Antiparallel Β-sheet Research Articles

Packing Structure of Antiparallel β-Sheet Polyalanine Region in a Sequential Model Peptide of Nephila clavipes Dragline Silk Studied Using 13C Solid-State NMR and MD Simulation.

Packing structures of polyalanine regions, which are considered to be the reason for the extremely high strength of spider dragline silks, were studied using a series of sequential peptides: (Glu)4GlyGlyLeuGlyGlyGlnGlyAlaGly(Ala)nGlyGlyAlaGlyGlnGlyGlyTyrGlyGly(Glu)4 (n = 3-8) using 13C solid-state NMR spectroscopy. The conformations of (Ala)n in the freeze-dried peptides changed gradually with increasing n from random coils to α-helices with partial antiparallel β-sheet (AP-β) structures. Conversely, all the insolubilized peptides, n = 6-8 after low-pH treatment and n = 4-8 after formic acid/methanol treatment, formed AP-β structures with significant amounts of staggered packing arrangements. These results are different from previously obtained results for pure alanine oligopeptides, that is, AP-β (Ala)n formed rectangular packing for less than n = 6 but staggered packings for n ≥ 7. The 13C-labeled peptides were also used to confirm the staggered packing arrangements from NMR dynamics. Furthermore, a MD simulation supported the observed results.

Gram-scale total synthesis of teixobactin promoting binding mode study and discovery of more potent antibiotics

Teixobactin represents a new class of antibiotics with novel structure and excellent activity against Gram-positive pathogens and Mycobacterium tuberculosis. Herein, we report a one-pot reaction to conveniently construct the key building block l-allo-Enduracidine in 30-gram scale in just one hour and a convergent strategy (3 + 2 + 6) to accomplish a gram-scale total synthesis of teixobactin. Several analogs are described, with 20 and 26 identified as the most efficacious analogs with 3~8-fold and 2~4-fold greater potency against vancomycin resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus respectively in comparison with teixobactin. In addition, they show high efficiency in Streptococcus pneumoniae septicemia mouse model and neutropenic mouse thigh infection model using methicillin-resistant Staphylococcus aureus. We also propose that the antiparallel β-sheet of teixobactin is important for its bioactivity and an antiparallel dimer of teixobactin is the minimal binding unit for lipid II via key amino acids variations and molecular docking.

Solution NMR structure of CGL2373, a polyketide cyclase-like protein from Corynebacterium glutamicum.

Protein CGL2373 from Corynebacterium glutamicum was previously proposed to be a member of the polyketide_cyc2 family, based on amino-acid sequence and secondary structure features derived from NMR chemical shift assignments. We report here the solution NMR structure of CGL2373, which contains three α-helices and one antiparallel β-sheet and adopts a helix-grip fold. This structure shows moderate similarities to the representative polyketide cyclases, TcmN, WhiE, and ZhuI. Nevertheless, unlike the structures of these homologs, CGL2373 structure looks like a half-open shell with a much larger pocket, and key residues in the representative polyketide cyclases for binding substrate and catalyzing aromatic ring formation are replaced with different residues in CGL2373. Also, the gene cluster where the CGL2373-encoding gene is located in C. glutamicum contains additional genes encoding nucleoside diphosphate kinase, folylpolyglutamate synthase, and valine-tRNA ligase, different from the typical gene cluster encoding polyketide cyclase in Streptomyces. Thus, although CGL2373 is structurally a polyketide cyclase-like protein, the function of CGL2373 may differ from the known polyketide cyclases and needs to be further investigated. The solution structure of CGL2373 lays a foundation for in silico ligand screening and binding site identifying in future functional study.

A knottin scaffold directs the CXC-chemokine–binding specificity of tick evasins

Tick evasins (EVAs) bind either CC- or CXC-chemokines by a poorly understood promiscuous or “one-to-many” mechanism to neutralize inflammation. Because EVAs potently inhibit inflammation in many preclinical models, highlighting their potential as biological therapeutics for inflammatory diseases, we sought to further unravel the CXC-chemokine–EVA interactions. Using yeast surface display, we identified and characterized 27 novel CXC-chemokine–binding evasins homologous to EVA3 and defined two functional classes. The first, which included EVA3, exclusively bound ELR+ CXC-chemokines, whereas the second class bound both ELR+ and ELR− CXC-chemokines, in several cases including CXC-motif chemokine ligand 10 (CXCL10) but, surprisingly, not CXCL8. The X-ray crystal structure of EVA3 at a resolution of 1.79 Å revealed a single antiparallel β-sheet with six conserved cysteine residues forming a disulfide-bonded knottin scaffold that creates a contiguous solvent-accessible surface. Swapping analyses identified distinct knottin scaffold segments necessary for different CXC-chemokine–binding activities, implying that differential ligand positioning, at least in part, plays a role in promiscuous binding. Swapping segments also transferred chemokine-binding activity, resulting in a hybrid EVA with dual CXCL10- and CXCL8-binding activities. The solvent-accessible surfaces of the knottin scaffold segments have distinctive shape and charge, which we suggest drives chemokine-binding specificity. These studies provide structural and mechanistic insight into how CXC-chemokine–binding tick EVAs achieve class specificity but also engage in promiscuous binding.

The Functional Mammalian CRES (Cystatin-Related Epididymal Spermatogenic) Amyloid is Antiparallel \u03b2-Sheet Rich and Forms a Metastable Oligomer During Assembly

An amyloid matrix composed of several family 2 cystatins, including the reproductive cystatin CRES, is an integral structure in the mouse epididymal lumen and has proposed functions in sperm maturation and protection. Understanding how CRES amyloid assembles in vitro may provide clues on how the epididymal amyloid matrix forms in vivo. We therefore purified full-length CRES under nondenaturing conditions and followed its aggregation from monomer to amyloid under conditions that may approximate those in the epididymal lumen. CRES transitioned into a metastable oligomer that was resistant to aggregation and only over extended time formed higher-ordered amyloids. High protein concentrations facilitated oligomer assembly and also were required to maintain the metastable state since following dilution the oligomer was no longer detected. Similar to other amyloid precursors, the formation of CRES amyloids correlated with a loss of α-helix and a gain of β-sheet content. However, CRES is unique in that its amyloids are rich in antiparallel β-sheets instead of the more common parallel β-sheets. Taken together, our studies suggest that early metastable oligomers may serve as building blocks for functional amyloid assembly and further reveal that antiparallel β-sheet-rich amyloids can be functional forms.

The peptide hormone glucagon forms amyloid fibrils with two coexisting β-strand conformations.

Glucagon and insulin maintain blood glucose homeostasis and are used to treat hypoglycemia and hyperglycemia in diabetic patients, respectively. Whereas insulin is stable for weeks in its solution formulation, glucagon fibrillizes rapidly at the acidic pH required for solubility, and is therefore formulated as a lyophilized powder that is reconstituted in acidic solution immediately before use. Here we use solid-state NMR to determine the atomic-resolution structure of fibrils of synthetic human glucagon grown at pharmaceutically relevant low pH. Unexpectedly, two sets of chemical shifts are observed, indicating the coexistence of two β-strand conformations. Those two conformations have distinct water accessibilities and intermolecular contacts, indicating that they alternate and hydrogen-bond in an antiparallel fashion along the fibril axis. Two antiparallel β-sheets assemble with symmetric homodimer cross sections. This amyloid structure is stabilized by numerous aromatic, cation-π, polar and hydrophobic interactions, suggesting mutagenesis approaches to inhibit fibrillization to improve this important drug.

Structures of three ependymin-related proteins suggest their function as a hydrophobic molecule binder.

Ependymin was first discovered as a predominant protein in brain extracellular fluid in fish and was suggested to be involved in functions mostly related to learning and memory. Orthologous proteins to ependymin called ependymin-related proteins (EPDRs) have been found to exist in various tissues from sea urchins to humans, yet their functional role remains to be revealed. In this study, the structures of EPDR1 from frog, mouse and human were determined and analyzed. All of the EPDR1s fold into a dimer using a monomeric subunit that is mostly made up of two stacking antiparallel β-sheets with a curvature on one side, resulting in the formation of a deep hydrophobic pocket. All six of the cysteine residues in the monomeric subunit participate in the formation of three intramolecular disulfide bonds. Other interesting features of EPDR1 include two asparagine residues with glycosylation and a Ca2+-binding site. The EPDR1 fold is very similar to the folds of bacterial VioE and LolA/LolB, which also use a similar hydrophobic pocket for their respective functions as a hydrophobic substrate-binding enzyme and a lipoprotein carrier, respectively. A further fatty-acid binding assay using EPDR1 suggests that it indeed binds to fatty acids, presumably via this pocket. Additional interactome analysis of EPDR1 showed that EPDR1 interacts with insulin-like growth factor 2 receptor and flotillin proteins, which are known to be involved in protein and vesicle translocation.

Identification of a natural ligand of the hazel allergen Cor a 1

Hazelnut is one of the most frequent causes of food allergy. The major hazel allergen in Northern Europe is Cor a 1, which is homologous to the major birch pollen allergen Bet v 1. Both allergens belong to the pathogenesis related class PR-10. We determined the solution structure of Cor a 1.0401 from hazelnut and identified a natural ligand of the protein. The structure reveals the protein fold characteristic for PR-10 family members, which consists of a seven-stranded antiparallel β-sheet, two short α-helices arranged in V-shape and a long C-terminal α-helix encompassing a hydrophobic pocket. However, despite the structural similarities between Cor a 1 and Bet v 1, they bind different ligands. We have shown previously that Bet v 1 binds to quercetin-3-O-sophoroside. Here, we isolated Cor a 1 from hazel pollen and identified the bound ligand, quercetin-3-O-(2“-O-β-D-glucopyranosyl)-β-D-galactopyranoside, by mass spectrometry and nuclear magnetic resonance spectroscopy (NMR). NMR experiments were performed to confirm binding. Remarkably, although it has been shown that PR-10 allergens show promiscuous binding behaviour in vitro, we can demonstrate that Cor a 1.0401 and Bet v 1.0101 exhibit highly selective binding for their specific ligand but not for the respective ligand of the other allergen.

Chiral Inversion of Amino Acids in Antiparallel β-Sheets at Interfaces Probed by Vibrational Sum Frequency Generation Spectroscopy.

A parallel study of protein variants with all (l-), all (d-), or mixed (l-)/(d-) amino acids can be used to assess how backbone architecture versus side chain identity determines protein structure. Here, we investigate the secondary structure and side chain orientation dynamics of the antiparallel β-sheet peptide LK7β (Ac-Leu-Lys-Leu-Lys-Leu-Lys-Leu-NH2) composed of all (l-), all (d-), or alternating (l-Leu)/(d-Lys) amino acids. Using interface-selective vibrational sum frequency generation spectroscopy (VSFG), we observe that the alternating (l-)/(d-) peptide lacks a resonant C-H stretching mode compared to the (l-) and (d-) variants and does not form antiparallel β-sheets. We rationalize our observations on the basis of density functional theory calculations and molecular dynamics (MD) simulations of LK7β at the air-water interface. Irrespective of the handedness of the amino acids, leucine side chains prefer to orient toward the hydrophobic air phase while lysine side chains prefer the hydrophilic water phase. These preferences dictate the backbone configuration of LK7β and thereby the folding of the peptide. Our MD simulations show that the preferred side chain orientations can force the backbone of a single strand of (l-) LK7β at the air-water interface to adopt β-sheet Ramachandran angles. However, denaturation of the β-sheets at pH = 2 results in a negligible chiral VSFG amide I response. The combined computational and experimental results lend critical support to the theory that a chiral VSFG response requires macroscopic chirality, such as in β-sheets. Our results can guide expectations about the VSFG optical responses of proteins and should improve understanding of how amino acid chirality modulates the structure and function of natural and de novo proteins at biological interfaces.

Functional and structural investigations of fibronectin-binding protein Apa from Mycobacterium tuberculosis

BackgroundAlanine and proline-rich protein (Apa) is a secreted antigen of Mycobacterium spp. which involves in stimulating immune responses and adhering to host cells by binding to fibronectin (Fn). Here, we report the crystal structure of Apa from Mycobacterium tuberculosis (Mtb) and its Fn-binding characteristics. MethodsThe crystal structure of Mtb Apa was determined at resolutions of 1.54 Å. The dissociation constants (KD) of Apa and individual modules of Fn were determined by surface plasmon resonance and enzyme-linked immunosorbent assay. Site-directed mutagenesis was performed to investigate the putative Fn-binding motif of Apa. ResultsMtb Apa folds into a large seven-stranded anti-parallel β-sheet which is flanked by three α-helices. The binding affinity of Mtb Apa to individual Fn modules was assessed and the results indicated that the Mtb Apa binds to FnIII-4 and FnIII-5 of Fn CBD segment. Notably, structure analysis suggested that the previously proposed Fn-binding motif 258RWFV261 is buried within the protein and may not be accessible to the binding counterpart. ConclusionsThe structural and Fn-binding characteristics we reported here provide molecular insights into the multifunctional protein Mtb Apa. FnIII-4 and FnIII-5 of CBD are the only two modules contributing to Apa-Fn interaction. General significanceThis is the first study to report the structure and Fn-binding characteristics of mycobacterial Apa. Since Apa plays a central role in stimulating immune responses and host cells adhesion, these results are of great importance in understanding the pathogenesis of mycobacterium. This information shall provide a guidance for the development of anti-mycobacteria regimen.

Role of Gln114 in Spectral Tuning of a Long-Wavelength Sensitive Visual Pigment.

Visual pigments of the long-wavelength sensitive opsin group (L group) are anion sensitive in nature. Their highly conserved amino acid residues, H197 and K200, exclusively interact with a chloride ion (Cl-) in the chromophore-binding pocket. Substitution of H197 completely abolishes Cl- binding and results in an ∼30 nm spectral blue-shift. Recent attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy studies of monkey green sensitive pigment have provided insights into the role of Cl- binding in stabilizing the antiparallel β-sheet at extracellular loop 2 (ECL2). In addition to maintaining the dark state of L opsins, Cl- binding is also believed to play a crucial role in spectral tuning. Here, we used a combination of site-directed mutagenesis in combination with UV-visible spectroscopy to show that Q1142.65 that is positioned far from ECL2 is also a crucial residue for the Cl- effect in L opsins. Comprehensive FTIR spectroscopic analyses on both ion-binding-induced and light-induced structural changes revealed that Q1142.65 contributes to the stability of β-sheet structure indirectly even though Q1142.65 is not located in ECL2. Overall, these structure-function studies are important for understanding the functional role of Cl- binding in L opsins.

Physico-chemical and nutritional properties of meat analogues based on Spirulina/lupin protein mixtures

The effect of the addition of Spirulina platensis flour and of extrusion parameters on texture, cooking yield, expressible moisture, total phenolic content (TPC), total flavonoid content (TFC), Trolox equivalent antioxidant activity (TEAC), in vitro protein digestibility (IVPD) and conformational changes of proteins using Fourier-transform infrared spectroscopy (FTIR) of lupin protein based meat analogues was studied. High moisture extrusion (HME) cooking was used to produce the meat analogues. The Spirulina concentration (15, 30 and 50%), extruder barrel temperature (145 °C, 160 °C and 170 °C), water feed (50, 55 and 60%), and screw speed (500, 800 and 1200 rpm) were varied. The Spirulina concentration and extrusion parameters significantly affected physical properties, such as texture, cooking yield and expressible moisture of the extrudates. The addition of Spirulina generally increased the TPC, TFC and TEAC values of the extrudates. Increased temperature and screw speed as well as decreased water feed slightly improved the content of TPC, TFC and TEAC, respectively. The addition of Spirulina at a level of 30% decreased the IVPD of the extrudates from 82 to 75.6%. However, increased water feed and screw speed partly counterbalanced this effect. Protein conformational analyses of the extrudates by FTIR showed that β-sheets were decreased, whereas α-helix, β-turn and antiparallel β-sheets were increased compared to the raw extrusion mixtures. On the whole, the HME process improved the values of TPC, TFC, TEAC and IVPD in the extrudates compared to the raw extrusion mixtures. The addition of Spirulina along with controlled extrusion parameters can deliver meat analogues with improved physico-chemical and nutritional properties.

Chiral Water Superstructures around Antiparallel β-Sheets Observed by Chiral Vibrational Sum Frequency Generation Spectroscopy.

Hydration modulates every aspect of protein structure and function. However, studying water structures in hydration shells remains challenging mostly due to overwhelming background from bulk water. We used vibrational sum frequency generation (SFG) spectroscopy to characterize hydrated films of an antiparallel β-sheet peptide (LK7β) adsorbed on glass slides. The hydrated films give chiral SFG response from water only when the peptide self-assembles into antiparallel β-sheets. Experiments of isotopic labeling, isotopic dilution of water, and H2O-D2O exchange kinetics corroborate the assignments of the chiral SFG response to water stretching modes. Because individual water molecules are achiral, the chiral SFG response indicates formation of chiral superstructures of water around the antiparallel β-sheet, implying that a protein secondary structure can imprint its chirality onto the surrounding water. This result demonstrates chiral SFG spectroscopy as a promising tool for probing water structures in protein hydration and addressing fundamental questions of protein structure-function.

Fourier transform infrared microspectroscopy detects biochemical changes during C. elegans lifespan

This study reports on the use of Fourier transform infrared (FTIR) microspectroscopy imaging to investigate biochemical changes occurring during C. elegans lifespan. C. elegans wild-type (N2) and the tub-1 mutant strain were cultured in agar plates. FTIR imaging was performed on single worms in transmission mode at day 8, 11, and 15. Principal component analysis was then performed to analyze the spectra acquired during C. elegans lifespan. The FTIR spectra were clustered in three groups corresponding to the spectra acquired from the worms at day 8, 11, and 15. The results showed major changes in lipids (vibration 1744 cm−1 assigned to CO stretching) and proteins (vibrations 1648 cm−1 assigned to CO stretching of amide I and 1548 cm−1 assigned to NH bending and C–N stretching in amid II). The vibration assigned to glycogen around 1155 cm−1 was present in the spectra acquired at day 8 and 11 but the peak area decreased by 49.3% and 73.3% in the spectra acquired at day 15 respectively from WT(N2) and tub-1 mutant strain. Furthermore, PC-1 loadings as a function of wavenumbers show that the presence of the vibration 1698 cm−1, attributed to antiparallel β-sheet, could indicate the formation of lipofuscin. The results obtained demonstrate that FTIR imaging could be used as a tool to monitor biochemical changes during lifespan studies, which could bring additional information to our understanding of longevity.

Aggregation of amylin: Spectroscopic investigation

Apart from its relevance to pathology, protein misfolding disease like Type-II Diabetes Mellitus, caused by amyloids of amylin protein has attracted more attention due to structural changes occurring during the aggregation process. We report extensive spectroscopy data of amylin during fibril formation through Raman, FTIR, CD, UV–vis absorption and photoluminescence (PL) spectroscopy. UV–vis and PL spectrum showed the sigmoidal growth of fibril with a lag time of ~2 days, which is consistent with earlier reported work using dynamic light scattering (DLS). Raman spectra revealed the formation of parallel and anti-parallel β-sheet from 0% to 20% with ageing (1st day to 21st day) at pH 6.5 ± 0.1. The results are corroborated by CD and FTIR data. These show the change in β-sheet by 23% at pH 6.5 ± 0.1, 26% at pH = 1.0 ± 0.1 and 30% at pH = 12 ± 0.1. It is also shown that the formation and conversion of other secondary structures into β-sheet is very sensitive towards the pH and ageing. The study may be used for the development of therapeutic strategies that could inhibit or even reverse the process of aggregation.

Secondary Structural Changes in Proteins as a Result of Electroadsorption at Aqueous-Organogel Interfaces.

The electroadsorption of proteins at aqueous-organic interfaces offers the possibility to examine protein structural rearrangements upon interaction with lipophilic phases, without modifying the bulk protein or relying on a solid support. The aqueous-organic interface has already provided a simple means of electrochemical protein detection, often involving adsorption and ion complexation; however, little is yet known about the protein structure at these electrified interfaces. This work focuses on the interaction between proteins and an electrified aqueous-organic interface via controlled protein electroadsorption. Four proteins known to be electroactive at such interfaces were studied: lysozyme, myoglobin, cytochrome c, and hemoglobin. Following controlled protein electroadsorption onto the interface, ex situ structural characterization of the proteins by FTIR spectroscopy was undertaken, focusing on secondary structural traits within the amide I band. The structural variations observed included unfolding to form aggregated antiparallel β-sheets, where the rearrangement was specifically dependent on the interaction with the organic phase. This was supported by MALDI ToF MS measurements, which showed the formation of protein-anion complexes for three of these proteins, and molecular dynamic simulations, which modeled the structure of lysozyme at an aqueous-organic interface. On the basis of these findings, the modulation of protein secondary structure by interfacial electrochemistry opens up unique prospects to selectively modify proteins.

Microstructural and conformational changes of gluten proteins in wheat-rye sourdough

Changes in the microstructure and secondary structure of sourdough gluten proteins were studied. Four strains of lactic acid bacteria (LAB) were used in fermentations, Lactobacillus delbrueckii subsp. bulgaricus (LB), Lactobacillus fermentum (LF), Lactobacillus plantarum (LP), and Pediococcus pentosaceus (PP). LAB fermentative profiles were performed by potenciometric measurements, microstructural analyses by scanning electron microscopy (SEM), and conformational changes by Raman spectroscopy. Gluten proteins experienced different degree of depolymerisation into defined microstructures, as fibril networks and laminar-like structures, and their occurrence was associated to an increase in β-sheets structures. These changes were dependent upon the distinctive acidifying kinetics of each strain. Gluten proteins of LF and LP sourdough were preferably arranged into laminar structures, and, in both cases, parallel β-sheet was the predominant conformation. Meanwhile for LB and PP sourdough, the development of three-dimensional fibril networks prevailed and it was accompanied by an increase of antiparallel β-sheets. LAB fermentative activity also lead to modifications in disulphide bonds and in the microenvironment concerning aromatic amino acids.

Diversity in protein secondary structure, molecular weight, mineral and amino acid composition of lentil and horse gram germplasm.

Lentil and horse gram germplasm was assessed for variety in seed and flour properties. Horsegram grains showed higher a* and b* and lower L* values as compared to lentil grains indicating lentil grains were lighter in color as compared to horse gram. Both the pulses showed significant differential accumulation of minerals. Flours from horse gram lines showed higher Mn, K, Mg, Na, Zn and Ca content and lower Cu and Fe content as compared to lentil lines. Polypeptide of 42kDa was present in IC94636 and IC139555 only and 35kDa PP subunit was absent in all the horse gram lines except IC94636. Major polymorphism among lentil lines was observed in 10, 35-37 and 55-49kDa PP subunits. Amount of β-sheets and β-turns was the highest whereas that of antiparallel β-sheets was the lowest. NIC17550, NIC17551 and NIC17552 showed higher content of antiparallel β-sheets and random coils among lentil lines. PL1 showed the highest portion of α-helixes and β-turns whereas PL57 showed the highest proportion of β-sheets among lentil lines. Lentil flours showed higher proportion of aspartic acid, glutamic acid, asparagine, serine, citrulline and serine and lower proportion of histidine, threonine, GABA, tyrosine and cystine as compared to horse gram.

Linking the Sequence, Anti-Tumor Function, and Shared Structural Features of Class Ib Hydrophobins

Hydrophobins are low molecular weight (5-20 kDa) self-assembling proteins secreted by fungi that accumulate at hydrophobic-hydrophilic interfaces and are extremely surface-active. Hydrophobins may undergo structural rearrangement and oligomerize to form rodlets, which are an insoluble functional amyloid that coats fungal spores to act as a water repellent, facilitate dispersal into the air, and prevent immune recognition. Due to their biochemical properties hydrophobins are a target a wide range of potential commercial applications. Hydrohphobins might also find use as a cancer treatment as they have also been shown to have anti-tumor properties, although the precise mechanism of this action is unclear. To better understand which sequence characteristics determine hydrophobin properties, we are characterizing the structure and properties of class IB hydrophobins from various fungal sources. Target proteins from Serpula lacrymans (SL1), Wallemia ichthyophaga (WI1), and Phanerochaete carnosa (PC1) were chosen for study due to their different sequence composition. We expressed uniformly 13C/15N-labeled protein in E. coli and then purified it to homogeneity using Ni2+ affinity and RP-HPLC and determined the high-resolution structure using NMR spectroscopy. We discovered that each hydrophobin contains shared structural features despite their dissimilar sequence compositions. The core feature is a four strand anti-parallel β-sheet that is connected by three loop sequences (L1-L3). In all hydrophobins the β-sheet folds to form a β-barrel-like structure, however the first loop is disordered in WI1 while it is an a-helix in SL1 and PC1. Spectroscopic amyloid formation assays indicate that these hydrophobins have differing propensities to form rodlets and are using AFM to characterize rodlet morphology. We are also examining which sequence and structure characteristics of hydrophobins influence their anti-tumour activity. Overall, this work will provide a fundamental correlation between the structure, function, and anti-tumour properties of hydrophobins.

Crystal structure of the Vibrio cholerae VqmA–ligand–DNA complex provides insight into ligand-binding mechanisms relevant for drug design

VqmA is a highly conserved transcriptional regulator of the quorum-sensing system of Vibrio cholerae, a major human pathogen that continues to imperil human health. VqmA represses biofilm formation and plays an important role in V. cholerae pathogenicity in the human host. Although VqmA's biological function is well understood, the molecular mechanisms by which its specific ligand (and effector), 3,5-dimethylpyrazine-2-ol (DPO), controls transcription of the target gene, vqmR, remain obscure. To elucidate the molecular mechanism of DPO binding, we used structural analyses and biochemical assays to study the V. cholerae VqmA-DPO-DNA complex. These analyses revealed that VqmA contains an N-terminal homodimer domain (PAS) and a C-terminal DNA-binding domain (DBD). We observed that VqmA directly binds to a DPO molecule via a compact hydrophobic pocket, consisting of a six-stranded antiparallel β-sheet and several α-helices. We also found that the VqmA dimer interacts with the quasi-palindromic sequence of the vqmR promoter through its DBD. The results of the biochemical studies indicated that a water atom and VqmA residues Phe-67 and Lys-101 play a key role in effector recognition, which is also assisted by Tyr-36 and Phe-99. This is the first molecular level view of the VqmA dimer bound to DPO and DNA. The structure-function analyses presented here improve our understanding of the complex mechanisms in the transcriptional regulation of VqmA in Vibrio spp. and may inform the design of drugs to manage V. cholerae infections.