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Crystallographic studies on the outer membrane heme receptor HasR from Serratia marcescens

Crystallographic studies on the outer membrane heme receptor HasR from Serratia marcescens

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BECKER, Stefanie, 2012. Crystallographic studies on the outer membrane heme receptor HasR from Serratia marcescens [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Becker2012Cryst-20814, title={Crystallographic studies on the outer membrane heme receptor HasR from Serratia marcescens}, year={2012}, author={Becker, Stefanie}, address={Konstanz}, school={Universität Konstanz} }

eng 2012 deposit-license Becker, Stefanie Becker, Stefanie The work presented here is focused on crystallographic studies on the outer membrane receptor HasR from Serratia marcescens. S. marcescens is an opportunistic pathogen which has become one of the prevalent organisms causing hospital-acquired infections. One of its main virulence factors is its ability to acquire iron from its host, primarily in form of heme. The receptor HasR can take up free heme or heme bound to the hemophore HasA, a soluble heme-binding protein secreted by S. marcescens. HasA can acquire heme from hemoglobin or hemoglobin-haptoglobin. The transport of heme through the outer membrane requires energy, which is provided by the periplasmic protein HasB. Here, HasR was studied alone as well as regarding its interactions with the hemophore HasA and the energy-providing protein HasB. When this work was started, no structures were known for any member of the heme receptor family.<br /><br />In this work, HasR and several mutants were crystallized in complex with HasA and heme and several 3D-structures determined providing insights into different stages of binding of the two proteins and heme transfer from HasA to HasR. Attempts to crystallize HasR alone and in complex with HasB have so far not been successful.<br /><br />HasR consists of a 22-stranded ß-barrel closed by an N-terminal globular domain. The ß-strands are connected by long and flexible extracellular loops and short periplasmic turns. The soluble extracellular hemophore HasA binds to HasR and transfers heme to the receptor. This process of binding and heme transfer occurs spontaneously and does not require energy.<br /><br />Based on the structures and in accord with functional data, we propose a model describing a four-step process of protein-protein binding coupled with heme transfer. In the first step, HasA contacts HasR mainly due to electrostatic interactions and binds in a first transition complex. This complex is seen in a mutant crystal structure. In the second step, the proteins rearrange and HasA closes in on HasR to cover a larger interface. In this step, one of the two heme coordinating loops of HasA becomes disordered, but the heme is still bound to HasA. This step is trapped in another mutant crystal structure. In the third step, a localized steric clash between heme and one residue of HasR triggers the movement of heme to its new binding site on HasR. This is the conformation that is seen in the crystal structure of the wild type complex. After the heme transfer, as a fourth step, a residue of HasA changes its conformation to prevent heme from sliding back to its binding site on HasA, which is confirmed by the HasA-HasR complex without heme. We think that this ensures close to 100% effciency of transfer from HasA to HasR without heme being lost to the environment.<br /><br />This model might be generally valid for ligand transfer from a protein with a higher to one with a lower ligand affnity, as is the case, for example, for receptors interacting directly with host proteins (e.g. hemoglobin, transferrin, lactoferrin).<br /><br />All studies were performed in tight cooperation with several collaborators: Philippe Delepelaire and coworkers in the Bacterial Membranes Unit at the Institut Pasteur in Paris did the genetic, micobiological and biochemical work which provide the functional examinations complementing our structural studies. Nadia Izadi-Pruneyre and coworkers in the Unit of NMR of Biomolecules at the Institut Pasteur in Paris provided NMR studies on HasB and ITC measurements on the interactions between HasB and HasR as well as HasA and HasR. Simon Becker in the group of Kay Diederichs (Fachbereich Biologie, Universität Konstanz) and Thomas Exner (Fachbereich Chemie, Universität Konstanz) performed molecular dynamics simulations based on our structures. 2012-11-07T10:17:32Z Crystallographic studies on the outer membrane heme receptor HasR from Serratia marcescens 2012-11-07T10:17:32Z Kristallographische Untersuchungen des Aussenmembranrezeptors HasR von Serratia marcescens

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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