Peter C. Maloney

Bioenergetics and Solute Transport in Lactococci

During the last few years the studies about the physiology and bioenergetics of lactic acid bacteria during growth and starvation have evolved from a descriptive level to an analysis of the molecular events in the regulation of various processes. Considerable progress has been made in the understanding of the modes of metabolic energy generation, the mechanism of homeostasis of the internal pH, and the mechanism and regulatory processes of transport systems for sugars, amino acids, peptides, and ions. Detailed studies of these transport processes have been performed in cytoplasmic membrane vesicles of these organisms in which a foreign proton pump has been introduced to generate a high proton motive force.

Structure and dynamics of NBD1 from CFTR characterized using crystallography and hydrogen/deuterium exchange mass spectrometry.

The DeltaF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and DeltaF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because DeltaF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and DeltaF508 constructs, and the DeltaF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide (1)H/(2)H exchange rates in matched F508 and DeltaF508 constructs reveal that DeltaF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the DeltaF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-DeltaF508 structures but completely solvent exposed in all DeltaF508 structures. These results reinforce the importance of the perturbation DeltaF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.

Energy coupling to ATP synthesis by the proton-translocating ATPase

SummaryThis review summarizes recent work on energy coupling to ATP synthesis by the reversible, proton-translocating ATPase of mitochondria, chloroplasts, and bacteria. In the first sections, this enzyme is distinguished from other ATP-linked ion transport systems, and progress in the biochemical analysis is discussed. There is at present a reasonably consistent idea of the overall structure of the enzyme, and one can begin to assign specific functional roles to individual subunits of the complex. The latter half of this review deals with mechanisms of energy coupling, about which there is clear divergence of opinion. An “indirect coupling” model would allow for the possibility that H+ translocation transmits energy for ATP synthesis by driving the enzyme through a sequence of conformational states, so that H+ translocated need not take part in the chemistry of ATP synthesis. By contrast, a “direct coupling” mechanism would specify that H+ translocated must participate in the chemical reaction by combining with oxygen from phosphate during the synthetic step. Such discussion is preceded by an outline of the “proton well”, since this idea forms the basis of one direct coupling model. In addition, it is suggested that the idea of a proton (ion) well may be of more general significance to the analysis of ion-coupled transport, because it includes the postulate that mechanistically significant ion binding can occur within the profile of the electric field. A proton (ion) well can be derived from both kinetic and equilibrium treatments, and from mechanistic considerations in fields as distinct as biochemistry and neurophysiology. As a result, it illustrates how further advances in formulating mechanisms of energy coupling might profit by a merger of technique and perspective from areas that have as a common goal an understanding of how large proteins catalyze movements of small molecules across a membrane.

Functional reconstitution of prokaryote and eukaryote membrane proteins.

A new strategy for the functional reconstitution of membrane proteins is described. This approach introduces a new class of protein stabilizing agents--osmolytes--whose presence at high concentration (10-20%) during detergent solubilization prevents the inactivations that normally occur when proteins are extracted from natural membranes. Osmolytes that act in this way include compounds such as glycerol and higher polyols (erythritol, xylitol, sorbitol), sugars (glucose, trehalose), and certain amino acids (glycine, proline, betaine). The beneficial effects of osmolytes are documented by reconstitution of a variety of prokaryote and eukaryote membrane proteins, including several proton- and calcium-motive ATPases, cation- and anion-linked solute carriers (symport and antiport), and a membrane-bound hydrolase from endoplasmic reticulum. In all cases, the presence of 20% glycerol or other osmolyte during detergent solubilization led to 10-fold or more increased specific activity in proteoliposomes. These positive effects did not depend on use of any specific detergent for protein solubilization, nor on any particular method of reconstitution, but for convenience most of the work reported here has used octylglucoside as the solubilizing agent, followed by detergent-dilution to form proteoliposomes. The overall approach outlined by these experiments is simple and flexible. It is now feasible to use reconstitution as an analytical tool to study the biochemical and physiological properties of membrane proteins.

Gout-causing Q141K mutation in ABCG2 leads to instability of the nucleotide-binding domain and can be corrected with small molecules

The multidrug ATP-binding cassette, subfamily G, 2 (ABCG2) transporter was recently identified as an important human urate transporter, and a common mutation, a Gln to Lys substitution at position 141 (Q141K), was shown to cause hyperuricemia and gout. The nature of the Q141K defect, however, remains undefined. Here we explore the Q141K ABCG2 mutation using a comparative approach, contrasting it with another disease-causing mutation in an ABC transporter, the deletion of Phe-508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR). We found, much like in ΔF508 CFTR, that the Q141K mutation leads to instability in the nucleotide-binding domain (NBD), a defect that translates to significantly decreased protein expression. However, unlike the CFTR mutant, the Q141K mutation does not interfere with the nucleotide-binding domain/intracellular loop interactions. This investigation has also led to the identification of critical residues involved in the protein–protein interactions necessary for the dimerization of ABCG2: Lys-473 (K473) and Phe-142 (F142). Finally, we have demonstrated the utility of using small molecules to correct the Q141K defect in expression and function as a possible therapeutic approach for hyperuricemia and gout.

Projection structure and molecular architecture of OxlT, a bacterial membrane transporter.

The major facilitator superfamily (MFS) represents the largest collection of evolutionarily related members within the class of membrane ‘carrier’ proteins. OxlT, a representative example of the MFS, is an oxalate‐transporting membrane protein in Oxalobacter formigenes. From an electron crystallographic analysis of two‐dimensional crystals of OxlT, we have determined the projection structure of this membrane transporter. The projection map at 6 Å resolution indicates the presence of 12 transmembrane helices in each monomer of OxlT, with one set of six helices related to the other set by an approximate internal two‐fold axis. The projection map reveals the existence of a central cavity, which we propose to be part of the pathway of oxalate transport. By combining information from the projection map with related biochemical data, we present probable models for the architectural arrangement of transmembrane helices in this protein superfamily.

The Evolution of Ion Pumps

The earliest problem faced by living cells was an unavoidable trend to swelling and lysis, as extracellular salts and water leaked through a semipermeable plasma membrane enclosing impermeant macromolecules. Two solutions countered this ever-present threat-a rigid cell wall to resist expansion and ion pumps to offset the passive influx with an active efflux. On the premise that ion pumps were the first solution, we have constructed an evolutionary sequence to account for the diversity of ion pumps found today.

Stoichiometry of proton movements coupled to ATP synthesis driven by a pH gradient in Streptococcus lactis.

SummaryAn electrochemical potential difference for H+ was established across the plasma membrane of the anaerobeStreptococcus lactis by addition of sulfuric acid to cells suspended in potassium phosphate at pH 8 along with valinomycin or permeant anions. Subsequent acidification of the cell was measured by the distribution of salicyclic acid. A comparison between cells treated or untreated with the inhibitor N,N′-dicyclohexylcarbodiimide was used to reveal that portion of net proton entry attributable to a direct coupling between H+ inflow and synthesis of ATP catalyzed by the reversible proton-translocating ATPase of this microorganism. When the imposed electrochemical proton gradient was below 180–190 mV, proton entry was at the rate expected of passive flux, for both control cells and cells treated with the ATPase inhibitor. However, at higher driving force acidification of control cells was markedly accelerated, coincident with ATP synthesis, while acidification of cells treated with the inhibitor continued at the rate characteristic of passive inflow. This observed threshold (180–190 mV) was identified as the reversal potential for this H+ “pump”. Parallel measurements showed that the free energy of hydrolysis for ATP in these washed cells was 8.4 kcal/mole (370 mV). The comparison between the reversal (threshold) potential and the free energy of hydrolysis for ATP indicates a stoichiometry of 2 H+/ATP for the coupling of proton movements to ATP formation in bacteria.

Voltage sensitivity of the proton-translocating adenosine 5′-triphosphatase in Streptococcus lactis

The reversible, membrane-bound ATPase of bacteria couples the movements of H’ to the synthesis of ATP [l-4]. Because this reaction is associated with the transfer of charge across a membrane, the bacterial ATPase catalyzes ATP formation when either electrical or chemical (pH) gradients are imposed [1,2,5-71, as does the ATPase of mitochondria and chloroplasts [8-l 11. In principle, kinetic responses to these two different driving forces might depend on a variety of factors. Thus, in attempting to decide between many possibilities, it seems important to characterize the synthetic reaction with regard to its voltage and pH sensitivity. Work with submitochondrial particles has shown that imposed electrical or pH gradients can initiate ATP formation at rates comparable to those found during oxidative phosphorylation [I 11. However, the rapid decay of these gradients made it difficult to compare their relative efficiencies in driving the reaction. Similarly, the relevant studies with chloroplasts have been performed under conditions where membrane capacitance was continually charged and discharged [ 121, or where only the pH gradient was measureable, and effects of membrane potential could not be examined [ 13,141. Here it has been possible to explore this topic using the anaerobic bacterium, Streptococcus Zactis. The results indicate that membrane potentials and pH gradients of equal thermodynamic value elicit identical rates of ATP formation. This suggests that inward moving protons can interact with the ATPase in a productive manner only after the full profile of the electric field has been crossed. Moreover, these results appear to exclude limiting versions of models for energy coupling in which membrane potential and pH

Identification and functional reconstitution of phosphate: Sugar phosphate antiport of Staphylococcus aureus

SummaryResting cells ofStaphylococcus aureus displayed a phosphate (Pi) exchange that was induced by growth with glucose 6-phosphate (G6P) orsn-glycerol 3-phosphate (G3P). Pi-loaded membrane vesicles from these cells accumulated32Pi, 2-deoxyglucose 6-phosphate (2DG6P) or G3P by an electroneutral exchange that required no external source of energy. On the other hand, when vesicles were loaded with morpholinopropane sulfonic acid (MOPS), only transport of32Pi (andl-histidine) was observed, and in that case transport depended on addition of an oxidizable substrate (dl-lactate). In such MOPS-loaded vesicles, accumulation of the organic phosphates, 2DG6P and G3P, could not be observed until vesicles were preincubated with both Pi anddl-lactate to establish an internal pool of Pi. Thistrans effect demonstrates that movement of 2DG6P or G3P is based on an antiport (exchange) with internal Pi.Reconstitution of membrane protein allowed a quantitative analysis of Pi-linked exchange. Pi-loaded proteoliposomes and membrane vesicles had comparable activities for the homologous32Pi∶Pi exchange (Kis of 2.2 and 1.4mm;Vmaxs of 180 and 83 nmol Pi/min per mg protein), indicating that the exchange reaction was recovered intact in the artificial system. Other work showed that heterologous exchange from either G6P- or G3P-grown cells had a preference for 2DG6P (Ki=27 μm) over G3P (Ki=1.3mm) and Pi (Ki=2.2mm), suggesting that the same antiporter was induced in both cases. We conclude that32Pi∶Pi exchange exhibited by resting cells reflects operation of an antiporter with high specificity for sugar 6-phosphate. In this respect, Pi-linked antiport inS. aureus resembles other examples in a newly described family of bacterial transporters that use anion exchange as the molecular basis of solute transport.