have been identified. Here, we use small angle neutron scattering and electron microscopy to investigate the FhuA-pb5 complex. Specific deuteration of one of the partners allows the complete masking in small angle neutron scattering of the surfactant and unlabeled proteins when the complex is solubilized in the fluorinated surfactant F₆-DigluM. Thus, individual structures within a membrane protein complex can be described. The solution structure of FhuA agrees with its crystal structure; that of pb5 shows an elongated shape. Neither displays significant conformational changes upon interaction. The mechanism of signal transduction within phage T5 thus appears different from that of phages binding cell wall saccharides, for which structural information is available.     

The Active-Site Cysteines of the Periplasmic Thioredoxin-Like Protein CcmG of Escherichia coli Are Important but Not Essential for Cytochrome c Maturation In Vivo

A new member of the family of periplasmic protein thiol:disulfide oxidoreductases, CcmG (also called DsbE), was characterized with regard to its role in cytochrome c maturation in Escherichia coli. The CcmG protein was shown to be membrane bound, facing the periplasm with its C-terminal, hydrophilic domain. A chromosomal, nonpolar in-frame deletion in ccmG resulted in the complete absence of all c-type cytochromes. Replacement of either one or both of the two cysteine residues of the predicted active site in CcmG (WCPTC) led to low but detectable levels of Bradyrhizobium japonicum holocytochrome c₅₅₀ expressed in E. coli. This defect, but not that of the ccmG null mutant, could be complemented by adding low-molecular-weight thiol compounds to growing cells, which is in agreement with a reducing function for CcmG.     

Identification of a New Site for Ferrichrome Transport by Comparison of the FhuA Proteins of Escherichia coli, Salmonella paratyphi B, Salmonella typhimurium, and Pantoea agglomerans

The fhuA genes of Salmonella paratyphi B, Salmonella typhimurium, and Pantoea agglomerans were sequenced and compared with the known fhuA sequence of Escherichia coli. The highly similar FhuA proteins displayed the largest difference in the predicted gating loop, which in E. coli controls the permeability of the FhuA channel and serves as the principal binding site for the phages T1, T5, and 80. All the FhuA proteins contained the region in the gating loops required in E. coli for ferrichrome and albomycin transport. The three subdomains required for phage binding were contained in the gating loop of S. paratyphi B which is infected by the E. coli phages, whereas two of the subdomains were deleted in S. typhimurium and P. agglomerans which are resistant to the E. coli phages. Small deletions in a surface loop adjacent to the gating loop, residues 236 to 243 and 236 to 248, inactivated E. coli FhuA with regard to transport of ferrichrome and albomycin, but sensitivity to T1 and T5 was fully retained and sensitivity to 80 and colicin M was reduced 10-fold. Full-size FhuA hybrid proteins of S. paratyphi B and S. typhimurium displayed S. paratyphi B FhuA activity when the hybrids contained two-thirds of either the N- or the C-terminal portions of S. paratyphi B and displayed S. typhimurium FhuA activity to phage ES18 when the hybrid contained two-thirds of the N-terminal region of the S. typhimurium FhuA. The central segment of the S. paratyphi B FhuA flanked on both sides by S. typhimurium FhuA regions conferred full sensitivity only to phage T5. The data support the essential role of the gating loop for the transport of ferrichrome and albomycin, identified an additional loop for ferrichrome and albomycin uptake, and suggest that several segments and their proper conformation, determined by the entire FhuA protein, contribute to the multiple FhuA activities.     

Site-directed Fluorescence Labeling Reveals a Revised N-terminal Membrane Topology and Functional Periplasmic Residues in the Escherichia coli Cell Division Protein FtsK

In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsK_N) is essential for division, and the C terminus (FtsK_C) is a well characterized DNA translocase. Although the function of FtsK_N is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsK_N and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsK_N is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsK_N was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsK_N also provides an important platform for future studies on essential interactions required for this process.     

Experimental Validation of the Predicted Binding Site of Escherichia coli K1 Outer Membrane Protein A to Human Brain Microvascular Endothelial Cells: IDENTIFICATION OF CRITICAL MUTATIONS THAT PREVENT E. COLI MENINGITIS*

Escherichia coli K1, the most common cause of meningitis in neonates, has been shown to interact with GlcNAc1–4GlcNAc epitopes of Ecgp96 on human brain microvascular endothelial cells (HBMECs) via OmpA (outer membrane protein A). However, the precise domains of extracellular loops of OmpA interacting with the chitobiose epitopes have not been elucidated. We report the loop-barrel model of these OmpA interactions with the carbohydrate moieties of Ecgp96 predicted from molecular modeling. To test this model experimentally, we generated E. coli K1 strains expressing OmpA with mutations of residues predicted to be critical for interaction with the HBMEC and tested E. coli invasion efficiency. For these same mutations, we predicted the interaction free energies (including explicit calculation of the entropy) from molecular dynamics (MD), finding excellent correlation (R² = 90%) with experimental invasion efficiency. Particularly important is that mutating specific residues in loops 1, 2, and 4 to alanines resulted in significant inhibition of E. coli K1 invasion in HBMECs, which is consistent with the complete lack of binding found in the MD simulations for these two cases. These studies suggest that inhibition of the interactions of these residues of Loop 1, 2, and 4 with Ecgp96 could provide a therapeutic strategy to prevent neonatal meningitis due to E. coli K1.