Steve W. Lockless

Structure of a Membrane-Embedded Prenyltransferase Homologous to UBIAD1.

A crystal structure of a member of the UbiA family of membrane-embedded prenyltransferases reveals the architecture of the active site and suggests a possible mechanism for catalysis.

Ion-binding properties of a K+ channel selectivity filter in different conformations.

Significance The selectivity filter of K+ channels is responsible for their exquisite ion selectivity. This region is also responsible for C-type inactivation, a regulatory process in many voltage-dependent K+ channels. Although the functional properties of inactivated channels have been known for decades, the first potential glimpse of their structure emerged from crystal structures of a constricted selectivity filter in an open channel. However, recent studies challenged the suggestion that the constricted selectivity filter is the inactivated structure, leaving open the question of what the inactivated structure looks like. Here, we provide evidence that the thermodynamic properties of the selectivity filter in an inactivated channel are more similar to properties of the conductive channel rather than the constricted open channel. K+ channels are membrane proteins that selectively conduct K+ ions across lipid bilayers. Many voltage-gated K+ (KV) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K+ channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to KV channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na+ or low [K+]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K+ ion binding to channels with an inactivated or conductive selectivity filter is different from K+ ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.

Live Imaging of Inorganic Phosphate in Plants with Cellular and Subcellular Resolution

Genetically encoded sensors enable dynamic monitoring of phosphate concentrations in cells and cell compartments of live plants. Despite variable and often scarce supplies of inorganic phosphate (Pi) from soils, plants must distribute appropriate amounts of Pi to each cell and subcellular compartment to sustain essential metabolic activities. The ability to monitor Pi dynamics with subcellular resolution in live plants is, therefore, critical for understanding how this essential nutrient is acquired, mobilized, recycled, and stored. Fluorescence indicator protein for inorganic phosphate (FLIPPi) sensors are genetically encoded fluorescence resonance energy transfer-based sensors that have been used to monitor Pi dynamics in cultured animal cells. Here, we present a series of Pi sensors optimized for use in plants. Substitution of the enhanced yellow fluorescent protein component of a FLIPPi sensor with a circularly permuted version of Venus enhanced sensor dynamic range nearly 2.5-fold. The resulting circularly permuted FLIPPi sensor was subjected to a high-efficiency mutagenesis strategy that relied on statistical coupling analysis to identify regions of the protein likely to influence Pi affinity. A series of affinity mutants was selected with dissociation constant values of 0.08 to 11 mm, which span the range for most plant cell compartments. The sensors were expressed in Arabidopsis (Arabidopsis thaliana), and ratiometric imaging was used to monitor cytosolic Pi dynamics in root cells in response to Pi deprivation and resupply. Moreover, plastid-targeted versions of the sensors expressed in the wild type and a mutant lacking the PHOSPHATE TRANSPORT4;2 plastidic Pi transporter confirmed a physiological role for this transporter in Pi export from root plastids. These circularly permuted FLIPPi sensors, therefore, enable detailed analysis of Pi dynamics with subcellular resolution in live plants.

Preferential binding of K+ ions in the selectivity filter at equilibrium explains high selectivity of K+ channels

K+ channels exhibit strong selectivity for K+ ions over Na+ ions based on electrophysiology experiments that measure ions competing for passage through the channel. During this conduction process, multiple ions interact within the region of the channel called the selectivity filter. Ion selectivity may arise from an equilibrium preference for K+ ions within the selectivity filter or from a kinetic mechanism whereby Na+ ions are precluded from entering the selectivity filter. Here, we measure the equilibrium affinity and selectivity of K+ and Na+ ions binding to two different K+ channels, KcsA and MthK, using isothermal titration calorimetry. Both channels exhibit a large preference for K+ over Na+ ions at equilibrium, in line with electrophysiology recordings of reversal potentials and Ba2+ block experiments used to measure the selectivity of the external-most ion-binding sites. These results suggest that the high selectivity observed during ion conduction can originate from a strong equilibrium preference for K+ ions in the selectivity filter, and that K+ selectivity is an intrinsic property of the filter. We hypothesize that the equilibrium preference for K+ ions originates in part through the optimal spacing between sites to accommodate multiple K+ ions within the selectivity filter.

A Clostridium difficile alanine racemase affects spore germination and accommodates serine as a substrate

Clostridium difficile has become one of the most common bacterial pathogens in hospital-acquired infections in the United States. Although C. difficile is strictly anaerobic, it survives in aerobic environments and transmits between hosts via spores. C. difficile spore germination is triggered in response to certain bile acids and glycine. Although glycine is the most effective co-germinant, other amino acids can substitute with varying efficiencies. Of these, l-alanine is an effective co-germinant and is also a germinant for most bacterial spores. Many endospore-forming bacteria embed alanine racemases into their spore coats, and these enzymes are thought to convert the l-alanine germinant into d-alanine, a spore germination inhibitor. Although the C. difficile Alr2 racemase is the sixth most highly expressed gene during C. difficile spore formation, a previous study reported that Alr2 has little to no role in germination of C. difficile spores in rich medium. Here, we hypothesized that Alr2 could affect C. difficile l-alanine-induced spore germination in a defined medium. We found that alr2 mutant spores more readily germinate in response to l-alanine as a co-germinant. Surprisingly, d-alanine also functioned as a co-germinant. Moreover, we found that Alr2 could interconvert l- and d-serine and that Alr2 bound to l- and d-serine with ∼2-fold weaker affinity to that of l- and d-alanine. Finally, we demonstrate that l- and d-serine are also co-germinants for C. difficile spores. These results suggest that C. difficile spores can respond to a diverse set of amino acid co-germinants and reveal that Alr2 can accommodate serine as a substrate.

Determinants of cation transport selectivity: Equilibrium binding and transport kinetics

The crystal structures of channels and transporters reveal the chemical nature of ion-binding sites and, thereby, constrain mechanistic models for their transport processes. However, these structures, in and of themselves, do not reveal equilibrium selectivity or transport preferences, which can be discerned only from various functional assays. In this Review, I explore the relationship between cation transport protein structures, equilibrium binding measurements, and ion transport selectivity. The primary focus is on K+-selective channels and nonselective cation channels because they have been extensively studied both functionally and structurally, but the principles discussed are relevant to other transport proteins and molecules.

Ion Binding to Transport Proteins using Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) is an emerging, label-free technology used to measure ligand binding to membrane proteins. This technology utilizes a titration calorimeter to measure the heat exchange upon ligands binding to proteins, the magnitude of which is based on the overall enthalpy of the reaction. In this protocol, the steps we and others use to measure ion binding to ion transport proteins are described.