Binding Hot Spot in the Weak Protein Complex of Physiological Redox Partners Yeast Cytochrome c and Cytochrome c Peroxidase

Transient protein interactions mediate many vital cellular processes such as signal transduction or intermolecular electron transfer. However, due to difficulties associated with their structural characterization, little is known about the principles governing recognition and binding in weak transient protein complexes. In particular, it has not been well established whether binding hot spots, which are frequently found in strong static complexes, also govern transient protein interactions. To address this issue, we have investigated an electron transfer complex of physiological partners from yeast: yeast iso-1-cytochrome c (Cc) and yeast cytochrome c peroxidase (CcP). Using isothermal titration calorimetry and NMR spectroscopy, we show that Cc R13 is a hot-spot residue, as R13A mutation has a strong destabilizing effect on binding. Furthermore, we employ a double-mutant cycle to illustrate that Cc R13 interacts with CcP Y39. The present results, in combination with those of earlier mutational studies, have enabled us to outline the extent of the energetically important Cc-CcP binding region. Based on our analysis, we propose that binding energy hot spots, which are prevalent in static protein complexes, could also govern transient protein interactions.     

Mycophagous beetle females do not behave competitively during intrasexual interactions in presence of a fungal resource

Intrasexual interactions can determine which individuals within a population have access to limited resources. Despite their potential importance on fitness generally and mating success especially, female–female interactions are not often measured in the same species where male–male interactions are well‐defined. In this study, we characterized female–female interactions in Bolitotherus cornutus, a mycophagous beetle species native to Northeastern North America. We used dyadic, behavioral assays to determine whether females perform directly aggressive or indirectly exclusionary competitive behaviors. Polypore shelf fungus, an important food and egg‐laying resource for B. cornutus females, is patchily distributed and of variable quality, so we tested for competition over fungus as a resource. Behavior of females was assessed in three sets of dyadic trials with randomly paired female partners. Overall, females did not behave aggressively toward their female partner or perform exclusionary behaviors over the fungal resource. None of the behaviors performed by females were individually repeatable. Two scenarios may explain our lack of observed competition: our trial context may not induce competition, or female B. cornutus simply may not behave competitively in the wild. We compare our results to a similar study on male–male interactions in the same species and propose future studies on female–female interactions under different competitive contexts to expand the understanding of female competition.     

Assessing SNP-SNP Interactions among DNA Repair,Modification and Metabolism Related Pathway Genes in Breast Cancer Susceptibility

Genome-wide association studies (GWASs) have identified low-penetrance common variants (i.e., single nucleotide polymorphisms, SNPs) associated with breast cancer susceptibility. Although GWASs are primarily focused on single-locus effects, gene-gene interactions (i.e., epistasis) are also assumed to contribute to the genetic risks for complex diseases including breast cancer. While it has been hypothesized that moderately ranked (P value based) weak single-locus effects in GWASs could potentially harbor valuable information for evaluating epistasis, we lack systematic efforts to investigate SNPs showing consistent associations with weak statistical significance across independent discovery and replication stages. The objectives of this study were i) to select SNPs showing single-locus effects with weak statistical significance for breast cancer in a GWAS and/or candidate-gene studies; ii) to replicate these SNPs in an independent set of breast cancer cases and controls; and iii) to explore their potential SNP-SNP interactions contributing to breast cancer susceptibility. A total of 17 SNPs related to DNA repair, modification and metabolism pathway genes were selected since these pathways offer a priori knowledge for potential epistatic interactions and an overall role in breast carcinogenesis. The study design included predominantly Caucasian women (2,795 cases and 4,505 controls) from Alberta, Canada. We observed two two-way SNP-SNP interactions (APEX1-rs1130409 and RPAP1-rs2297381; MLH1-rs1799977 and MDM2-rs769412) in logistic regression that conferred elevated risks for breast cancer (P _interaction<7.3×10⁻³). Logic regression identified an interaction involving four SNPs (MBD2-rs4041245, MLH1-rs1799977, MDM2-rs769412, BRCA2-rs1799943) (P _permutation = 2.4×10⁻³). SNPs involved in SNP-SNP interactions also showed single-locus effects with weak statistical significance, while BRCA2-rs1799943 showed stronger statistical significance (P _{correlation/trend} = 3.2×10⁻⁴) than the others. These single-locus effects were independent of body mass index. Our results provide a framework for evaluating SNPs showing statistically weak but reproducible single-locus effects for epistatic effects contributing to disease susceptibility.     

Novel structural insights into rotavirus recognition of ganglioside glycan receptors

Rotaviruses ubiquitously infect children under the age of 5, being responsible for more than half a million diarrhoeal deaths each year worldwide. Host cell oligosaccharides containing sialic acid(s) are critical for attachment by rotaviruses. However, to date, no detailed three-dimensional atomic model showing the exact rotavirus interactions with these glycoconjugate receptors has been reported. Here, we present the first crystallographic structures of the rotavirus carbohydrate-recognizing protein VP8^? in complex with ganglioside G_M3 glycans. In combination with assessment of the inhibition of rotavirus infectivity by N-acetyl and N-glycolyl forms of this ganglioside, our results reveal key details of rotavirus-ganglioside G_M3 glycan recognition. In addition, they show a direct correlation between the carbohydrate specificities exhibited by VP8^? from porcine and by monkey rotaviruses and the respective infectious virus particles. These novel results also indicate the potential binding interactions of rotavirus VP8^? with other sialic acid-containing gangliosides.     

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets^1-3. Since they function in complexes⁴, methods to identify and characterize their interactions are necessary⁵. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE⁶. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.     

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions ¹. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage ^{2, 3}. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle ^4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection ⁷. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization ⁸. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.     

Trypanosoma brucei: Nearest neighbor analysis on the major variable surface coat glycoprotein—Crosslinking patterns with intact cells

Cleavable Crosslinking reagents were used to study interactions among proteins of the surface coat of Trypanosoma brucei. The proteins were resolved by two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When intact cells were treated with dithiobis(succinimidylpropionate), we obtained extensive intermolecular Crosslinking of major variable surface coat glycoprotein (VSCG) molecules. This reagent generated no apparent crosslinks between VSCG and other membrane-associated proteins. Complete conversion to oligomers equal to or greater than octamers occurred within 20 min. When purified VSCG in solution was treated with dithiobis(succimidylpropionate), dimers were found. A complex of Cu²⁺ and 1,10-phenanthroline was used to catalyze air oxidation of adjacent sulfhydryls to disulfide bonds; however, no crosslinking among VSCG molecules nor between VSCG and other proteins was observed. The results presented indicate that VSCG in solution exists predominately in the form of dimers. Whether VSCG in situ also occurred as dimers could not be determined; however, since we observed trimeric and tetrameric forms of VSCG when untreated cells were analyzed, it is likely that weak interactions occur among the protein molecules. These interactions are less stable than the dimer association observed with purified VSCG. Finally, the analysis indicated that VSCGs of this stock of T. brucei, derived from UGANDA/ 60/TREU/164[ETat3], contained at least one intramolecular disulfide bond. We examined T. brucei stocks 427 and EATRO 110 and obtained similar results. Thus, it appears that intramolecular disulfide bonding is a general feature of T. brucei VSCGs.     

Molecular determinants for the interaction of human neutrophil alpha defensin 1 with its propeptide

Human neutrophil α-defensins (HNPs) are cationic antimicrobial peptides that are synthesized in vivo as inactive precursors (proHNPs). Activation requires proteolytic excision of their anionic N-terminal inhibitory pro peptide. The pro peptide of proHNP1 also interacts specifically with and inhibits the antimicrobial activity of HNP1 inter-molecularly. In the light of the opposite net charges segregated in proHNP1, functional inhibition of the C-terminal defensin domain by its propeptide is generally thought to be of electrostatic nature. Using a battery of analogs of the propeptide and of proHNP1, we identified residues in the propeptide region important for HNP1 binding and inhibition. Only three anionic residues in the propeptide, Glu¹⁵, Asp²⁰ and Glu²³, were modestly important for interactions with HNP1. By contrast, the hydrophobic residues in the central part of the propeptide, and the conserved hydrophobic motif Val²⁴Val²⁵Val²⁶Leu²⁸ in particular, were critical for HNP1 binding and inhibition. Neutralization of all negative charges in the propeptide only partially activated the bactericidal activity of proHNP1. Our data indicate that hydrophobic forces have a dominant role in mediating the interactions between HNP1 and its propeptide — a finding largely contrasting the commonly held view that the interactions are of an electrostatic nature.     

Carboxyl pKa Values and Acid Denaturation of BBL

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK_a values of all the carboxylic residues in this pH range. We employed ¹³C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK_a values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK_a values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK_a, and, using thermodynamic cycles, we could calculate their pK_as in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured ¹³C^α chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.     

Evolutionary Optimization of Computationally Designed Enzymes: Kemp Eliminases of the KE07 Series

Understanding enzyme catalysis through the analysis of natural enzymes is a daunting challenge—their active sites are complex and combine numerous interactions and catalytic forces that are finely coordinated. Study of more rudimentary (wo)man-made enzymes provides a unique opportunity for better understanding of enzymatic catalysis. KE07, a computationally designed Kemp eliminase that employs a glutamate side chain as the catalytic base for the critical proton abstraction step and an apolar binding site to guide substrate binding, was optimized by seven rounds of random mutagenesis and selection, resulting in a > 200-fold increase in catalytic efficiency. Here, we describe the directed evolution process in detail and the biophysical and crystallographic studies of the designed KE07 and its evolved variants. The optimization of KE07's activity to give a k_cat/K_M value of ∼ 2600 s^− 1 M^− 1 and an ∼ 10⁶-fold rate acceleration (k_cat/k_uncat) involved the incorporation of up to eight mutations. These mutations led to a marked decrease in the overall thermodynamic stability of the evolved KE07s and in the configurational stability of their active sites. We identified two primary contributions of the mutations to KE07's improved activity: (i) the introduction of new salt bridges to correct a mistake in the original design that placed a lysine for leaving-group protonation without consideration of its “quenching” interactions with the catalytic glutamate, and (ii) the tuning of the environment, the pK_a of the catalytic base, and its interactions with the substrate through the evolution of a network of hydrogen bonds consisting of several charged residues surrounding the active site.     

New examples of low-dimensional metal-pydco complexes: Syntheses, structures and magnetic properties

This work presents a systematic investigation on coordination chemistry of a novel pyridine-2,6-dicarboxylic acid N-oxide (pydco), and also reveals the significant function of supramolecular interactions in dominating the resultant crystalline nets. Assemblies of pydco with transition-metal ions under similar conditions yield a series of polymers in the absence/presence of the organonitrogen ligands {[Cu(pydco)(L)_0.5(H₂O)] · 2H₂O}_n (L = bipy (1), bpa (2) and bpe (3)), {[M(pydco)(bpp)(H₂O)] · 2H₂O}_n (M = Cu (4) and Ni (5)), [Ag₂(pydco)]_n (6) and [Ag₂Cu(pydco)₂]_n (7) (bipy = 4,4′-bipyridine, bpa = 1,2-bis(4-pyridyl)ethane, bpe = 1,2-bis(4-pyridyl)ethene, bpp = 1,3-bis(4-pyridyl)propane). 1-5 feature different structural characteristics, although they exhibit analogous chain networks. Remarkably, extended architectures are further constructed with the aid of weak interactions. Reaction of pydco with AgAc yields a 2-D polymer 6, which was reported in our recent Communication. A mixed-metal coordination polymer 7 was obtained by the self-assembly of AgAc, Cu(Ac)₂ · H₂O and pydco.In 7, two left- and right-hand helical chains are constructed by carboxylic groups of pydco and Cu centers, which are further connected by [AgCO₂]₂ cores into a 2-D network. Structural evolution under the co-ligand intervention in this series of compounds, as well as the general coordination rule of pydco, has been further discussed. Furthermore, variable temperature magnetic properties of 1, 3 and 7 are also studied. The magnetic measurements of 1 and 3 reveal the existence of weak antiferromagnetic interactions with J₁ = −4.59 cm⁻¹ and J₂ = −4.63 cm⁻¹, respectively. Whereas 7 displays weak ferromagnetic interactions with J₃ = 1.81 cm⁻¹.     

Inhibitory and stimulatory micropeptides preferentially bind to different conformations of the cardiac calcium pump

The ATP-dependent ion pump sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) sequesters Ca²⁺ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, the activity of this transporter is tightly controlled via direct interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, the micropeptide phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca²⁺ signals that drive the contraction/relaxation cycle. We hypothesized that the functions of these peptides may relate to their reciprocal preferences for SERCA binding; SERCA binds PLB more avidly at low cytoplasmic [Ca²⁺] but binds DWORF better when [Ca²⁺] is high. In the present study, we demonstrated this opposing Ca²⁺ sensitivity is due to preferential binding of DWORF and PLB to different intermediate states that SERCA samples during the Ca²⁺ transport cycle. We show PLB binds best to the SERCA E1-ATP state, which prevails at low [Ca²⁺]. In contrast, DWORF binds most avidly to E1P and E2P states that are more populated when Ca²⁺ is elevated. Moreover, FRET microscopy revealed dynamic shifts in SERCA–micropeptide binding equilibria during cellular Ca²⁺ elevations. A computational model showed that DWORF exaggerates changes in PLB–SERCA binding during the cardiac cycle. These results suggest a mechanistic basis for inhibitory versus stimulatory micropeptide function, as well as a new role for DWORF as a modulator of dynamic oscillations of PLB–SERCA regulatory interactions.     

Long-Lived, High-Strength States of ICAM-1 Bonds to β2 Integrin, I: Lifetimes of Bonds to Recombinant αLβ2 Under Force

Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant α_Lβ₂ immobilized on microspheres and β₂ integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with integrin activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling in leukocytes. Taking advantage of very rare events, we used repeated measurements of bond lifetimes under steady ramps of force to achieve a direct assay for the off-rates of ICAM-1 from β₂ integrin in each experiment. Of fundamental importance, the assay for off-rates does not depend on how the force is applied over time, and remains valid when the rates of dissociation change with different levels of force. In this first article, we present results from tests of a monovalent ICAM-1 probe against immobilized α_Lβ₂ in environments of divalent cations (Ca²⁺, Mg²⁺, and Mn²⁺) and demonstrate in detail the method for assay of off-rates. When extrapolated to zero force, the force-free values for the off-rates are found to be consistent with published solution-based assays of soluble ICAM-1 dissociation from immobilized LFA-1, i.e., ∼10⁻²/s in Mg²⁺ or Mn²⁺ and ∼1/s in Ca²⁺. At the same time, as expected for adhesive function, we find that the β₂ integrin bonds activated in Mn²⁺ or Mg²⁺ possess significant and persistent mechanical strength (e.g., >20 pN for >1 s) even when subjected to slow force ramps (<10 pN/s). As discussed in our companion article, using the same assay, we find that although the rates of dissociation for diICAM-1fc bonds to LFA-1 on neutrophils in Mn²⁺ are similar to those for mICAM-1 bonds to recombinant α_Lβ₂ on microspheres, they appear to represent a dimeric attachment to a pair of tightly clustered integrin heterodimers. The mechanical strengths and lifetimes of the dimeric interactions increase dramatically when the neutrophils are stimulated by the chemokine IL-8 or are bound with an allosterically activating (anti-CD18) monoclonal antibody, demonstrating the major impact of cell signaling on LFA-1.     

Probing non-specific interactions of Ca2+-calmodulin in E. coli lysate

The biological environment in which a protein performs its function is a crowded milieu containing millions of molecules that can potentially lead to a great many transient, non-specific interactions. NMR spectroscopy is especially well suited to study these weak molecular contacts. Here, non-specific interactions between the Ca²⁺-bound form of calmodulin (CaM) and non-cognate proteins in Escherichia coli lysate are explored using Ile, Leu, Val and Met methyl probes. Changes in CaM methyl chemical shifts as a function of added E. coli lysate are measured to determine a minimum ‘average’ dissociation constant for interactions between Ca²⁺-CaM and E. coli lysate proteins. ²H R ₂ and ¹³C R ₁ spin relaxation rates report on the binding reaction as well. Our results further highlight the power of methyl containing side-chains for characterizing biomolecular interactions, even in complex in-cell like environments.     

Thermodynamic Characterization of ppGpp Binding to EF-G or IF2 and of Initiator tRNA Binding to Free IF2 in the Presence of GDP, GTP, or ppGpp

In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5-25 °C. We find that ppGpp has a higher affinity for IF2 than for EF-G (1.7-2.8 μM K_dversus 9.1-13.9 μM K_d at 10-25 °C), suggesting that during stringent response in vivo, IF2 is more responsive to ppGpp than to EF-G. We investigated the effects of ppGpp, GDP, and GTP on IF2 interactions with fMet-tRNA^fMet demonstrating that IF2 binds to initiator tRNA with submicromolar K_d and that affinity is altered by the G nucleotides only slightly. This—in conjunction with earlier reports on IF2 interactions with fMet-tRNA^fMet in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNA^fMet binding and GTP was suggested to strongly promote fMet-tRNA^fMet binding—sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNA^fMet selection.     

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ^1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications ^3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications ^9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment ^12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.     

Selectivity mechanisms in MscS-like channels: From structure to function

The E. coli mechanosensitive (MS) channel of small conductance (EcMscS) is the prototype of a diverse family of channels present in all domains of life. While EcMscS has been extensively studied, recent developments show that MscS may display some characteristics not widely conserved in this protein subfamily. With numerous members now electrophysiologically characterized, this subfamily of channels displays a breadth of ion selectivity with both anion and cation selective members. The selectivity of these channels may be relatively weak in comparison to voltage-gated channels but their selectivity mechanisms represent great novelty. Recent studies have identified unexpected residues important for selectivity in these homologs revealing different selectivity mechanisms than those employed by voltage gated K⁺, Na⁺, Ca²⁺ and Cl^- channels whose selectivity filters are housed within their transmembrane pores. This commentary looks at what is currently known about MscS subfamily selectivity and begins to unravel the potential physiological relevance of these differences.     

A fusion of the Bacteroides fragilis ferrous iron import proteins reveals a role for FeoA in stabilizing GTP-bound FeoB

Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe²⁺ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system is the primary prokaryotic Fe²⁺ import machinery, and two constituent proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remains enigmatic. In this work, we explored the distribution of feoAB operons encoding a fusion of FeoA tethered to the N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion poises FeoA to interact with FeoB to affect function. To test this hypothesis, we characterized the soluble NFeoAB fusion protein from Bacteroides fragilis, a commensal organism implicated in drug-resistant infections. Using X-ray crystallography, we determined the 1.50-Å resolution structure of BfFeoA, which adopts an SH3-like fold implicated in protein–protein interactions. Using a combination of structural modeling, small-angle X-ray scattering, and hydrogen–deuterium exchange mass spectrometry, we show that FeoA and NFeoB interact in a nucleotide-dependent manner, and we mapped the protein–protein interaction interface. Finally, using guanosine triphosphate (GTP) hydrolysis assays, we demonstrate that BfNFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis that is not potassium-stimulated. Importantly, truncation of FeoA from this fusion demonstrates that FeoA–NFeoB interactions function to stabilize the GTP-bound form of FeoB. Taken together, our work reveals a role for FeoA function in the fused FeoAB system and suggests a function for FeoA among prokaryotes.     

A Novel Assay for Assessing Juxtamembrane and Transmembrane Domain Interactions Important for Receptor Heterodimerization

Understanding the basis of specificity in receptor homodimerization versus heterodimerization is essential in determining the role receptor plays in signal transduction. Specificity in each of the interfaces formed during signal transduction involves cooperative interactions between receptor extracellular, transmembrane (TM), and cytoplasmic domains. While methods exist for studying receptor heterodimerization in cell membranes, they are limited to either TM domains expressed in an inverted orientation or capture only heterodimerization in a single assay. To address this limitation, we have developed an assay (DN-AraTM) that enables simultaneous measurement of homodimerization and heterodimerization of type I receptor domains in their native orientation, including both soluble and TM domains. Using integrin α_IIb and RAGE (receptor for advanced glycation end products) as model type I receptor systems, we demonstrate both specificity and sensitivity of our approach, which will provide a novel tool to identify specific domain interactions that are important in regulating signal transduction.