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Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins <em>In Vivo</em>

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

Universität Osnabrück

The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK(2)) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK(2) complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a V(max) of 1.25 micromol P(i)/min/mg and a K(m) of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIA(Glc), a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes.

Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK(2)) of Salmonella typhimurium.

We previously characterized a defective-folding variant of the periplasmic maltose-binding protein, MalE31. To examine the alternative folding pathways open to the MalE31 precursor, we have analyzed the cellular fates of this aggregation-prone protein carrying altered signal sequences. Our results are most easily interpreted by a kinetic competition between exportation, folding, and degradation.

Folding and aggregation of export-defective mutants of the maltose-binding protein.

We previously characterized a defective-folding mutant of maltose-binding protein of Escherichia coli, MalE31, which formed periplasmic inclusion bodies. Here, we show that MalE31 aggregation does not affect bacterial growth at 30 degrees C but is lethal at 37 degrees C. Surprisingly, under mild heat shock conditions at 42 degrees C, inclusion bodies are degraded and bacterial growth is restored. One physiological consequence for the cells overproducing MalE31 was to induce an extracytoplasmic stress response by increasing the expression of the heat shock protease DegP via the CpxA/CpxR two-component signalling pathway. Furthermore, we show that the Cpx response is required to rescue the cells from the toxicity mediated by MalE31. Finally, expression of highly destabilized MalE variants that do not aggregate in the periplasm also induces the Cpx pathway, indicating that inclusion body formation is not necessary to activate this specific extracytoplasmic stress regulatory system.

Temperature effect on inclusion body formation and stress response in the periplasm of Escherichia coli.

In Escherichia coli the Cpx sensor regulator system senses different kinds of envelope stress and responds by triggering the expression of periplasmic folding factors and proteases. It consists of the membrane-anchored sensor kinase CpxA, the response regulator CpxR, and the periplasmic protein CpxP. The Cpx pathway is induced in vivo by a variety of signals including pH variation, osmotic stress, and misfolded envelope proteins and is inhibited by overproduced CpxP. Because it is not clear how the Cpx pathway is able to recognize and correspond to so many different signals we overproduced, solubilized, purified, and incorporated the complete membrane-integral CpxA protein into proteoliposomes to analyze its biochemical properties in more detail. Autokinase and phosphotransfer activities of the reconstituted CpxA-His6 protein were stimulated by KCl. NaCl also stimulated the activities but to a lesser extent. Other osmotic active solutes as glycine betaine, sucrose, and proline had no effect. The system was further characterized by testing for susceptibility to sensor kinase inhibitors. Among these, Closantel inhibited the activities of solubilized but not of the reconstituted CpxA-His6 protein. We further analyzed the effect of CpxP on CpxA activities. Purified tagless CpxP protein reduced the phosphorylation status of CpxA to 50% but had no effect on CpxA phosphotransfer or phosphatase activities. As the in vitro system excludes the involvement of other factors our finding is the first biochemical evidence for direct protein-protein interaction between the sensor kinase CpxA and the periplasmic protein CpxP resulting in a down-regulation of the autokinase activity of CpxA.

Purification, reconstitution, and characterization of the CpxRAP envelope stress system of Escherichia coli.

In Escherichia coli, the CpxRA sensor regulator system is induced by a variety of signals, including pH, osmolarity, metals and misfolded envelope proteins. Here, we analyzed the effect of the folding defective maltose binding protein MalE219 on the reconstituted Cpx signalling pathway in detail. Surprisingly, autokinase and phosphatase activities of the reconstituted CpxA-6His protein remained unaffected, whereas phosphotransfer to CpxR became activated by MalE219. Since stimulation occurred only in CpxA-containing proteoliposomes, our data provide the first biochemical indication of allosteric stimulation due to direct contact between MalE219 and the sensor kinase CpxA. Consequently, we suggest that this direct interaction is a new mechanism enabling the Cpx pathway to sense misfolded proteins.

Misfolded maltose binding protein MalE219 induces the CpxRA envelope stress response by stimulating phosphoryl transfer from CpxA to CpxR.

Bacteria are equipped with two-component systems to cope with environmental changes, and auxiliary proteins provide response to additional stimuli. The Cpx two-component system is the global modulator of cell envelope stress in gram-negative bacteria that integrates very different signals and consists of the kinase CpxA, the regulator CpxR, and the dual function auxiliary protein CpxP. CpxP both inhibits activation of CpxA and is indispensable for the quality control system of P pili that are crucial for uropathogenic Escherichia coli during kidney colonization. How these two essential biological functions of CpxP are linked is not known. Here, we report the crystal structure of CpxP at 1.45 Å resolution with two monomers being interdigitated like "left hands" forming a cap-shaped dimer. Our combined structural and functional studies suggest that CpxP inhibits the kinase CpxA through direct interaction between its concave polar surface and the negatively charged sensor domain on CpxA. Moreover, an extended hydrophobic cleft on the convex surface suggests a potent substrate recognition site for misfolded pilus subunits. Altogether, the structural details of CpxP provide a first insight how a periplasmic two-component system inhibitor blocks its cognate kinase and is released from it.

Structural basis for two-component system inhibition and pilus sensing by the auxiliary CpxP protein.

In Gram-negative bacteria, the Cpx signal transduction pathway (CpxAR) has an overall regulatory function, but the mechanism of its activation is still poorly understood. Here we investigated the impact of the periplasmic sensor domain of CpxA on signalling by mutational analysis in vitro. Substitutions (R33C and L38P) in the N-terminal region significantly increased CpxA autophosphorylation, whereas a substitution in a predicted beta sheet (E92K), which impaired the inhibitory effect of the auxiliary CpxP protein, had no significant effect on catalytic activity. Thus, our data suggest different regions on the periplasmic domain of CpxA, which impact to modulate signal transduction.

Different regulatory regions are located on the sensor domain of CpxA to fine-tune signal transduction.

Membrane proteins are crucial for many essential cellular processes. As membrane proteins function in complexes, methods to detect and to characterize membrane protein-protein interactions are undoubtedly required. Therefore, we developed the "Membrane-Strep-tagged protein interaction experiment" (Membrane-SPINE) that combines the specific purification of a Strep-tagged membrane protein with the reversible fixation of protein complexes by formaldehyde cross-linking. In combination with MS analysis, we suggest Membrane-SPINE as a powerful tool to identify unknown interaction partners of membrane proteins in vivo.

Membrane-SPINE: an improved method to identify protein-protein interaction partners of membrane proteins in vivo.

The Cpx-envelope stress system coordinates the expression and assembly of surface structures important for the virulence of Gram-negative pathogenic bacteria. It is comprised of the membrane-anchored sensor kinase CpxA, the cytosolic response regulator CpxR and the accessory protein CpxP. Characteristic of the group of two-component systems, the Cpx system responds to a broad range of stimuli including pH, salt, metals, lipids and misfolded proteins that cause perturbation in the envelope. Moreover, the Cpx system has been linked to inter-kingdom signalling and bacterial cell death. However, although signal specificity has been assumed, for most signals the mechanism of signal integration is not understood. Recent structural and functional studies provide the first insights into how CpxP inhibits CpxA and serves as sensor for misfolded pilus subunits, pH and salt. Here, we summarize and reflect on the current knowledge on signal integration by the Cpx-envelope stress system.

Sabine Hunke

Molekulare Mikrobiologie

Universität Osnabrück

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo