Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer

A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP–HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin β₄ can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI–MS (electrospray ionization–mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin β₄ did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin β₄ as well as of other proteins and peptides.     

The Conserved PFT1 Tandem Repeat Is Crucial for Proper Flowering in Arabidopsis thaliana

It is widely appreciated that short tandem repeat (STR) variation underlies substantial phenotypic variation in organisms. Some propose that the high mutation rates of STRs in functional genomic regions facilitate evolutionary adaptation. Despite their high mutation rate, some STRs show little to no variation in populations. One such STR occurs in the Arabidopsis thaliana gene PFT1 (MED25), where it encodes an interrupted polyglutamine tract. Although the PFT1 STR is large (∼270 bp), and thus expected to be extremely variable, it shows only minuscule variation across A. thaliana strains. We hypothesized that the PFT1 STR is under selective constraint, due to previously undescribed roles in PFT1 function. We investigated this hypothesis using plants expressing transgenic PFT1 constructs with either an endogenous STR or synthetic STRs of varying length. Transgenic plants carrying the endogenous PFT1 STR generally performed best in complementing a pft1 null mutant across adult PFT1-dependent traits. In stark contrast, transgenic plants carrying a PFT1 transgene lacking the STR phenocopied a pft1 loss-of-function mutant for flowering time phenotypes and were generally hypomorphic for other traits, establishing the functional importance of this domain. Transgenic plants carrying various synthetic constructs occupied the phenotypic space between wild-type and pft1 loss-of-function mutants. By varying PFT1 STR length, we discovered that PFT1 can act as either an activator or repressor of flowering in a photoperiod-dependent manner. We conclude that the PFT1 STR is constrained to its approximate wild-type length by its various functional requirements. Our study implies that there is strong selection on STRs not only to generate allelic diversity, but also to maintain certain lengths pursuant to optimal molecular function.     

Open and Closed Conformations of the Isolated Transmembrane Domain of Death Receptor 5 Support a New Model of Activation

It has long been presumed that activation of the apoptosis-initiating Death Receptor 5, as well as other structurally homologous members of the TNF-receptor superfamily, relies on ligand-stabilized trimerization of noninteracting receptor monomers. We and others have proposed an alternate model in which the TNF-receptor dimer—sitting at the vertices of a large supramolecular receptor network of ligand-bound receptor trimers—undergoes a closed-to-open transition, propagated through a scissorslike conformational change in a tightly bundled transmembrane (TM) domain dimer. Here we have combined electron paramagnetic resonance spectroscopy and potential-of-mean force calculations on the isolated TM domain of the long isoform of DR5. The experiments and calculations both independently validate that the opening transition is intrinsic to the physical character of the TM domain dimer, with a significant energy barrier separating the open and closed states.Death receptor 5 (DR5) is a member of the tumor necrosis factor receptor (TNFR) superfamily that mediates apoptosis when bound by its cognate ligand, TNF-related apoptosis-inducing ligand (1). Upregulated in cancer cells, DR5 is among the most actively pursued anticancer targets (2). TNF-related apoptosis-inducing ligand binds to preassembled DR5 trimers at their extracellular domains, causing the formation of oligomeric ligand-receptor networks that are held together by receptor dimers (3). In the long-isoform of DR5, this dimer is crosslinked via ligand-induced disulfide bond formation between two transmembrane (TM) domain α-helices at Cys-209, and is further stabilized by a GxxxG motif one helix-turn downstream (3).Our recent study of the structurally homologous TNFR1 showed that receptor activation involves a conformational change that propagates from the extracellular domain to the cytosolic domain through a separation (or opening) of the TM domains of the dimer (4). We have therefore hypothesized that the activation of DR5, and indeed all structurally homologous TNF-receptors, involves a scissorslike opening of the TM domain dimer (Fig. 1).Open in a separate windowFigure 1Activation model of the DR5-L TM dimer. The sequence and positions of the disulfide bond and TOAC spin label (top), along with our previously published model (bottom, left) are shown. We propose an activation model (bottom, right) in which the transmembrane dimer pivots at its disulfide bond to reach an active open conformation.Using electron paramagnetic resonance (EPR) spectroscopy, a technique that has been used previously to study TM helix architecture and dynamics (5,6), and potential-of-mean force (PMF) calculations (7,8), this study addresses the question of whether the isolated disulfide-linked DR5-L TM domain dimer occupies distinct open and closed states (Fig. 1), and how its dynamic behavior contributes to the free-energy landscape of the opening transition of the full-length receptor.The DR5-L TM domain was synthesized with TOAC, an amino acid with a nitroxide spin label rigidly fixed to the α-carbon (9), incorporated at position 32 (Fig. 1), with some minor modification to facilitate EPR measurements. Previous work confirmed that this peptide forms disulfide-linked dimers (e.g., via comparison to 2-ME treated sample) and a negligible population of higher-order oligomers (further supported by model fitting of the EPR data below). For peptide work, residues were renumbered such that Thr-204 corresponds to Thr-1, and so on. The cytosolic Cys-29 (which we previously showed does not participate in a disulfide bond in cells) was replaced with serine to prevent the formation of antiparallel disulfide-linked dimers, and Trp-34 was replaced with tyrosine to prevent intrinsic fluorescence in fluorescence studies (not published). Continuous-wave (CW) dipolar EPR (sensitive only to spin-spin distances <25 Å) was used to measure TOAC-TOAC distances within the TM dimers and revealed an ordered Gaussian distribution centered at 16 Å (full width half-maximum (FWHM) = 4 Å), corresponding to a closed state (Fig. 2 A). Double electron-electron resonance (DEER) (sensitive to spin-spin distances from 15 to 60 Å) also detected a short distance consistent with the dipolar EPR data, along with a longer, disordered component (32.9 Å, FWHM = 28 Å) (Fig. 2 B). Together, these measurements indicate the presence of a compact, ordered closed state and a broader, disordered open state. EPR on oriented membranes also indicated two structural states. Global fitting revealed two populations of spin-label tilt angles (orientation of the nitroxide principal axis relative to the membrane normal): a narrow conformation (24°, FWHM = 20°), and a disordered conformation (50°, FWHM = 48°) (Fig. 2 C). This bimodal orientational distribution (Fig. 2 C) is remarkably consistent with the bimodal distance distribution (Fig. 2 B).Open in a separate windowFigure 2EPR spectra (left) of 32-TOAC-DR5 in lipid, and resulting structural distributions (right). (A) CW dipolar EPR spectra (left) of dimer (1 mM diamide) and monomer (1 mM 2-mercaptoethanol). Best-fit spin-spin distance distribution was a single Gaussian centered at 16 ± 2 Å (right). (B) The DEER waveform (left) of 32-TOAC-DR5 dimer was best fit (right) to a two-Gaussian distribution. The short distance was constrained to agree with the CW data, because DEER has poor sensitivity for distances <20 Å. The long-distance distribution is centered at 32.9 Å and is much broader. (C) CW EPR spectra (left) of 32-TOAC-DR5, with the membrane-normal oriented parallel (red) and perpendicular (blue) to the field. Simultaneous (global) fitting of these spectra reveals narrow and broad components (right). (In panels B and C, the overall distribution is plotted as black, while the closed and open components are plotted as green and magenta, respectively.)We subsequently conducted a PMF calculation (10) using the DR5-L TM dimer starting configuration developed by our group previously (3), embedded in a DMPC bilayer, with the Leu-32/Leu-32 C_α distance as the reaction coordinate. Three calculations were run from independent starting configurations, each using 50 windows spaced in 0.5° increments, and run for 20 ns at each window (totaling 3 μs). Each of the calculations yielded a similar result, and the averaged free energy curve (Fig. 3 A) agrees remarkably well with our EPR measurements: a narrow distribution at the closed conformation (∼16 Å, Fig. 3 B) separated by an ∼3 kcal/mol energy barrier from a broad distribution of accessible open conformations at ∼27 Å, (Fig. 3 C). Each of the three individual PMF plots can be found in Fig. S1 in the Supporting Material.Open in a separate windowFigure 3(A) PMF calculation of the DR5 TM domain dimer along the Leu-32/Leu-32 distance reaction coordinate. The PMF calculation reveals a narrow closed state and a broader open state separated by a free energy barrier. Representative snapshots of the (B) closed state and (C) open state.In the closed state, the helices are tightly packed at the GxxxG interfacial motif and all the way down the juxtaposed helix faces at residues Ala-18, Leu-22, Ala-25, and Val-26. The tight packing is aided by kinking and twisting of the two helices around their common axis, increasing the interacting surface area. In the open conformations, the Ala-18, Leu-22, Ala-25, and Val-26 pairs are dissociated and, interestingly, the GxxxG motif at Gly-10 and Gly-14 remains tightly packed. The open state energy well is only slightly less favorable than the closed state (by ∼2 kcal/mol), and its free energy profile is relatively broad and flat. The increased crossing angle in the open state is facilitated by straightening of the helix kink and is not accommodated by a change in bilayer thickness (see Fig. S3, A and B).The observed change in helix-helix distance (11 Å between the two minima in the PMF) is extremely close to that observed previously in live-cell FRET studies of a constitutively active form of TNFR1 (∼8 Å change between states using large fluorescence probes at the cytosolic domains) (4). The change observed in the EPR data (17 Å) may be an overestimate because the measurement is made between TOAC spin labels that likely protrude from the two helices, depending on rotational orientation. These results collectively show that activation of these receptors requires a small, but clearly significant conformational opening of the TM domains. One important note is that our EPR experiments recapitulate the equilibrium distribution of the two states despite there being no driving force to traverse the barrier between them (∼3 kcal/mol in the closed-to-open transition and ∼1 kcal/mol in the open-to-closed transition, Fig. 3). We do not interpret the results to mean that the dimer necessarily traverses these barriers at 4°C. Rather, there likely exist multiple reaction paths for dimerization of the abstracted TM domains. Finally, in the context of the full-length receptor, how the ligand induces a conformational change capable of overcoming the closed-to-open barrier remains an important question.Whether the observed structural transition in the TM domain dimer of the long-isoform of DR5 is a ubiquitous conformational switch that acts over the entire TNFR superfamily remains unknown. Vilar et al. (11) first proposed a similar scissors-model for activation of p75 neurotrophin receptor, which has a cysteine at the center of its TM helix. The short isoform of DR5 lacks a TM domain cysteine, but does form noncovalent dimers in cells, with likely TM domain dimer contacts (3). Among the other closely related and structurally homologous members of the TNFR superfamily, TNFR1 contains a cysteine at the center of the TM domain, but lacks any discernible small residue motifs (e.g., GxxxG). TNFR2 lacks a TM cysteine on the extracellular side, but does have a GxxxG motif positioned similarly to that of DR5. On the other hand, Death Receptor 4, whose functional distinction from DR5 has remained somewhat elusive, lacks both a cysteine and any recognizable small-residue hydrophobic motif.In summary, we have extended recent findings that point to the TM domain of DR5 as an essential structural component in the conformational change associated with activation. Our findings that the DR5-L TM domain occupies distinct open and closed states, separated by a substantial energy barrier, points the way to further studies across the TNF-receptor superfamily.     

A Systematic Approach to Species–Level Identification of Chile Pepper (Capsicum spp.) Seeds: Establishing the Groundwork for Tracking the Domestication and Movement of Chile Peppers through the Americas and Beyond

A Systematic Approach to Species–Level Identification of Chile Pepper (Capsicum spp.) Seeds: Establishing the Groundwork for Tracking the Domestication and Movement of Chile Peppers through the Americas and Beyond The chile pepper (Capsicum spp.), a plant held in great esteem throughout history, was independently domesticated in a series of places including highland Bolivia, central Mexico, the Amazon, the Caribbean, and other locales with a particularly long history of cultivation and use in the central Andes of South America. Though identification of chile pepper species through fruit morphology is possible and has been utilized by botanists studying modern and archaeological specimens, species–level identification of Capsicum seeds has remained undetermined. Given the greater abundance of seed remains in the archaeological record due to the higher likelihood of preservation, the ability to identify specific Capsicum domesticates has profound implications for tracking the domestication and spread of chile peppers prehistorically through the Americas and historically through trade and exchange to the rest of the world. This article presents a systematic procedure to identify Capsicum seeds to the species level created by adopting a morphometric approach to compare attributes of modern Capsicum seeds to archaeological seeds.     

INCIPIENT SPECIATION OF SEA STAR POPULATIONS BY ADAPTIVE GAMETE RECOGNITION COEVOLUTION

Reproductive isolation—the key event in speciation—can evolve when sexual conflict causes selection favoring different combinations of male and female adaptations in different populations. Likely targets of such selection include genes that encode proteins on the surfaces of sperm and eggs, but no previous study has demonstrated intraspecific coevolution of interacting gamete recognition genes under selection. Here, we show that selection drives coevolution between an egg receptor for sperm (OBi1) and a sperm acrosomal protein (bindin) in diverging populations of a sea star (Patiria miniata). We found positive selection on OBi1 in an exon encoding part of its predicted substrate‐binding protein domain, the ligand for which is found in bindin. Gene flow was zero for the parts of bindin and OBi1 in which selection for high rates of amino acid substitution was detected; higher gene flow for other parts of the genome indicated selection against immigrant alleles at bindin and OBi1. Populations differed in allele frequencies at two key positively selected sites (one in each gene), and differences at those sites predicted fertilization rate variation among male–female pairs. These patterns suggest adaptively evolving loci that influence reproductive isolation between populations.     

Interference competition in entomopathogenic nematodes: male Steinernema kill members of their own and other species

There is evidence of competition within and between helminth species, but the mechanisms involved are not well described. In interference competition, organisms prevent each other from using the contested resource through direct negative interactions, either chemical or physical. Steinernema spp. are entomopathogenic nematodes; they enter a living insect host which they kill and consume with the aid of symbiotic bacteria. Several studies have demonstrated intra- and interspecific competition in Steinernema, mediated by a scramble for resources and by incompatibility of the bacterial symbiont. Here we describe a mechanism by which male Steinernema may compete directly for resources, both food (host) and females, by physically injuring or killing members of another species as well as males of their own species. A series of experiments was conducted in hanging drops of insect haemolymph. Males of each of four species (Steinernemalongicaudum, Steinernemacarpocapsae, Steinernemakraussei and Steinernemafeltiae), representing three of the five phylogenetic clades of the genus, killed each other. Within 48 h, up to 86% of pairs included at least one dead male, compared with negligible mortality in single male controls. There was evidence of intraspecific difference: one strain of S. feltiae (4CFMO) killed while another (UK76) did not. Males also killed both females and males of other Steinernema spp. There was evidence of a hierarchy of killing, with highest mortality due to S. longicaudum followed by S. carpocapsae, S. kraussei and S. feltiae. Wax moth larvae were co-infected with members of two Steinernema spp. to confirm that killing also takes place in the natural environment of an insect cadaver. When insects were co-infected with one infective juvenile of each species, S. longicaudum males killed both S. feltiae UK76 and Steinernema hermaphroditum. Wax moths co-infected with larger, equal numbers of S. longicaudum and S. feltiae UK76 produced mainly S. longicaudum progeny, as expected based on hanging drop experiments.