real-time-measurements-membrane-proteinreceptor-interactions-using

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

The Technion-Israel Institute of Technology

In all kingdoms of life, ATP Binding Cassette (ABC) transporters participate in many physiological and pathological processes. Despite the diversity of their functions, they have been considered to operate by a largely conserved mechanism. One deviant is the vitamin B12 transporter BtuCD that has been shown to operate by a distinct mechanism. However, it is unknown if this deviation is an exotic example, perhaps arising from the nature of the transported moiety. Here we compared two ABC importers of identical substrate specificity (molybdate/tungstate), and find that their interactions with their substrate binding proteins are utterly different. One system forms a high-affinity, slow-dissociating complex that is destabilized by nucleotide and substrate binding. The other forms a low-affinity, transient complex that is stabilized by ligands. The results highlight significant mechanistic divergence among ABC transporters, even when they share the same substrate specificity. We propose that these differences are correlated with the different folds of the transmembrane domains of ABC transporters.

Two molybdate/tungstate ABC transporters that interact very differently with their substrate binding proteins.

Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7-/- mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.

Elena Vigonsky

Department of Biochemistry,
The Bruce and Ruth Rappaport Faculty of Medicine,
Department of Biochemistry, The Bruce and Ruth Rappaport Faculty of Medicine

The Technion-Israel Institute of Technology

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Elena Vigonsky

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry,The Bruce and Ruth Rappaport Faculty of Medicine,Department of Biochemistry, The Bruce and Ruth Rappaport Faculty of Medicine

The Technion-Israel Institute of Technology

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Department of Biochemistry,
The Bruce and Ruth Rappaport Faculty of Medicine,
Department of Biochemistry, The Bruce and Ruth Rappaport Faculty of Medicine