Conformational Dynamics in the Binding-Protein-Independent Mutant of the Escherichia coli Maltose Transporter, MalG511, and Its Interaction with Maltose Binding Protein.

MalG511 is a genetically selected binding-protein-independent mutant of the Escherichia coli maltose transporter MalFGK2, which retains specificity for maltose and shows a high basal ATPase activity in the absence of maltose binding protein (MBP). It shows an intriguing biphasic behavior in maltose transport assays in the presence of MBP, with low levels of MBP stimulating the activity and higher levels (>50 µM) inhibiting the transport activity. Remarkably, the rescuing effect of the MBP suppressor mutant, MBPG13D, turns it into an attractive model for studying regulatory mechanisms in the ABC transporter superfamily. It is hypothesized that the special characteristics of MalG511 result from mutations that shift its equilibrium toward the transition state of MalFGK2. We tested this hypothesis by using site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy, which showed conformational changes in MalG511 and its interaction with MBP and MBPG13D during its catalytic cycle. We found that MalG511 utilizes the same alternate access mechanism as MalFGK2, including all three open, semi-open, and closed states of the MalK dimer, to transport maltose across the membrane. However, the equilibrium of this mutant is shifted toward the semi-open state in its resting state and interacts with MBP with high affinity, providing an explanation for the inhibition of MalG511 by MBP at higher concentrations. In contrast, the mutant binding protein, MBPG13D, interacts with lower affinity and could restore MalG511 to a normal catalytic cycle.

Biochemical characterization of essential cell division proteins FtsX and FtsE that mediate peptidoglycan hydrolysis by PcsB in Streptococcus pneumoniae.

The FtsEX:PcsB complex forms a molecular machine that carries out peptidoglycan (PG) hydrolysis during normal cell division of the major respiratory pathogenic bacterium, Streptococcus pneumoniae (pneumococcus). FtsX is an integral membrane protein and FtsE is a cytoplasmic ATPase that together structurally resemble ABC transporters. Instead of transport, FtsEX transduces signals from the cell division apparatus to stimulate PG hydrolysis by PcsB, which interacts with extracellular domains of FtsX. Structural studies of PcsB and one extracellular domain of FtsX have recently appeared, but little is known about the biochemical properties of the FtsE ATPase or the intact FtsX transducer protein. We report here purifications and characterizations of tagged FtsX and FtsE proteins. Pneumococcal FtsX-GFP-His and FtsX-His could be overexpressed in Escherichia coli without toxicity, and FtsE-His remained soluble during purification. FtsX-His dimerizes in detergent micelles and when reconstituted in phospholipid nanodiscs. FtsE-His binds an ATP analog with an affinity comparable to that of ATPase subunits of ABC transporters, and FtsE-His preparations have a low, detectable ATPase activity. However, attempts to detect complexes of purified FtsX-His, FtsE-His, and PcsB-His or coexpressed tagged FtsX and FtsE were not successful with the constructs and conditions tested so far. In working with nanodiscs, we found that PcsB-His has an affinity for charged phospholipids, mediated partly by interactions with its coiled-coil domain. Together, these findings represent first steps toward reconstituting the FtsEX:PcsB complex biochemically and provide information that may be relevant to the assembly of the complex on the surface of pneumococcal cells.

Full engagement of liganded maltose-binding protein stabilizes a semi-open ATP-binding cassette dimer in the maltose transporter.

MalFGK2 is an ATP-binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose-binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide-binding subunits (MalK dimer). This MBP-stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent. We found that full engagement of both lobes of maltose-bound MBP unto MalFGK2 is facilitated by nucleotides and stabilizes a semi-open MalK dimer. Maltose-bound MBP promotes the transition to the semi-open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi-open MalK2 conformation by maltose-bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi-open conformation, from which it can proceed to hydrolyze ATP.

Effects of lipid environment on the conformational changes of an ABC importer.

In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.

Small substrate transport and mechanism of a molybdate ATP binding cassette transporter in a lipid environment.

Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. To understand the mechanism of transport in the biological membrane as a whole, the effects of the lipid bilayer on transport needed to be addressed. Continuous wave-electron paramagnetic resonance and in vivo molybdate uptake studies were used to test the impact of the lipid environment on the mechanism and function of MolBC-A. Working with the bacterium Haemophilus influenzae, we found that MolBC-A functions as a low affinity molybdate transporter in its native environment. In periods of high extracellular molybdate concentration, H. influenzae makes use of parallel molybdate transport systems (MolBC-A and ModBC-A) to take up a greater amount of molybdate than a strain with ModBC-A alone. In addition, the movement of the translocation pathway in response to nucleotide binding and hydrolysis in a lipid environment is conserved when compared with in-detergent analysis. However, electron paramagnetic resonance spectroscopy indicates that a lipid environment restricts the flexibility of the MolBC translocation pathway. By combining continuous wave-electron paramagnetic resonance spectroscopy and substrate uptake studies, we reveal details of molybdate transport and the logistics of uptake systems that employ multiple transporters for the same substrate, offering insight into the mechanisms of nutrient uptake in bacteria.

Carbon catabolite repression of the maltose transporter revealed by X-ray crystallography.

Efficient carbon utilization is critical to the survival of microorganisms in competitive environments. To optimize energy usage, bacteria have developed an integrated control system to preferentially uptake carbohydrates that support rapid growth. The availability of a preferred carbon source, such as glucose, represses the synthesis and activities of proteins necessary for the transport and metabolism of secondary carbon sources. This regulatory phenomenon is defined as carbon catabolite repression. In enteric bacteria, the key player of carbon catabolite repression is a component of the glucose-specific phosphotransferase system, enzyme IIA (EIIA(Glc)). It is known that unphosphorylated EIIA(Glc) binds to and inhibits a variety of transporters when glucose is available. However, understanding the underlying molecular mechanism has been hindered by the complete absence of structures for any EIIA(Glc)-transporter complexes. Here we present the 3.9 Å crystal structure of Escherichia coli EIIA(Glc) in complex with the maltose transporter, an ATP-binding cassette (ABC) transporter. The structure shows that two EIIA(Glc) molecules bind to the cytoplasmic ATPase subunits, stabilizing the transporter in an inward-facing conformation and preventing the structural rearrangements necessary for ATP hydrolysis. We also show that the half-maximal inhibitory concentrations of the full-length EIIA(Glc) and an amino-terminal truncation mutant differ by 60-fold, consistent with the hypothesis that the amino-terminal region, disordered in the crystal structure, functions as a membrane anchor to increase the effective EIIA(Glc) concentration at the membrane. Together these data suggest a model of how the central regulatory protein EIIA(Glc) allosterically inhibits maltose uptake in E. coli.

EPR spectroscopy of MolB2C2-a reveals mechanism of transport for a bacterial type II molybdate importer.

In bacteria, ATP-binding cassette (ABC) transporters are vital for the uptake of nutrients and cofactors. Based on differences in structure and activity, ABC importers are divided into two types. Type I transporters have been well studied and employ a tightly regulated alternating access mechanism. Less is known about Type II importers, but much of what we do know has been observed in studies of the vitamin B12 importer BtuC2D2. MolB2C2 (formally known as HI1470/71) is also a Type II importer, but its substrate, molybdate, is ∼10-fold smaller than vitamin B12. To understand mechanistic differences among Type II importers, we focused our studies on MolBC, for which alternative conformations may be required to transport its relatively small substrate. To investigate the mechanism of MolBC, we employed disulfide cross-linking and EPR spectroscopy. From these studies, we found that nucleotide binding is coupled to a conformational shift at the periplasmic gate. Unlike the larger conformational changes in BtuCD-F, this shift in MolBC-A is akin to unlocking a swinging door: allowing just enough space for molybdate to slip into the cell. The lower cytoplasmic gate, identified in BtuCD-F as "gate I," remains open throughout the MolBC-A mechanism, and cytoplasmic gate II closes in the presence of nucleotide. Combining our results, we propose a peristaltic mechanism for MolBC-A, which gives new insight in the transport of small substrates by a Type II importer.

The ABC transporter MalFGK(2) sequesters the MalT transcription factor at the membrane in the absence of cognate substrate.

MalK, the cytoplasmic component of the maltose ABC transporter from Escherichia coli is known to control negatively the activity of MalT, the activator of the maltose regulon, through complex formation. Here we further investigate this regulatory process by monitoring MalT activity and performing fluorescence microscopy analyses under various conditions. We establish that, under physiological conditions, the molecular entity that interacts with MalT is not free MalK, but the maltose transporter, MalFGK(2) , which sequesters MalT to the membrane. Furthermore, we provide compelling evidence that the transporter's ability to bind MalT is not constitutive, but strongly diminished when MalFGK(2) is engaged in sugar transport. Notably, the outward-facing transporter, i.e. the catalytic intermediate, is ineffective in inhibiting MalT compared to the inward-facing state, i.e. the resting form. Analyses of available genetic and structural data suggest how the interaction between one inactive MalT molecule and MalFGK(2) would be sensitive to the transporter state, thereby allowing MalT release upon maltose entrance. A related mechanism may underpin signalling by other ABC transporters.

ABC solute importers in bacteria.

The bacterial ABC (ATP-binding cassette) importers mediate nutrient uptake and some are essential for survival in environments where nutrients are limited, such as in the human body. Although ABC importers exhibit remarkable versatility in the substrates that they can transport, they appear to share a similar multisubunit architecture and mechanism of energization by ATP hydrolysis. This chapter will provide both basic understanding and up-to-date information on the structure, mechanism and regulation of this important family of proteins.

Dynamics of alpha-helical subdomain rotation in the intact maltose ATP-binding cassette transporter.

ATP-binding cassette (ABC) transporters are powered by a nucleotide-binding domain dimer that opens and closes during cycles of ATP hydrolysis. These domains consist of a RecA-like subdomain and an α-helical subdomain that is specific to the family. Many studies on isolated domains suggest that the helical subdomain rotates toward the RecA-like subdomain in response to ATP binding, moving the family signature motif into a favorable position to interact with the nucleotide across the dimer interface. Moreover, the transmembrane domains are docked into a cleft at the interface between these subdomains, suggesting a putative role of the rotation in interdomain communication. Electron paramagnetic resonance spectroscopy was used to study the dynamics of this rotation in the intact Escherichia coli maltose transporter MalFGK(2). This importer requires a periplasmic maltose-binding protein (MBP) that activates ATP hydrolysis by promoting the closure of the cassette dimer (MalK(2)). Whereas this rotation occurred during the transport cycle, it required not only trinucleotide, but also MBP, suggesting it is part of a global conformational change in the transporter. Interaction of AMP-PNP-Mg(2+) and a MBP that is locked in a closed conformation induced a transition from open MalK(2) to semiopen MalK(2) without significant subdomain rotation. Inward rotation of the helical subdomain and complete closure of MalK(2) therefore appear to be coupled to the reorientation of transmembrane helices and the opening of MBP, events that promote transfer of maltose into the transporter. After ATP hydrolysis, the helical subdomain rotates out as MalK(2) opens, resetting the transporter in an inward-facing conformation.

Uncoupling substrate transport from ATP hydrolysis in the Escherichia coli maltose transporter.

Members of the ATP-binding cassette superfamily couple the energy from ATP hydrolysis to the active transport of substrates across the membrane. The maltose transporter, a well characterized model system, consists of a periplasmic maltose-binding protein (MBP) and a multisubunit membrane transporter, MalFGK(2). On the basis of the structure of the MBP-MalFGK(2) complex in an outward-facing conformation (Oldham, M. L., Khare, D., Quiocho, F. A., Davidson, A. L., and Chen, J. (2007) Nature 450, 515-521), we identified two mutants in transmembrane domains MalF and MalG that generated futile cycling; although interaction with MBP stimulated the ATPase activity of the transporter, maltose was not transported. Both mutants appeared to disrupt the normal transfer of maltose from MBP to MalFGK(2). In the first case, substitution of aspartate for glycine in the maltose-binding site of MalF likely generated a futile cycle by preventing maltose from binding to MalFGK(2) during the catalytic cycle. In the second case, a four-residue deletion of a periplasmic loop of MalG limited its reach into the maltose-binding pocket of MBP, allowing maltose to remain associated with MBP during the catalytic cycle. Retention of maltose in the MBP binding site in the deletion mutant, as well as insertion of this loop into the binding site in the wild type, was detected by EPR as a change in mobility of a nitroxide spin label positioned near the maltose-binding pocket of MBP.

Functional reconstitution of an ABC transporter in nanodiscs for use in electron paramagnetic resonance spectroscopy.

Electron paramagnetic resonance (EPR) spectroscopy is a powerful biophysical technique for study of the structural dynamics of membrane proteins. Many of these proteins interact with ligands or proteins on one or both sides of the membrane. Membrane proteins are typically reconstituted in proteoliposomes to observe their function in a physiologically relevant environment. However, membrane proteins can insert into liposomes in two different orientations, and surfaces facing the lumen of the vesicle can be inaccessible to ligands. This heterogeneity can lead to subpopulations that do not respond to ligand binding, complicating EPR spectral analysis, particularly for distance measurements. Using the well-characterized maltose transporter, an ATP binding cassette (ABC) transporter that interacts with ligands on both sides of the membrane, we provide evidence that reconstitution into nanodiscs, which are soluble disk-shaped phospholipid bilayers, is an ideal solution to these problems. We describe the functional reconstitution of the maltose transporter into nanodiscs and demonstrate that this system is ideally suited to study conformational changes and intramolecular distances by EPR.

Glycosylation regulates prestin cellular activity.

Glycosylation is a common post-translational modification of proteins and is implicated in a variety of cellular functions including protein folding, degradation, sorting and trafficking, and membrane protein recycling. The membrane protein prestin is an essential component of the membrane-based motor driving electromotility changes (electromotility) in the outer hair cell (OHC), a central process in auditory transduction. Prestin was earlier identified to possess two N-glycosylation sites (N163, N166) that, when mutated, marginally affect prestin nonlinear capacitance (NLC) function in cultured cells. Here, we show that the double mutant prestin(NN163/166AA) is not glycosylated and shows the expected NLC properties in the untreated and cholesterol-depleted HEK 293 cell model. In addition, unlike WT prestin that readily forms oligomers, prestin(NN163/166AA) is enriched as monomers and more mobile in the plasma membrane, suggesting that oligomerization of prestin is dependent on glycosylation but is not essential for the generation of NLC in HEK 293 cells. However, in the presence of increased membrane cholesterol, unlike the hyperpolarizing shift in NLC seen with WT prestin, cells expressing prestin(NN163/166AA) exhibit a linear capacitance function. In an attempt to explain this finding, we discovered that both WT prestin and prestin(NN163/166AA) participate in cholesterol-dependent cellular trafficking. In contrast to WT prestin, prestin(NN163/166AA) shows a significant cholesterol-dependent decrease in cell-surface expression, which may explain the loss of NLC function. Based on our observations, we conclude that glycosylation regulates self-association and cellular trafficking of prestin(NN163/166AA). These observations are the first to implicate a regulatory role for cellular trafficking and sorting in prestin function. We speculate that the cholesterol regulation of prestin occurs through localization to and internalization from membrane microdomains by clathrin- and caveolin-dependent mechanisms.

Binding Protein-Dependent Uptake of Maltose into Cells via an ATP-Binding Cassette Transporter.

Maltose and maltodextrins are actively transported across the cytoplasmic membrane of Escherichia coli and Salmonella by a periplasmic binding protein (BP)- dependent transport system. Since 1996, there have been many advances in the understanding of the structure and mechanism of the maltose transporter, in the assembly of the membrane-associated transporter complex, and in the mechanism of regulation of transport both at the DNA and the protein level. The transporter has been studied in detergent and reconstituted in liposome vesicles, and while many features, including the ability of maltose-binding protein (MBP) to stimulate ATPase activity, are retained in detergent, it has been noted that the basal ATPase activity of the transporter is elevated in detergent compared with liposomes. This review focuses on these recent developments, which have culminated in a high resolution structure of MBP in a complex with the MalFGK2 transporter. While this review focuses on the maltose system, complementary work has been carried out on many different ATP binding cassette (ABC) transporters, all of which has contributed in important ways to the understanding of the maltose transport system. The regulation of the maltose transport system, at the DNA level, is implemented by the synergistic action of MalT and cAMP/CAP complex and, at the protein level, by interactions of MalK with unphosphorylated EIIAglc, a signal-transducing component of the phosphoenolpyruvate-glucose phosphotransferase system.

Alternating access in maltose transporter mediated by rigid-body rotations.

ATP-binding cassette transporters couple ATP hydrolysis to substrate translocation through an alternating access mechanism, but the nature of the conformational changes in a transport cycle remains elusive. Previously we reported the structure of the maltose transporter MalFGK(2) in an outward-facing conformation in which the transmembrane (TM) helices outline a substrate-binding pocket open toward the periplasmic surface and ATP is poised for hydrolysis along the closed nucleotide-binding dimer interface. Here we report the structure of the nucleotide-free maltose transporter in which the substrate binding pocket is only accessible from the cytoplasm and the nucleotide-binding interface is open. Comparison of the same transporter crystallized in two different conformations reveals that alternating access involves rigid-body rotations of the TM subdomains that are coupled to the closure and opening of the nucleotide-binding domain interface. The comparison also reveals that point mutations enabling binding protein-independent transport line dynamic interfaces in the TM region.

Structural insights into ABC transporter mechanism.

ATP-binding cassette (ABC) transporters utilize the energy from ATP hydrolysis to transport substances across the membrane. In recent years, crystal structures of several ABC transporters have become available. These structures show that both importers and exporters oscillate between two conformations: an inward-facing conformation with the substrate translocation pathway open to the cytoplasm and an outward-facing conformation with the translocation pathway facing the opposite side of the membrane. In this review, conformational differences found in the structures of homologous ABC transporters are analyzed to understand how alternating-access is achieved. It appears that rigid-body rotations of the transmembrane subunits, coinciding with the opening and closing of the nucleotide-binding subunits, couples ATP hydrolysis to substrate translocation.

Both maltose-binding protein and ATP are required for nucleotide-binding domain closure in the intact maltose ABC transporter.

The maltose transporter MalFGK(2) of Escherichia coli is a member of the ATP-binding cassette superfamily. A periplasmic maltose-binding protein (MBP) delivers maltose to MalFGK(2) and stimulates its ATPase activity. Site-directed spin labeling EPR spectroscopy was used to study the opening and closing of the nucleotide-binding interface of MalFGK(2) during the catalytic cycle. In the intact transporter, closure of the interface coincides not just with the binding of ATP, as seen with isolated nucleotide-binding domains, but requires both MBP and ATP, implying that MBP stimulates ATPase activity by promoting the closure of the nucleotide-binding interface. After ATP hydrolysis, with MgADP and MBP bound, the nucleotide-binding interface resides in a semi-open configuration distinct from the fully open configuration seen in the absence of any ligand. We propose that P(i) release coincides with the reorientation of transmembrane helices to an inward-facing conformation and the final step of maltose translocation into the cell.

Structure, function, and evolution of bacterial ATP-binding cassette systems.

SUMMARY: ATP-binding cassette (ABC) systems are universally distributed among living organisms and function in many different aspects of bacterial physiology. ABC transporters are best known for their role in the import of essential nutrients and the export of toxic molecules, but they can also mediate the transport of many other physiological substrates. In a classical transport reaction, two highly conserved ATP-binding domains or subunits couple the binding/hydrolysis of ATP to the translocation of particular substrates across the membrane, through interactions with membrane-spanning domains of the transporter. Variations on this basic theme involve soluble ABC ATP-binding proteins that couple ATP hydrolysis to nontransport processes, such as DNA repair and gene expression regulation. Insights into the structure, function, and mechanism of action of bacterial ABC proteins are reported, based on phylogenetic comparisons as well as classic biochemical and genetic approaches. The availability of an increasing number of high-resolution structures has provided a valuable framework for interpretation of recent studies, and realistic models have been proposed to explain how these fascinating molecular machines use complex dynamic processes to fulfill their numerous biological functions. These advances are also important for elucidating the mechanism of action of eukaryotic ABC proteins, because functional defects in many of them are responsible for severe human inherited diseases.

Crystal structure of a catalytic intermediate of the maltose transporter.

The maltose uptake system of Escherichia coli is a well-characterized member of the ATP-binding cassette transporter superfamily. Here we present the 2.8-A crystal structure of the intact maltose transporter in complex with the maltose-binding protein, maltose and ATP. This structure, stabilized by a mutation that prevents ATP hydrolysis, captures the ATP-binding cassette dimer in a closed, ATP-bound conformation. Maltose is occluded within a solvent-filled cavity at the interface of the two transmembrane subunits, about halfway into the lipid bilayer. The binding protein docks onto the entrance of the cavity in an open conformation and serves as a cap to ensure unidirectional translocation of the sugar molecule. These results provide direct evidence for a concerted mechanism of transport in which solute is transferred from the binding protein to the transmembrane subunits when the cassette dimer closes to hydrolyse ATP.

Tuning of the outer hair cell motor by membrane cholesterol.

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. We observed that alterations of cochlear cholesterol modulate hearing in mice. Mammalian hearing is powered by outer hair cell (OHC) electromotility, a membrane-based motor mechanism that resides in the OHC lateral wall. We show that membrane cholesterol decreases during maturation of OHCs. To study the effects of cholesterol on hearing at the molecular level, we altered cholesterol levels in the OHC wall, which contains the membrane protein prestin. We show a dynamic and reversible relationship between membrane cholesterol levels and voltage dependence of prestin-associated charge movement in both OHCs and prestin-transfected HEK 293 cells. Cholesterol levels also modulate the distribution of prestin within plasma membrane microdomains and affect prestin self-association in HEK 293 cells. These findings indicate that alterations in membrane cholesterol affect prestin function and functionally tune the outer hair cell.