ABSTRACT
The identification of human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem revitalized hopes of developing a universal influenza vaccine. Using a rational design and library approach, we engineered stable HA stem antigens ("mini-HAs") based on an H1 subtype sequence. Our most advanced candidate exhibits structural and bnAb binding properties comparable to those of full-length HA, completely protects mice in lethal heterologous and heterosubtypic challenge models, and reduces fever after sublethal challenge in cynomolgus monkeys. Antibodies elicited by this mini-HA in mice and nonhuman primates bound a wide range of HAs, competed with human bnAbs for HA stem binding, neutralized H5N1 viruses, and mediated antibody-dependent effector activity. These results represent a proof of concept for the design of HA stem mimics that elicit bnAbs against influenza A group 1 viruses.
Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H5N1 Subtype/immunology , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Humans , Mice , Protein Multimerization , Protein Structure, SecondaryABSTRACT
Hybrid antimicrobials containing an antibacterial linked to a multidrug resistance (MDR) pump inhibitor make up a promising new class of agents for countering efflux-mediated bacterial drug resistance. This study explores the effects of varying the relative orientation of the antibacterial and efflux pump inhibitor components in three isomeric hybrids (SS14, SS14-M, and SS14-P) which link the antibacterial alkaloid and known substrate for the NorA MDR pump berberine to different positions on INF55 (5-nitro-2-phenylindole), an inhibitor of NorA. The MICs for all three hybrids against wild-type, NorA-knockout, and NorA-overexpressing Staphylococcus aureus cells were found to be similar (9.4 to 40.2 microM), indicating that these compounds are not effectively effluxed by NorA. The three hybrids were also found to have similar curing effects in a Caenorhabditis elegans live infection model. Each hybrid was shown to accumulate in S. aureus cells to a greater extent than either berberine or berberine in the presence of INF55, and the uptake kinetics of SS14 were found to differ from those of SS14-M and SS14-P. The effects on the uptake and efflux of the NorA substrate ethidium bromide into S. aureus cells in the presence or absence of the hybrids were used to confirm MDR inhibition by the hybrids. MDR-inhibitory activity was confirmed for SS14-M and SS14-P but not for SS14. Molecular dynamics simulations revealed that SS14 prefers to adopt a conformation that is not prevalent in either SS14-M or SS14-P, which may explain why some properties of SS14 diverge from those of its two isomers. In summary, subtle repositioning of the pump-blocking INF55 moiety in berberine-INF55 hybrids was found to have a minimal effect on their antibacterial activities but to significantly alter their effects on MDR pumps.
Subject(s)
Anti-Bacterial Agents , Berberine , Enterococcus faecalis/drug effects , Indoles , Staphylococcus aureus/drug effects , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Berberine/chemistry , Berberine/pharmacology , Caenorhabditis elegans/drug effects , Caenorhabditis elegans/microbiology , Drug Design , Drug Resistance, Multiple, Bacterial , Ethidium , Indoles/chemistry , Indoles/pharmacology , Microbial Sensitivity Tests , Molecular Structure , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Conjugation of the NorA substrate berberine and the NorA inhibitor 5-nitro-2-phenyl-1H-indole via a methylene ether linking group gave the 13-substituted berberine-NorA inhibitor hybrid, 3. A series of simpler arylmethyl ether hybrid structures were also synthesized. The hybrid 3 showed excellent antibacterial activity (MIC Staphylococcus aureus, 1.7 microM), which was over 382-fold more active than the parent antibacterial berberine, against this bacterium. This compound was also shown to block the NorA efflux pump in S. aureus.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Berberine/chemistry , Enterococcus faecalis/drug effects , Indoles/pharmacology , Intercalating Agents/pharmacology , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Staphylococcus aureus/drug effects , Anti-Bacterial Agents/chemistry , Ether/chemistry , Indoles/chemistry , Intercalating Agents/chemistry , Molecular StructureABSTRACT
Protein translocation in Escherichia coli is mediated by the translocase that, in its minimal form, comprises a protein-conducting pore (SecYEG) and a motor protein (SecA). The SecYEG complex forms a narrow channel in the membrane that allows passage of secretory proteins (preproteins) in an unfolded state only. It has been suggested that the SecA requirement for translocation depends on the folding stability of the mature preprotein domain. Here we studied the effects of the signal sequence and SecB on the folding and translocation of folding stabilizing and destabilizing mutants of the mature maltose binding protein (MBP). Although the mutations affect the folding of the precursor form of MBP, these are drastically overruled by the combined unfolding stabilization of the signal sequence and SecB. Consequently, the translocation kinetics, the energetics and the SecA and SecB dependence of the folding mutants are indistinguishable from those of wild-type preMBP. These data indicate that unfolding of the mature domain of preMBP is likely not a rate-determining step in translocation when the protein is targeted to the translocase via SecB.
Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation/genetics , Periplasmic Binding Proteins/chemistry , Periplasmic Binding Proteins/metabolism , Protein Folding , Adenosine Triphosphatases/metabolism , Bacterial Proteins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Endopeptidase K/metabolism , Escherichia coli Proteins/isolation & purification , Kinetics , Membrane Transport Proteins/metabolism , Mutagenesis , Mutant Proteins/isolation & purification , Periplasmic Binding Proteins/isolation & purification , Protein Precursors/chemistry , Protein Precursors/isolation & purification , Protein Precursors/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Transport , SEC Translocation Channels , SecA Proteins , Spectrometry, Fluorescence , Thermodynamics , TryptophanABSTRACT
How chaperone interactions affect protein folding pathways is a central problem in biology. With the use of optical tweezers and all-atom molecular dynamics simulations, we studied the effect of chaperone SecB on the folding and unfolding pathways of maltose binding protein (MBP) at the single-molecule level. In the absence of SecB, we find that the MBP polypeptide first collapses into a molten globulelike compacted state and then folds into a stable core structure onto which several alpha helices are finally wrapped. Interactions with SecB completely prevent stable tertiary contacts in the core structure but have no detectable effect on the folding of the external alpha helices. It appears that SecB only binds to the extended or molten globulelike structure and retains MBP in this latter state. Thus during MBP translocation, no energy is required to disrupt stable tertiary interactions.
Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins/chemistry , Periplasmic Binding Proteins/chemistry , Protein Folding , Computer Simulation , Escherichia coli Proteins/metabolism , Models, Molecular , Optical Tweezers , Periplasmic Binding Proteins/metabolism , Protein Conformation , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
Protein translocation across the cellular membranes is an ubiquitous and crucial activity of cells. This process is mediated by translocases that consist of a protein conducting channel and an associated motor protein. Motor proteins interact with protein substrates and utilize the free energy of ATP binding and hydrolysis for protein unfolding, translocation and unbinding. Since motor proteins are found either at the cis- or trans-side of the membrane, different mechanisms for translocation have been proposed. In the Power stroke model, cis-acting motors are thought to push, while trans-motors pull on the substrate protein during translocation. In the Brownian ratchet model, translocation occurs by diffusion of the unfolded polypeptide through the translocation pore while directionality is achieved by trapping and refolding. Recent insights in the structure and function of the molecular motors suggest that different mechanisms can be employed simultaneously.
Subject(s)
Molecular Motor Proteins/metabolism , Protein Transport/physiology , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Endoplasmic Reticulum Chaperone BiP , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/metabolism , Models, Biological , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Molecular Motor Proteins/chemistry , Protein Folding , SEC Translocation Channels , SecA ProteinsABSTRACT
In Escherichia coli, secretory proteins (preproteins) are translocated across the cytoplasmic membrane by the Sec system composed of a protein-conducting channel, SecYEG, and an ATP-dependent motor protein, SecA. After binding of the preprotein to SecYEG-bound SecA, cycles of ATP binding and hydrolysis by SecA are thought to drive the stepwise translocation of the preprotein across the membrane. To address how the length of a preprotein substrate affects the SecA-driven translocation process, we constructed derivatives of the precursor of the outer membrane protein A (proOmpA) with 2, 4, 6, and 8 in-tandem repeats of the periplasmic domain. With increasing polypeptide length, an increasing delay in the time before full-length translocation was observed, but the translocation rate expressed as amino acid translocation per minute remained constant. These data indicate that in the ATP-dependent reaction, SecA drives a constant rate of preprotein translocation consistent with a stepping mechanism of translocation.
Subject(s)
Adenosine Triphosphatases/physiology , Bacterial Proteins/physiology , Membrane Transport Proteins/physiology , Adenosine Triphosphate/metabolism , Hydrolysis , Kinetics , Protein Precursors/metabolism , Protein Transport , SEC Translocation Channels , SecA ProteinsABSTRACT
Binding-protein-dependent secondary transporters make up a unique transport protein family. They use a solute-binding protein in proton-motive-force-driven transport. Only a few systems have been functionally analysed. The yiaMNO genes of Escherichia coli K-12 encode one family member that transports the rare pentose l-xylulose. Its physiological role is unknown, since wild-type E. coli K-12 does not utilize l-xylulose as sole carbon source. Deletion of the yiaMNO genes in E. coli K-12 strain MC4100 resulted in remarkable changes in the transition from exponential growth to the stationary phase, high-salt survival and biofilm formation.
