Native membrane proteins vs. yeast recombinant: an example: the mitochondrial ADP/ATP carrier.

The mitochondrial ADP/ATP carrier (Ancp) has long been a paradigm for studies of the mitochondrial carrier family due to, among other properties, its natural abundance and the existence of specific inhibitors, namely, carboxyatractyloside (CATR) and bongkrekic acid (BA), which lock the carrier under distinct and stable conformations. Bovine Anc1p isolated in complex with CATR in the presence of an aminoxyde detergent (LAPAO) was crystallized and its 3D structure determined. It is the first mitochondrial carrier structure resolved at high resolution (2.

PBP active site flexibility as the key mechanism for beta-lactam resistance in pneumococci.

Penicillin-binding proteins (PBPs), the main targets of beta-lactam antibiotics, are membrane-associated enzymes that catalyze the two last steps in the biosynthesis of peptidoglycan. In Streptococcus pneumoniae, a major human pathogen, the surge in resistance to such antibiotics is a direct consequence of the proliferation of mosaic PBP-encoding genes, which give rise to proteins containing tens of mutations. PBP2b is a major drug resistance target, and its modification is essential for the development of high levels of resistance to piperacillin.

Molecular, functional, and pathological aspects of the mitochondrial ADP/ATP carrier.

In providing the cell with ATP generated by oxidative phosphorylation, the mitochondrial ADP/ATP carrier plays a central role in aerobic eukaryotic cells. Combining biochemical, genetic, and structural approaches contributes to understanding the molecular mechanism of this essential transport system, the dysfunction of which is implicated in neuromuscular diseases.

Relations between structure and function of the mitochondrial ADP/ATP carrier.

Import and export of metabolites through mitochondrial membranes are vital processes that are highly controlled and regulated at the level of the inner membrane. Proteins of the mitochondrial carrier family ( MCF ) are embedded in this membrane, and each member of the family achieves the selective transport of a specific metabolite. Among these, the ADP/ATP carrier transports ADP into the mitochondrial matrix and exports ATP toward the cytosol after its synthesis.

Conformation-dependent swinging of the matrix loop m2 of the mitochondrial Saccharomyces cerevisiae ADP/ATP carrier.

Structure-function relationships of the membrane-embedded Saccharomyces cerevisiae mitochondrial ADP/ATP carrier were investigated through two independent approaches, namely, limited proteolysis and cysteine labeling. Experiments were carried out in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors of the ADP/ATP transport that bind to two distinct conformers involved in the translocation process. The proteolysis approach allowed us to demonstrate (i) that N- and C-terminal extremities of ADP/ATP carrier are facing the intermembrane space and (ii) that the central region of the carrier corresponding to the matrix loop m2 is accessible to externally added trypsin in a conformation-sensitive manner, being cleaved at the Lys163-Gly164 and Lys178-Thr179 bonds in the carrier-CATR and the carrier-BA complexes, respectively.

Structural basis for lipid-mediated interactions between mitochondrial ADP/ATP carrier monomers.

The oligomerization state of the ADP/ATP carrier is an important issue in understanding the mechanism underlying nucleotide exchange across the inner mitochondrial membrane. The first high resolution structure obtained in the presence of carboxyatractyloside revealed a large cavity formed within a monomer in which the inhibitor is strongly bound. Whereas the protein-protein interactions implicated in the first crystal form are not biologically relevant, the new crystal form described herein, highlights favorable protein-protein interactions.

Functional characterization and purification of a Saccharomyces cerevisiae ADP/ATP carrier-iso 1 cytochrome c fusion protein.

A recombinant fusion protein combining the mitochondrial ADP/ATP carrier (Anc2p) and the iso-1-cytochrome c (Cyc1p), both from Saccharomyces cerevisiae, has been genetically elaborated with the aim of increasing the polar surface area of the carrier to facilitate its crystallization. The gene encoding the his-tagged fusion protein was expressed in yeast under the control of the regulatory sequences of ScANC2. The chimeric carrier, Anc2-Cyc1(His6)p, was able to restore growth on a non-fermentable carbon source of a yeast strain devoid of functional ADP/ATP carrier, which demonstrated its transport activity.

Crystallization of the bovine ADP/ATP carrier is critically dependent upon the detergent-to-protein ratio.

The ADP/ATP carrier is an integral membrane protein located in the mitochondrial inner membrane. It mediates the exchange of cytosolic ADP for ATP generated in the mitochondrial matrix. Here, the purification and crystallization of the bovine ADP/ATP carrier in complex with the inhibitor carboxyatractyloside in the presence of LAPAO, an aminoxide detergent, is reported.

Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside.

ATP, the principal energy currency of the cell, fuels most biosynthetic reactions in the cytoplasm by its hydrolysis into ADP and inorganic phosphate. Because resynthesis of ATP occurs in the mitochondrial matrix, ATP is exported into the cytoplasm while ADP is imported into the matrix. The exchange is accomplished by a single protein, the ADP/ATP carrier.