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Small heat-shock proteins (sHSPs) are ubiquitously expressed molecular chaperones that inhibit amyloid fibril formation; however, their mechanisms of action remain poorly understood. sHSPs comprise a conserved α-crystallin domain flanked by variable N- and C-terminal regions. To investigate the functional contributions of these three regions, we compared the chaperone activities of various constructs of human αB-crystallin (HSPB5) and heat-shock 27-kDa protein (Hsp27, HSPB1) during amyloid formation by α-synuclein and apolipoprotein C-II. Using an array of approaches, including thioflavin T fluorescence assays and sedimentation analysis, we found that the N-terminal region of Hsp27 and the terminal regions of αB-crystallin are important for delaying amyloid fibril nucleation and for disaggregating mature apolipoprotein C-II fibrils. We further show that the terminal regions are required for stable fibril binding by both sHSPs and for mediating lateral fibril–fibril association, which sequesters preformed fibrils into large aggregates and is believed to have a cytoprotective function. We conclude that although the isolated α-crystallin domain retains some chaperone activity against amyloid formation, the flanking domains contribute additional and important chaperone activities, both in delaying amyloid formation and in mediating interactions of sHSPs with amyloid aggregates. Both these chaperone activities have significant implications for the pathogenesis and progression of diseases associated with amyloid deposition, such as Parkinson's and Alzheimer's diseases.

Interleukin (IL) 11 activates multiple intracellular signaling pathways by forming a complex with its cell surface α-receptor, IL-11Rα, and the β-subunit receptor, gp130. Dysregulated IL-11 signaling has been implicated in several diseases, including some cancers and fibrosis. Mutations in IL-11Rα that reduce signaling are also associated with hereditary cranial malformations. Here we present the first crystal structure of the extracellular domains of human IL-11Rα and a structure of human IL-11 that reveals previously unresolved detail. Disease-associated mutations in IL-11Rα are generally distal to putative ligand-binding sites. Molecular dynamics simulations showed that specific mutations destabilize IL-11Rα and may have indirect effects on the cytokine-binding region. We show that IL-11 and IL-11Rα form a 1:1 complex with nanomolar affinity and present a model of the complex. Our results suggest that the thermodynamic and structural mechanisms of complex formation between IL-11 and IL-11Rα differ substantially from those previously reported for similar cytokines. This work reveals key determinants of the engagement of IL-11 by IL-11Rα that may be exploited in the development of strategies to modulate formation of the IL-11-IL-11Rα complex.

G protein–coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α1A-adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [13CϵH3]methionine-labeled α1A-adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [13CϵH3]Methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α1A-adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells.

The cell surfaces of many bacteria carry filamentous polypeptides termed adhesins that enable binding to both biotic and abiotic surfaces. Surface adherence is facilitated by the exquisite selectivity of the adhesins for their cognate ligands or receptors and is a key step in niche or host colonization and pathogenicity. Streptococcus gordonii is a primary colonizer of the human oral cavity and an opportunistic pathogen, as well as a leading cause of infective endocarditis in humans. The fibrillar adhesin CshA is an important determinant of S. gordonii adherence, forming peritrichous fibrils on its surface that bind host cells and other microorganisms. CshA possesses a distinctive multidomain architecture comprising an N-terminal target-binding region fused to 17 repeat domains (RDs) that are each ∼100 amino acids long. Here, using structural and biophysical methods, we demonstrate that the intact CshA repeat region (CshA_RD1–17, domains 1–17) forms an extended polymeric monomer in solution. We recombinantly produced a subset of CshA RDs and found that they differ in stability and unfolding behavior. The NMR structure of CshA_RD13 revealed a hitherto unreported all β-fold, flanked by disordered interdomain linkers. These findings, in tandem with complementary hydrodynamic studies of CshA_RD1–17, indicate that this polypeptide possesses a highly unusual dynamic transitory structure characterized by alternating regions of order and disorder. This architecture provides flexibility for the adhesive tip of the CshA fibril to maintain bacterial attachment that withstands shear forces within the human host. It may also help mitigate deleterious folding events between neighboring RDs that share significant structural identity without compromising mechanical stability.

The Natural Product Domain Seeker (NaPDoS) webtool detects and classifies ketosynthase (KS) and condensation domains from genomic, metagenomic, and amplicon sequence data. Unlike other tools, a phylogeny-based classification scheme is used to make broader predictions about the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes in which these domains are found. NaPDoS is particularly useful for the analysis of incomplete biosynthetic genes or gene clusters, as are often observed inpoorly assembled genomes and metagenomes, or when loci are not clustered, as in eukaryotic genomes. To help support the growing interest in sequence-based analyses ofnatural product biosynthetic diversity, here we introduceversion 2 of the webtool, NaPDoS2, available at http://napdos.ucsd.edu/napdos2. This update includes the additionof 1417 KS sequences, representing a major expansion of the taxonomic and functional diversity represented in the webtool database. The phylogeny-based KS classification scheme now recognizes 41 class and subclass assignments, including new type II PKS subclasses. Workflow modifications accelerate run times, allowing larger datasets to be analyzed. In addition, default parameters were established using statistical validation tests to maximize KS detection and classification accuracy while minimizing false positives. We further demonstrate the applications of NaPDoS2 to assess PKS biosynthetic potential using genomic, metagenomic, and PCR amplicon datasets. These examples illustrate how NaPDoS2 can be used to predict biosynthetic potential and detect genes involved in the biosynthesis of specific structure classes or new biosynthetic mechanisms.

RNA stem loop structures have been frequently shown to regulate essential cellular processes. The selenocysteine insertion sequence (SECIS) element, found in the 3′ UTRs of all selenoprotein mRNAs, is an example of such a structure, as it is required for the incorporation of the 21st amino acid, selenocysteine (Sec). Selenoprotein synthesis poses a mechanistic challenge because Sec is incorporated during translation in response to a stop codon (UGA). Although it is known that a SECIS-binding protein (SBP2) is required for Sec insertion, the mechanism of action remains elusive. Additional complexity is present in the synthesis of selenoprotein P (SELENOP), which is the only selenoprotein that contains multiple UGA codons and possesses two SECIS elements in its 3′ UTR. Thus, full-length SELENOP synthesis requires processive Sec incorporation. Using zebrafish Selenop, in vitro translation assays, and (75)Se labeling in HEK293 cells, we found here that processive Sec incorporation is an intrinsic property of the SECIS elements. Specifically, we identified critical features of SECIS elements that are required for processive Sec incorporation. A screen of the human SECIS elements revealed that most of these elements support processive Sec incorporation in vitro; however, we also found that the processivity of Sec incorporation into Selenop in cells is tightly regulated. We propose a model for processive Sec incorporation that involves differential recruitment of SECIS-binding proteins.

PHR (PAM/Highwire/RPM-1) proteins are conserved RING E3 ubiquitin ligases that function in developmental processes, such as axon termination and synapse formation, as well as axon degeneration. At present, our understanding of how PHR proteins form ubiquitin ligase complexes remains incomplete. Although genetic studies indicate NMNAT2 is an important mediator of PHR protein function in axon degeneration, it remains unknown how PHR proteins inhibit NMNAT2. Here, we decipher the biochemical basis for how the human PHR protein PAM, also called MYCBP2, forms a noncanonical Skp/Cullin/F-box (SCF) complex that contains the F-box protein FBXO45 and SKP1 but lacks CUL1. We show FBXO45 does not simply function in substrate recognition but is important for assembly of the PAM/FBXO45/SKP1 complex. Interestingly, we demonstrate a novel role for SKP1 as an auxiliary component of the target recognition module that enhances binding of FBXO45 to NMNAT2. Finally, we provide biochemical evidence that PAM polyubiquitinates NMNAT2 and regulates NMNAT2 protein stability and degradation by the proteasome.

Chloramphenicol (Cam) is a broad-spectrum antibiotic used to combat bacterial infections in humans and animals. Cam export from bacterial cells is one of the mechanisms by which pathogens resist Cam's antibacterial effects, and several different proteins are known to facilitate this process. However, to date no report exists on any specific transport protein that facilitates Cam uptake. The proton-coupled oligopeptide transporter (POT) YdgR from Escherichia coli is a prototypical member of the POT family, functioning in proton-coupled uptake of di- and tripeptides. By following bacterial growth and conducting LC-MS–based assays we show here that YdgR facilitates Cam uptake. Some YdgR variants displaying reduced peptide uptake also exhibited reduced Cam uptake, indicating that peptides and Cam bind YdgR at similar regions. Homology modeling of YdgR, Cam docking, and mutational studies suggested a binding mode that resembles that of Cam binding to the multidrug resistance transporter MdfA. To our knowledge, this is the first report of Cam uptake into bacterial cells mediated by a specific transporter protein. Our findings suggest a specific bacterial transporter for drug uptake that might be targeted to promote greater antibiotic influx to increase cytoplasmic antibiotic concentration for enhanced cytotoxicity.

Inflammatory bowel diseases (IBDs) are a set of complex and debilitating diseases for which there is no satisfactory treatment. Recent studies have shown that small peptides show promise for reducing inflammation in models of IBD. However, these small peptides are likely to be unstable and rapidly cleared from the circulation, and therefore, if not modified for better stability, represent non-viable drug leads. We hypothesized that improving the stability of these peptides by grafting them into a stable cyclic peptide scaffold may enhance their therapeutic potential. Using this approach, we have designed a novel cyclic peptide that comprises a small bioactive peptide from the annexin A1 protein grafted into a sunflower trypsin inhibitor cyclic scaffold. We used native chemical ligation to synthesize the grafted cyclic peptide. This engineered cyclic peptide maintained the overall fold of the naturally occurring cyclic peptide, was more effective at reducing inflammation in a mouse model of acute colitis than the bioactive peptide alone, and showed enhanced stability in human serum. Our findings suggest that the use of cyclic peptides as structural backbones offers a promising approach for the treatment of IBD and potentially other chronic inflammatory conditions.

The ubiquitous second messenger cAMP mediates signal transduction processes in the malarial parasite that regulate host erythrocyte invasion and the proliferation of merozoites. In Plasmodium falciparum, the central receptor for cAMP is the single regulatory subunit (R) of protein kinase A (PKA). To aid the development of compounds that can selectively dysregulate parasite PKA signaling, we solved the structure of the PKA regulatory subunit in complex with cAMP and a related analogue that displays antimalarial activity, (Sp)-2-Cl-cAMPS. Prior to signaling, PKA-R holds the kinase's catalytic subunit (C) in an inactive state by exerting an allosteric inhibitory effect. When two cAMP molecules bind to PKA-R, they stabilize a structural conformation that facilitates its dissociation, freeing PKA-C to phosphorylate downstream substrates such as apical membrane antigen 1. Although PKA activity was known to be necessary for erythrocytic proliferation, we show that uncontrolled induction of PKA activity using membrane-permeable agonists is equally disruptive to growth.

Mycobacterium tuberculosis DosS is critical for the induction of M. tuberculosis dormancy genes in response to nitric oxide (NO), carbon monoxide (CO), or hypoxia. These environmental stimuli, which are sensed by the DosS heme group, result in autophosphorylation of a DosS His residue, followed by phosphotransfer to an Asp residue of the response regulator DosR. To clarify the mechanism of gaseous ligand recognition and signaling, we investigated the hydrogen-bonding interactions of the iron-bound CO and NO ligands by site-directed mutagenesis of Glu-87 and His-89. Autophosphorylation assays and molecular dynamics simulations suggest that Glu-87 has an important role in ligand recognition, whereas His-89 is essential for signal transduction to the kinase domain, a process for which Arg-204 is important. Mutation of Glu-87 to Ala or Gly rendered the protein constitutively active as a kinase, but with lower autophosphorylation activity than the wild-type in the Fe(II) and the Fe(II)-CO states, whereas the E87D mutant had little kinase activity except for the Fe(II)-NO complex. The H89R mutant exhibited attenuated autophosphorylation activity, although the H89A and R204A mutants were inactive as kinases, emphasizing the importance of these residues in communication to the kinase core. Resonance Raman spectroscopy of the wild-type and H89A mutant indicates the mutation does not alter the heme coordination number, spin state, or porphyrin deformation state, but it suggests that interdomain interactions are disrupted by the mutation. Overall, these results confirm the importance of the distal hydrogen-bonding network in ligand recognition and communication to the kinase domain and reveal the sensitivity of the system to subtle differences in the binding of gaseous ligands.

CXCR4 is a G protein-coupled receptor with excellent potential as a therapeutic target for a range of clinical conditions, including stem cell mobilization, cancer prognosis and treatment, fibrosis therapy, and HIV infection. We report here the development of a fully human single-domain antibody-like scaffold termed an "i-body," the engineering of which produces an i-body library possessing a long complementarity determining region binding loop, and the isolation and characterization of a panel of i-bodies with activity against human CXCR4. The CXCR4-specific i-bodies show antagonistic activity in a range of in vitro and in vivo assays, including inhibition of HIV infection, cell migration, and leukocyte recruitment but, importantly, not the mobilization of hematopoietic stem cells. Epitope mapping of the three CXCR4 i-bodies AM3-114, AM4-272, and AM3-523 revealed binding deep in the binding pocket of the receptor.

Mutations affecting the SECISBP2 protein necessary for selenocysteine incorporation are linked to human disease, but with a wide range of clinical outcomes. To gain insight into this diversity, Zhao et al. dissect the phenotypic and molecular consequences of two specific mutations in the Secisbp2 gene that partially disrupt selenoprotein synthesis. They observe surprising tissue-dependent effects, emphasizing the complexities of translational science.

Selenoproteins are unique as they contain selenium in their active site in the form of the 21st amino acid selenocysteine (Sec), which is encoded by an in-frame UGA stop codon. Sec incorporation requires both cis- and trans-acting factors, which are known to be sufficient for Sec incorporation in vitro, albeit with low efficiency. However, the abundance of the naturally occurring selenoprotein that contains 10 Sec residues (SEPP1) suggests that processive and efficient Sec incorporation occurs in vivo. Here, we set out to study native SEPP1 synthesis in vitro to identify factors that regulate processivity and efficiency. Deletion analysis of the long and conserved 3'-UTR has revealed that the incorporation of multiple Sec residues is inherently processive requiring only the SECIS elements but surprisingly responsive to the selenium concentration. We provide evidence that processive Sec incorporation is linked to selenium utilization and that reconstitution of known Sec incorporation factors in a wheat germ lysate does not permit multiple Sec incorporation events, thus suggesting a role for yet unidentified mammalian-specific processes or factors. The relationship between our findings and the channeling theory of translational efficiency is discussed.

HIV-1 protease is an essential enzyme for viral particle maturation and is a target in the fight against HIV-1 infection worldwide. Several natural polymorphisms are also associated with drug resistance. Here, we utilized both pulsed electron double resonance, also called double electron-electron resonance, and NMR (15)N relaxation measurements to characterize equilibrium conformational sampling and backbone dynamics of an HIV-1 protease construct containing four specific natural polymorphisms commonly found in subtypes A, F, and CRF_01 A/E. Results show enhanced backbone dynamics, particularly in the flap region, and the persistence of a novel conformational ensemble that we hypothesize is an alternative flap orientation of a curled open state or an asymmetric configuration when interacting with inhibitors.

Serotonin (5-hydroxytryptamine; 5-HT) is a CNS neurotransmitter increasingly recognized to exert immunomodulatory effects outside the CNS that contribute to the pathogenesis of autoimmune and chronic inflammatory diseases. 5-HT signals to activate the RhoA/Rho kinase (ROCK) pathway, a pathway known for its ability to regulate phagocytosis. The clearance of apoptotic cells (i.e. efferocytosis) is a key modulator of the immune response that is inhibited by the RhoA/ROCK pathway. Because efferocytosis is defective in many of the same illnesses where 5-HT has been implicated in disease pathogenesis, we hypothesized that 5-HT would suppress efferocytosis via activation of RhoA/ROCK. The effect of 5-HT on efferocytosis was examined in murine peritoneal and human alveolar macrophages, and its mechanisms were investigated using pharmacologic blockade and genetic deletion. 5-HT impaired efferocytosis by murine peritoneal macrophages and human alveolar macrophages. 5-HT increased phosphorylation of myosin phosphatase subunit 1 (Mypt-1), a known ROCK target, and inhibitors of RhoA and ROCK reversed the suppressive effect of 5-HT on efferocytosis. Peritoneal macrophages expressed the 5-HT transporter and 5-HT receptors (R) 2a, 2b, but not 2c. Inhibition of 5-HTR2a and 5-HTR2b had no effect on efferocytosis, but blockade of the 5-HT transporter prevented 5-HT-impaired efferocytosis. Genetic deletion of the 5-HT transporter inhibited 5-HT uptake into peritoneal macrophages, prevented 5-HT-induced phosphorylation of Mypt-1, reversed the inhibitory effect of 5-HT on efferocytosis, and decreased cellular peritoneal inflammation. These results suggest a novel mechanism by which 5-HT might disrupt efferocytosis and contribute to the pathogenesis of autoimmune and chronic inflammatory diseases.

Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPI-anchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys6) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity.

Disruptions in microtubule motor transport are associated with a variety of neurodegenerative diseases. Post-translational modification of the cargo-binding domain of the light and heavy chains of kinesin has been shown to regulate transport, but less is known about how modifications of the motor domain affect transport. Here we report on the effects of phosphorylation of a mammalian kinesin motor domain by the kinase JNK3 at a conserved serine residue (Ser-175 in the B isoform and Ser-176 in the A and C isoforms). Phosphorylation of this residue has been implicated in Huntington disease, but the mechanism by which Ser-175 phosphorylation affects transport is unclear. The ATPase, microtubule-binding affinity, and processivity are unchanged between a phosphomimetic S175D and a nonphosphorylatable S175A construct. However, we find that application of force differentiates between the two. Placement of negative charge at Ser-175, through phosphorylation or mutation, leads to a lower stall force and decreased velocity under a load of 1 piconewton or greater. Sedimentation velocity experiments also show that addition of a negative charge at Ser-175 favors the autoinhibited conformation of kinesin. These observations imply that when cargo is transported by both dynein and phosphorylated kinesin, a common occurrence in the cell, there may be a bias that favors motion toward the minus-end of microtubules. Such bias could be used to tune transport in healthy cells when properly regulated but contribute to a disease state when misregulated.