Protein encapsulation in liposomes: efficiency depends on interactions between protein and phospholipid bilayer.

Background  

We investigated the encapsulation mechanism of enzymes into liposomes. The existing protocols to achieve high encapsulation efficiencies are basically optimized for chemically stable molecules. Enzymes, however, are fragile and encapsulation requires in addition the preservation of their functionality. Using acetylcholinesterase as a model, we found that most protocols lead to a rapid denaturation of the enzyme with loss in the functionality and therefore inappropriate for such an application. The most appropriate method is based on lipid film hydration but had a very low efficiency.     

Staphylococcus aureus ClpC ATPase is a late growth phase effector of metabolism and persistence

Staphylococcus aureus Clp ATPases (molecular chaperones) alter normal physiological functions including an aconitase‐mediated effect on post‐stationary growth, acetate catabolism, and entry into death phase (Chatterjee et al., J. Bacteriol. 2005, 187, 4488–4496). In the present study, the global function of ClpC in physiology, metabolism, and late‐stationary phase survival was examined using DNA microarrays and 2‐D PAGE followed by MALDI‐TOF MS. The results suggest that ClpC is involved in regulating the expression of genes and/or proteins of gluconeogenesis, the pentose‐phosphate pathway, pyruvate metabolism, the electron transport chain, nucleotide metabolism, oxidative stress, metal ion homeostasis, stringent response, and programmed cell death. Thus, one major function of ClpC is balancing late growth phase carbon metabolism. Furthermore, these changes in carbon metabolism result in alterations of the intracellular concentration of free NADH, the amount of cell‐associated iron, and fatty acid metabolism. This study provides strong evidence for ClpC as a critical factor in staphylococcal energy metabolism, stress regulation, and late‐stationary phase survival; therefore, these data provide important insight into the adaptation of S. aureus toward a persister state in chronic infections.     

The MalF P2 Loop of the ATP-Binding Cassette Transporter MalFGK2 from Escherichia coli and Salmonella enterica Serovar Typhimurium Interacts with Maltose Binding Protein (MalE) throughout the Catalytic Cycle

We have investigated the interaction of the uncommonly large periplasmic P2 loop of the MalF subunit of the maltose ATP-binding cassette transporter (MalFGK₂) from Escherichia coli and Salmonella enterica serovar Typhimurium with maltose binding protein (MalE) by site-specific chemical cross-linking in the assembled transport complex. We focused on possible distance changes between two pairs of residues of the P2 loop and MalE during the transport cycle. The distance between MalF(S205C) and MalE(T80C) (∼5 Å) remained unchanged under all conditions tested. Cross-linking did not affect the ATPase activity of the complex. The distance between MalF(T177C) and MalE(T31C) changed from ∼10 Å to ∼5 Å upon binding of ATP (or maltose, with a less pronounced result) and was reset to ∼10 Å after hydrolysis of one ATP. A cross-link (∼25 Å) between MalF(S205C) and MalE(T31C) was observed only when the transporter resided in a transition state-like conformation, as was the case after vanadate trapping or in a binding protein-independent mutant, both of which are characterized by tight binding of unliganded MalE to the transporter. Thus, we propose that the observed cross-link is indicative of catalytic intermediates of the transporter. Together, our results strengthen the notion that the MalF P2 loop plays an important role in intersubunit communication. In particular, this loop is involved in keeping MalE in close contact with the transporter. The data are discussed with respect to a crystal structure and current transport models.ATP-binding cassette (ABC) transporters utilize the free energy of ATP hydrolysis to translocate substrates across biological membranes and can function as import or export systems (17). ABC transporters are generally composed of two hydrophobic, pore-forming transmembrane subunits and transmembrane domains (TMDs) and two hydrophilic nucleotide-binding (or ABC) subunits and nucleotide-binding domains (NBDs) that hydrolyze ATP (9). The crystal structures of isolated NBDs (6, 23, 34, 43) revealed that NBDs can be divided into a RecA-like subdomain comprising both the Walker A and the Walker B motifs, which are involved in nucleotide binding, and a helical subdomain harboring the unique LSGGQ motif (35). Furthermore, in the physiologically relevant NBD dimer, the nucleotide is complexed between the Walker A and B sites of one monomer and the LSGGQ motif of the opposing monomer. Both subdomains are joined by the “Q loop” containing a conserved glutamine residue that binds to the Mg²⁺ ion and attacking water and is likely to be involved in communicating ATP binding to the TMDs (10, 20, 29). ATP-dependent closing of the NBD dimer is thought to provide one possibility of the power stroke of ABC transporters (38).ABC importers that are confined to prokaryotes mediate the uptake of a large variety of solutes, including inorganic ions, amino acids, sugars, vitamins, oligopeptides, and polyamines (5). They require an additional protein, the extracytoplasmic solute binding protein (SBP), in order to capture the substrate and to deliver it to the cognate ABC transporter (37). SBPs typically consist of two lobes that are connected by a linker region. The interface between the two lobes forms the substrate binding site. Upon binding of the ligand, the proteins undergo a conformational change from an open toward a closed state (33) which, by interaction with extracytoplasmic peptide regions of TMDs of the cognate ABC transporter, initiates the transport process (31). The molecular events by which binding of ATP to the NBDs and interaction of liganded binding proteins with the TMDs are communicated to eventually trigger substrate translocation are still poorly understood.The maltose ABC transporter of Escherichia coli and Salmonella enterica serovar Typhimurium is one of the best-characterized transporters and thus serves as a model system for studying the mechanism by which ABC transporters exert their functions in general (15). The transporter is composed of the extracytoplasmic (periplasmic) maltose binding protein (MalE), the membrane-spanning subunits MalF and MalG, and two copies of the ATP-hydrolyzing subunit (MalK) (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.(A) Structure of the catalytic intermediate of the maltose transporter [MalFGK(E159Q)₂-E]. The complex is shown in a ribbon diagram. White horizontal bars mark the boundaries of the membrane. Color code: yellow, MalE; cyan, MalF; red, MalG; green and magenta, MalK dimer. (B) Close-up view of the contact site between MalF P2 and the N-terminal lobe of MalE. The color code is the same as that for panel A. Residues from regions I and II that were replaced by cysteines are indicated in pink (MalF) and green (MalE). Residue MalE-K179, which was used as a control, is shown in green. The figure was drawn with DS ViewerPro 6.0 (Accelrys, Cambridge, United Kingdom), using the coordinates from entry 2R6G in the Brookhaven Protein Data Bank.Recently, suppressor mutational analysis provided a first hint that substrate availability is communicated from MalE to the MalK dimer via periplasmic loop regions of MalFG (11). Moreover, by site-directed cross-linking based on previous genetic evidence (19, 40), we demonstrated a close proximity of MalE G13 to Pro-78 in the first periplasmic loop (P1) of MalG, independently of cofactors such as maltose or ATP. Interaction of both residues was also observed in intact cells (11). These findings led us to propose that a copy of MalE is permanently associated with the transporter throughout the catalytic cycle. Furthermore, we have found that a region of the large, periplasmic P2 loop of MalF around Ser-205 (Fig. ​(Fig.1)1) is in cross-linking distance from MalE in the presence of maltose and MgATP only or when the transporter resides in the vanadate-trapped transition state. These results were perfectly confirmed by the subsequently published crystal structure of the MalFGK(E159Q)₂-E complex, which represents a transport intermediate (32). Here, the MalK dimer is complexed with two ATP molecules, and MalE is tightly associated with MalFG, but maltose has already been released into a binding pocket formed by MalF only. In particular, the N-terminal lobe of MalE is in close contact with the P2 loop of MalF (Fig. ​(Fig.1A1A).In this communication, we have taken advantage of this structural information to gain further insight into the MalF P2-MalE interaction during the transport cycle. We demonstrate ATP- and maltose-dependent distance changes between selected pairs of residues of the loop and MalE in the assembled complex by site-specific cross-linking. Our data demonstrate for the first time that the MalF P2 loop is in close contact to MalE throughout the catalytic cycle.     

Characterization of the amino acids involved in substrate specificity of methionine sulfoxide reductase A

Methionine sulfoxide reductases (Msrs) are ubiquitous enzymes that catalyze the thioredoxin-dependent reduction of methionine sulfoxide (MetSO) back to methionine. In vivo, Msrs are essential in protecting cells against oxidative damages on proteins and in the virulence of some bacteria. There exists two structurally unrelated classes of Msrs. MsrAs are stereo-specific toward the S epimer on the sulfur of the sulfoxide, whereas MsrBs are specific toward the R isomer. Both classes of Msrs display a similar catalytic mechanism of sulfoxide reduction by thiols via the sulfenic acid chemistry and a better affinity for protein-bound MetSO than for free MetSO. Recently, the role of the amino acids implicated in the catalysis of the reductase step of Neisseria meningitidis MsrA was determined. In the present study, the invariant amino acids potentially involved in substrate binding, i.e. Phe-52, Trp-53, Asp-129, His-186, Tyr-189, and Tyr-197, were substituted. The catalytic parameters under steady-state conditions and of the reductase step of the mutated MsrAs were determined and compared with those of the wild type. Altogether, the results support the presence of at least two binding subsites. The first one, whose contribution is major in the efficiency of the reductase step and in which the epsilon-methyl group of MetSO binds, is the hydrophobic pocket formed by Phe-52 and Trp-53, the position of the indole ring being stabilized by interactions with His-186 and Tyr-189. The second subsite composed of Asp-129 and Tyr-197 contributes to the binding of the main chain of the substrate but to a lesser extent.     

Surfactant protein A (SP-A)-mediated clearance of Staphylococcus aureus involves binding of SP-A to the staphylococcal adhesin eap and the macrophage receptors SP-A receptor 210 and scavenger receptor class A

Staphylococcus aureus causes life-threatening pneumonia in hospitals and deadly superinfection during viral influenza. The current study investigated the role of surfactant protein A (SP-A) in opsonization and clearance of S. aureus. Previous studies showed that SP-A mediates phagocytosis via the SP-A receptor 210 (SP-R210). Here, we show that SP-R210 mediates binding and control of SP-A-opsonized S. aureus by macrophages. We determined that SP-A binds S. aureus through the extracellular adhesin Eap. Consequently, SP-A enhanced macrophage uptake of Eap-expressing (Eap(+)) but not Eap-deficient (Eap(-)) S. aureus. In a reciprocal fashion, SP-A failed to enhance uptake of Eap(+) S. aureus in peritoneal Raw264.7 macrophages with a dominant negative mutation (SP-R210(DN)) blocking surface expression of SP-R210. Accordingly, WT mice cleared infection with Eap(+) but succumbed to sublethal infection with Eap- S. aureus. However, SP-R210(DN) cells compensated by increasing non-opsonic phagocytosis of Eap(+) S. aureus via the scavenger receptor scavenger receptor class A (SR-A), while non-opsonic uptake of Eap(-) S. aureus was impaired. Macrophages express two isoforms: SP-R210(L) and SP-R210(S). The results show that WT alveolar macrophages are distinguished by expression of SP-R210(L), whereas SR-A(-/-) alveolar macrophages are deficient in SP-R210(L) expressing only SP-R210(S). Accordingly, SR-A(-/-) mice were highly susceptible to both Eap(+) and Eap(-) S. aureus. The lungs of susceptible mice generated abnormal inflammatory responses that were associated with impaired killing and persistence of S. aureus infection in the lung. In conclusion, alveolar macrophage SP-R210(L) mediates recognition and killing of SP-A-opsonized S. aureus in vivo, coordinating inflammatory responses and resolution of S. aureus pneumonia through interaction with SR-A.     

Recognition of gram-positive intestinal bacteria by hybridoma- and colostrum-derived secretory immunoglobulin A is mediated by carbohydrates

Humans live in symbiosis with 10(14) commensal bacteria among which >99% resides in their gastrointestinal tract. The molecular bases pertaining to the interaction between mucosal secretory IgA (SIgA) and bacteria residing in the intestine are not known. Previous studies have demonstrated that commensals are naturally coated by SIgA in the gut lumen. Thus, understanding how natural SIgA interacts with commensal bacteria can provide new clues on its multiple functions at mucosal surfaces. Using fluorescently labeled, nonspecific SIgA or secretory component (SC), we visualized by confocal microscopy the interaction with various commensal bacteria, including Lactobacillus, Bifidobacteria, Escherichia coli, and Bacteroides strains. These experiments revealed that the interaction between SIgA and commensal bacteria involves Fab- and Fc-independent structural motifs, featuring SC as a crucial partner. Removal of glycans present on free SC or bound in SIgA resulted in a drastic drop in the interaction with gram-positive bacteria, indicating the essential role of carbohydrates in the process. In contrast, poor binding of gram-positive bacteria by control IgG was observed. The interaction with gram-negative bacteria was preserved whatever the molecular form of protein partner used, suggesting the involvement of different binding motifs. Purified SIgA and SC from either mouse hybridoma cells or human colostrum exhibited identical patterns of recognition for gram-positive bacteria, emphasizing conserved plasticity between species. Thus, sugar-mediated binding of commensals by SIgA highlights the currently underappreciated role of glycans in mediating the interaction between a highly diverse microbiota and the mucosal immune system.     

Photoinduced reactivity of the HIV-1 envelope glycoprotein with a membrane-embedded probe reveals insertion of portions of the HIV-1 Gp41 cytoplasmic tail into the viral membrane

The interactions of HIV-1 Env (gp120-gp41) with CD4 and coreceptors trigger a barrage of conformational changes in Env that drive the membrane fusion process. Various regions of gp41 have profound effects on HIV entry and budding. However, the precise interactions between gp41 and the membrane have not been elucidated. To examine portions of membrane proteins that are embedded in membrane lipids, we have studied photoinduced chemical reactions in membranes using the lipid bilayer specific probe iodonaphthyl azide (INA). Here we show that in addition to the transmembrane anchor, amphipatic sequences in the cytoplasmic tail (CT) of HIV-1 gp41 are labeled by INA. INA labeling of the HIV-1 gp41 CT was similar whether wild-type or a mutant HIV-1 was used with uncleaved p55 Gag, which does not allow entry. These results shed light on the disposition of the HIV-1 gp41 CT with respect to the membrane. Moreover, our data have general implications for topology of membrane proteins and their in situ interactions with the lipid bilayer.     

A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products

The capacity to produce large amounts of protein in mammalian cells is important in several contexts, including large-scale generation of biologically useful proteins, gene therapy, and transdominant genetics in cultured cells. For transdominant genetics, retroviral vectors are especially useful for delivery of expression libraries. However, even the potent CMV promoter is often unable to stimulate single-copy production of protein beyond the 1 microM level. We have adapted the HIV2/Tat expression system to retroviral vectors to boost expression above levels attainable with CMV promoters. We show that the system produces protein levels in four cell types tested which exceed levels attained by wild-type CMV or modified CMV promoters. In one cell line, the increase is 10-fold above CMV. Coupled with a stable expressed protein, levels of about 4 microM can be produced from presumptive single-copy retroviral transductants, and 30 microM from multicopy transductants.