Antimicrobial Peptide Recognition Motif of the Substrate Binding Protein SapA from Nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen associated with respiratory diseases, including otitis media and exacerbations of chronic obstructive pulmonary disease. NTHi exhibits resistance to killing by host antimicrobial peptides (AMPs) mediated by SapA, the substrate binding protein of the sensitivity to antimicrobial peptides (Sap) transporter. However, the specific mechanisms by which SapA selectively binds various AMPs such as defensins and cathelicidin are unknown. In this study, we report mutational analyses of both defensin AMPs and the SapA binding pocket to define the specificity of AMP recognition. Bactericidal assays revealed that NTHi lacking SapA are more susceptible to human beta defensins and LL-37, while remaining highly resistant to a human alpha defensin. In contrast to homologues, our research underscores the distinct specificity of NTHi SapA, which selectively recognizes and binds to peptides containing the charged-hydrophobic motif PKE and RRY. These findings provide valuable insight into the divergence of SapA among bacterial species and NTHi SapA's ability to selectively interact with specific AMPs to mediate resistance.

Activity of the pleiotropic drug resistance transcription factors Pdr1p and Pdr3p is modulated by binding site flanking sequences.

The transcription factors Pdr1p and Pdr3p regulate pleiotropic drug resistance (PDR) in Saccharomyces cerevisiae via the PDR responsive elements (PDREs) to modulate gene expression. However, the exact mechanisms underlying the differences in their regulons remain unclear. Employing genomic occupancy profiling (CUT&RUN), binding assays, and transcription studies, we characterized the differences in sequence specificity between transcription factors. Findings reveal distinct preferences for core PDRE sequences and the flanking sequences for both proteins. While flanking sequences moderately alter DNA binding affinity, they significantly impact Pdr1/3p transcriptional activity. Notably, both proteins demonstrated the ability to bind half sites, showing potential enhancement of transcription from adjacent PDREs. This insight sheds light on ways Pdr1/3p can differentially regulate PDR.

Unraveling the Half and Full Site Sequence Specificity of the Saccharomyces cerevisiae Pdr1p and Pdr3p Transcription Factors.

The transcription factors Pdr1p and Pdr3p regulate pleotropic drug resistance (PDR) in Saccharomyces cerevisiae , via the PDR responsive elements (PDREs) to modulate gene expression. However, the exact mechanisms underlying the differences in their regulons remain unclear. Employing genomic occupancy profiling (CUT&RUN), binding assays, and transcription studies, we characterized the differences in sequence specificity between transcription factors. Findings reveal distinct preferences for core PDRE sequences and the flanking sequences for both proteins. While flanking sequences moderately alter DNA binding affinity, they significantly impact Pdr1/3p transcriptional activity. Notably, both proteins demonstrated the ability to bind half sites, showing potential enhancement of transcription from adjacent PDREs. This insight sheds light on ways Pdr1/3 can differentially regulate PDR.

Detergent Alternatives: Membrane Protein Purification Using Synthetic Nanodisc Polymers.

The development of styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers provides an alternative to traditional detergent extraction of integral membrane proteins. By inserting into the membrane, these polymers can extract membrane proteins along with lipids in the form of native nanodiscs made by poly(styrene co-maleic anhydride) derivatives. Unlike detergent solubilization, where membrane proteins may lose annular lipids necessary for proper folding and stability, native nanodiscs allow for proteins to reside in the natural lipid environment. In addition, polymer-based nanodiscs can be purified using common chromatography methods similar to protocols established with detergent solubilization purification. Here we describe the solubilization screening and purification of an integral membrane protein using several commercial copolymers.

Transcription factors and ABC transporters: from pleiotropic drug resistance to cellular signaling in yeast.

Budding yeast Saccharomyces cerevisiae survives in microenvironments utilizing networks of regulators and ATP-binding cassette (ABC) transporters to circumvent toxins and a variety of drugs. Our understanding of transcriptional regulation of ABC transporters in yeast is mainly derived from the study of multidrug resistance protein networks. Over the past two decades, this research has not only expanded the role of transcriptional regulators in pleiotropic drug resistance (PDR) but evolved to include the role that regulators play in cellular signaling and environmental adaptation. Inspection of the gene networks of the transcriptional regulators and characterization of the ABC transporters has clarified that they also contribute to environmental adaptation by controlling plasma membrane composition, toxic-metal sequestration, and oxidative stress adaptation. Additionally, ABC transporters and their regulators appear to be involved in cellular signaling for adaptation of S. cerevisiae populations to nutrient availability. In this review, we summarize the current understanding of the S. cerevisiae transcriptional regulatory networks and highlight recent work in other notable fungal organisms, underlining the expansion of the study of these gene networks across the kingdom fungi.

Structural and functional diversity calls for a new classification of ABC transporters.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.

Oligopeptide-binding protein from nontypeable Haemophilus influenzae has ligand-specific sites to accommodate peptides and heme in the binding pocket.

In nontypeable Haemophilus influenzae (NTHi), the oligopeptide-binding protein (OppA) serves as the substrate-binding protein (SBP) of the oligopeptide transport system responsible for the import of peptides. We solved the crystal structure of nthiOppA in complex with hydrophobic peptides of various sizes. Our novel hexapeptide complex demonstrates the flexibility of the nthiOppA-binding cavity to expand and accommodate the longer peptide while maintaining similar protein-peptide interactions of smaller peptide complexes. In addition to acquiring peptides from the host environment, as a heme auxotroph NTHi utilizes host hemoproteins as a source of essential iron. OppA is a member of the Cluster C SBP family, and unlike other SBP families, some members recognize two distinctly different substrates. DppA (dipeptide), MppA (murein tripeptide), and SapA (antimicrobial peptides) are Cluster C proteins known to also transport heme. We observed nthiOppA shares this heme-binding characteristic and established heme specificity and affinity by surface plasmon resonance (SPR) of the four Cluster C proteins in NTHi. Ligand-docking studies predicted a distinct heme-specific cleft in the binding pocket, and using SPR competition assays, we observed that heme does not directly compete with peptide in the substrate-binding pocket. Additionally, we identified that the individual nthiOppA domains differentially contribute to substrate binding, with one domain playing a dominant role in heme binding and the other in peptide binding. Our results demonstrate the multisubstrate specificity of nthiOppA and the role of NTHi Cluster C proteins in the heme-uptake pathway for this pathogen.

Microevolution in response to transient heme-iron restriction enhances intracellular bacterial community development and persistence.

Bacterial pathogens must sense, respond and adapt to a myriad of dynamic microenvironmental stressors to survive. Adaptation is key for colonization and long-term ability to endure fluctuations in nutrient availability and inflammatory processes. We hypothesize that strains adapted to survive nutrient deprivation are more adept for colonization and establishment of chronic infection. In this study, we detected microevolution in response to transient nutrient limitation through mutation of icc. The mutation results in decreased 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in nontypeable Haemophilus influenzae (NTHI). In a preclinical model of NTHI-induced otitis media (OM), we observed a significant decrease in the recovery of effusion from ears infected with the icc mutant strain. Clinically, resolution of OM coincides with the clearance of middle ear fluid. In contrast to this clinical paradigm, we observed that the icc mutant strain formed significantly more intracellular bacterial communities (IBCs) than the parental strain early during experimental OM. Although the number of IBCs formed by the parental strain was low at early stages of OM, we observed a significant increase at later stages that coincided with absence of recoverable effusion, suggesting the presence of a mucosal reservoir following resolution of clinical disease. These data provide the first insight into NTHI microevolution during nutritional limitation and provide the first demonstration of IBCs in a preclinical model of chronic OM.

Selective substrate uptake: The role of ATP-binding cassette (ABC) importers in pathogenesis.

The uptake of nutrients, including metals, amino acids and peptides are required for many biological processes. Pathogenic bacteria scavenge these essential nutrients from microenvironments to survive within the host. Pathogens must utilize a myriad of mechanisms to acquire these essential nutrients from the host while mediating the effects of toxicity. Bacteria utilize several transport proteins, including ATP-binding cassette (ABC) transporters to import and expel substrates. ABC transporters, conserved across all organisms, are powered by the energy from ATP to move substrates across cellular membranes. In this review, we will focus on nutrient uptake, the role of ABC importers at the host-pathogen interface, and explore emerging therapies to combat pathogenesis. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Diversity in ABC transporters: type I, II and III importers.

ATP-binding cassette transporters are multi-subunit membrane pumps that transport substrates across membranes. While significant in the transport process, transporter architecture exhibits a range of diversity that we are only beginning to recognize. This divergence may provide insight into the mechanisms of substrate transport and homeostasis. Until recently, ABC importers have been classified into two types, but with the emergence of energy-coupling factor (ECF) transporters there are potentially three types of ABC importers. In this review, we summarize an expansive body of research on the three types of importers with an emphasis on the basics that underlie ABC importers, such as structure, subunit composition and mechanism.

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.

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.

Classification of a Haemophilus influenzae ABC transporter HI1470/71 through its cognate molybdate periplasmic binding protein, MolA.

molA (HI1472) from H. influenzae encodes a periplasmic binding protein (PBP) that delivers substrate to the ABC transporter MolB(2)C(2) (formerly HI1470/71). The structures of MolA with molybdate and tungstate in the binding pocket were solved to 1.6 and 1.7 Å resolution, respectively. The MolA-binding protein binds molybdate and tungstate, but not other oxyanions such as sulfate and phosphate, making it the first class III molybdate-binding protein structurally solved. The ∼100 µM binding affinity for tungstate and molybdate is significantly lower than observed for the class II ModA molybdate-binding proteins that have nanomolar to low micromolar affinity for molybdate. The presence of two molybdate loci in H. influenzae suggests multiple transport systems for one substrate, with molABC constituting a low-affinity molybdate locus.

The structural basis of cooperative regulation at an alternate genetic switch.

Bacteriophage lambda is a paradigm for understanding the role of cooperativity in gene regulation. Comparison of the regulatory regions of lambda and the unrelated temperate bacteriophage 186 provides insight into alternate ways to assemble functional genetic switches. The structure of the C-terminal domain of the 186 repressor, determined at 2.7 A resolution, reveals an unusual heptamer of dimers, consistent with presented genetic studies. In addition, the structure of a cooperativity mutant of the full-length 186 repressor, identified by genetic screens, was solved to 1.95 A resolution. These structures provide a molecular basis for understanding lysogenic regulation in 186. Whereas the overall fold of the 186 and lambda repressor monomers is remarkably similar, the way the two repressors cooperatively assemble is quite different and explains in part the differences in their regulatory activity.

Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia.

To gain insight into melanoma pathogenesis, we characterized an insertional mouse mutant, TG3, that is predisposed to develop multiple melanomas. Physical mapping identified multiple tandem insertions of the transgene into intron 3 of Grm1 (encoding metabotropic glutamate receptor 1) with concomitant deletion of 70 kb of intronic sequence. To assess whether this insertional mutagenesis event results in alteration of transcriptional regulation, we analyzed Grm1 and two flanking genes for aberrant expression in melanomas from TG3 mice. We observed aberrant expression of only Grm1. Although we did not detect its expression in normal mouse melanocytes, Grm1 was ectopically expressed in the melanomas from TG3 mice. To confirm the involvement of Grm1 in melanocytic neoplasia, we created an additional transgenic line with Grm1 expression driven by the dopachrome tautomerase promoter. Similar to the original TG3, the Tg(Grm1)EPv line was susceptible to melanoma. In contrast to human melanoma, these transgenic mice had a generalized hyperproliferation of melanocytes with limited transformation to fully malignant metastasis. We detected expression of GRM1 in a number of human melanoma biopsies and cell lines but not in benign nevi and melanocytes. This study provides compelling evidence for the importance of metabotropic glutamate signaling in melanocytic neoplasia.