Novel variants provide differential stabilisation of human equilibrative nucleoside transporter 1 states.

Human equilibrative nucleoside transporters represent a major pharmaceutical target for cardiac, cancer and viral therapies. Understanding the molecular basis for transport is crucial for the development of improved therapeutics through structure-based drug design. ENTs have been proposed to utilise an alternating access mechanism of action, similar to that of the major facilitator superfamily.

Thermostability Assays: a Generic and Versatile Tool for Studying the Functional and Structural Properties of Membrane Proteins in Detergents.

There are very few generic methods to assess the stability and functional properties of membrane proteins solubilized in detergent. For this purpose, a thiol-reactive fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) can be used. An unfolding profile is obtained when the fluorochrome becomes fluorescent on reaction with cysteine residues that have been exposed during thermal denaturation of the protein population.

IMPROvER: the Integral Membrane Protein Stability Selector.

Identifying stabilising variants of membrane protein targets is often required for structure determination. Our new computational pipeline, the Integral Membrane Protein Stability Selector (IMPROvER) provides a rational approach to variant selection by employing three independent approaches: deep-sequence, model-based and data-driven. In silico tests using known stability data, and in vitro tests using three membrane protein targets with 7, 11 and 16 transmembrane helices provided measures of success.

Calcium-regulated mitochondrial ATP-Mg/P carriers evolved from a fusion of an EF-hand regulatory domain with a mitochondrial ADP/ATP carrier-like domain.

The mitochondrial ATP-Mg/P carrier is responsible for the calcium-dependent regulation of adenosine nucleotide concentrations in the mitochondrial matrix, which allows mitochondria to respond to changing energy requirements of the cell. The carrier is expressed in mitochondria of fungi, plants and animals and belongs to the family of mitochondrial carriers. The carrier is unusual as it consists of three separate domains: (i) an N-terminal regulatory domain with four calcium-binding EF-hands similar to calmodulin, (ii) a loop domain containing an amphipathic α-helix and (iii) a mitochondrial carrier domain related to the mitochondrial ADP/ATP carrier.

Defining Dynamics of Membrane-Bound Pyrophosphatases by Experimental and Computational Single-Molecule FRET.

Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular animals, making them a viable target for drug design against protozoan parasites such as Plasmodium falciparum. An excellent understanding of many of the catalytic states throughout the enzymatic cycle has already been afforded by crystallography.

A Simple Strategy to Determine the Dependence of Membrane-Bound Pyrophosphatases on K as a Cofactor.

Membrane-bound pyrophosphatases (mPPases) couple pyrophosphate hydrolysis to H and/or Na pumping across membranes and are found in all domains of life except for multicellular animals including humans. They are important for development and stress resistance in plants. Furthermore, mPPases play a role in virulence of human pathogens that cause severe diseases such as malaria and African sleeping sickness.

Calcium regulation of the human mitochondrial ATP-Mg/Pi carrier SLC25A24 uses a locking pin mechanism.

Mitochondrial ATP-Mg/Pi carriers import adenine nucleotides into the mitochondrial matrix and export phosphate to the cytosol. They are calcium-regulated to control the size of the matrix adenine nucleotide pool in response to cellular energetic demands. They consist of three domains: an N-terminal regulatory domain containing four calcium-binding EF-hands, a linker loop domain with an amphipathic α-helix and a C-terminal mitochondrial carrier domain for the transport of substrates.

Insights into the mechanism of membrane pyrophosphatases by combining experiment and computer simulation.

Membrane-integral pyrophosphatases (mPPases) couple the hydrolysis of pyrophosphate (PP) to the pumping of Na, H, or both these ions across a membrane. Recently solved structures of the Na-pumping mPPase (TmPPase) and H-pumping mPPase revealed the basis of ion selectivity between these enzymes and provided evidence for the mechanisms of substrate hydrolysis and ion-pumping. Our atomistic molecular dynamics (MD) simulations of TmPPase demonstrate that loop 5-6 is mobile in the absence of the substrate or substrate-analogue bound to the active site, explaining the lack of electron density for this loop in resting state structures.

Calcium-induced conformational changes in the regulatory domain of the human mitochondrial ATP-Mg/Pi carrier.

The mitochondrial ATP-Mg/Pi carrier imports adenine nucleotides from the cytosol into the mitochondrial matrix and exports phosphate. The carrier is regulated by the concentration of cytosolic calcium, altering the size of the adenine nucleotide pool in the mitochondrial matrix in response to energetic demands. The protein consists of three domains; (i) the N-terminal regulatory domain, which is formed of two pairs of fused calcium-binding EF-hands, (ii) the C-terminal mitochondrial carrier domain, which is involved in transport, and (iii) a linker region with an amphipathic α-helix of unknown function.