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LonA proteases and ClpB chaperones are key components of the protein quality control system in bacterial cells. LonA proteases form a unique family of ATPases associated with diverse cellular activities (AAA+ ) proteins due to the presence of an unusual N-terminal region comprised of two domains: a β-structured N domain and an α-helical domain, including the coiled-coil fragment, which is referred to as HI(CC). The arrangement of helices in the HI(CC) domain is reminiscent of the structure of the H1 domain of the first AAA+ module of ClpB chaperones. It has been hypothesized that LonA proteases with a single AAA+ module may also contain a part of another AAA+ module, the full version of which is present in ClpB. Here, we established and tested the structural basis of this hypothesis using the known crystal structures of various fragments of LonA proteases and ClpB chaperones, as well as the newly determined structure of the Escherichia coli LonA fragment (235-584). The similarities and differences in the corresponding domains of LonA proteases and ClpB chaperones were examined in structural terms. The results of our analysis, complemented by the finding of a singular match in the location of the most conserved axial pore-1 loop between the LonA NB domain and the NB2 domain of ClpB, support our hypothesis that there is a structural and functional relationship between two coiled-coil fragments and implies a similar mechanism of engagement of the pore-1 loops in the AAA+ modules of LonAs and ClpBs.

Small intestine adenocarcinoma is a rare intestinal malignancy with distinct clinical, pathological, and molecular characteristics. Recently, a fusion of the intestinal stem-cell marker olfactomedin 4 (OLFM4) and the proto-oncogene RET has been identified in a small intestine adenocarcinoma patient. Here we investigated the biological effects of OLFM4-RET fusion and whether it can initiate tumorigenesis in small intestine. OLFM4 expression was found to be frequently lost or reduced in human small intestine adenocarcinoma, and its downregulation correlated with high tumor grade and advanced tumor stage. Expression of OLFM4-RET fusion-induced cellular transformation in HEK293 cells and blocked RET-induced inhibition of colony growth in HuTu 80 small intestine adenocarcinoma cells. Further, expression of OLFM4-RET activated the RAS-RAF-MAPK and STAT3 cell signaling pathways in both HEK293 cells and HuTu 80 cells. OLFM4-RET expression in HEK293 cells upregulated multiple families of genes related to carcinogenesis, cancer progression, and metastasis. Targeted expression of OLFM4-RET in the small intestine led to the development of hyperplasia, adenoma, or adenocarcinoma in transgenic mice. Our study suggests that OLFM4-RET is an oncogenic driver of small intestine tumorigenesis. Therefore, the small intestine adenocarcinoma patients with OLFM4-RET fusion may benefit from treatment with RET kinase inhibitor.

β-barrel proteins are folded and inserted into outer membranes by multi-subunit protein complexes that are conserved across different types of outer membranes. In Gram-negative bacteria this complex is the barrel-assembly machinery (BAM), in mitochondria it is the sorting and assembly machinery (SAM) complex, and in chloroplasts it is the outer envelope protein Oep80. Mitochondrial β-barrel precursor proteins are translocated from the cytoplasm to the intermembrane space by the translocase of the outer membrane (TOM) complex, and stabilized by molecular chaperones before interaction with the assembly machinery. Outer membrane bacterial BamA interacts with four periplasmic accessory proteins, whereas mitochondrial Sam50 interacts with two cytoplasmic accessory proteins. Despite these major architectural differences between BAM and SAM complexes, their core proteins, BamA and Sam50, seem to function the same way. Based on the new SAM complex structures, we propose that the mitochondrial β-barrel folding mechanism follows the budding model with barrel-switching aiding in the release of new barrels. We also built a new molecular model for Tom22 interacting with Sam37 to identify regions that could mediate TOM-SAM supercomplex formation.

Bacterial contact-dependent growth inhibition (CDI) systems use a type Vb secretion mechanism to export large CdiA toxins across the outer membrane by dedicated outer membrane transporters called CdiB. Here, we report the first crystal structures of two CdiB transporters from Acinetobacter baumannii and Escherichia coli. CdiB transporters adopt a TpsB fold, containing a 16-stranded transmembrane β-barrel connected to two periplasmic domains. The lumen of the CdiB pore is occluded by an N-terminal α-helix and the conserved extracellular loop 6; these two elements adopt different conformations in the structures. We identified a conserved DxxG motif located on strand β1 that connects loop 6 through different networks of interactions. Structural modifications of DxxG induce rearrangement of extracellular loops and alter interactions with the N-terminal α-helix, preparing the system for α-helix ejection. Using structural biology, functional assays, and molecular dynamics simulations, we show how the barrel pore is primed for CdiA toxin secretion.

The TonB–ExbB–ExbD molecular motor harnesses the proton motive force across the bacterial inner membrane to couple energy to transporters at the outer membrane, facilitating uptake of essential nutrients such as iron and cobalamine. TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Until recently, no high-resolution structural information was available for this unique molecular motor. We published the first crystal structure of ExbB–ExbD in 2016 and showed that five copies of ExbB are arranged as a pentamer around a single copy of ExbD. However, our spectroscopic experiments clearly indicated that two copies of ExbD are present in the complex. To resolve this ambiguity, we used single-particle cryo-electron microscopy to show that the ExbB pentamer encloses a dimer of ExbD in its transmembrane pore, and not a monomer as previously reported. The revised stoichiometry has implications for motor function., Herve Celia et al. report the single-particle cryo-EM structure of the bacterial ExbB–ExbD subcomplex reconstituted in lipid nanodiscs. They show that the ExbB pentamer encloses the ExbD dimer in the transmembrane pore, with implications for motor function.

Paralogous enzymes arise from gene duplication events that confer a novel function, although it is unclear how cross-reaction between the original and duplicate protein interaction network is minimized. We investigated HPr:EIsugar and NPr:EINtr, the initial complexes of paralogous phosphorylation cascades involved in sugar import and nitrogen regulation in bacteria, respectively. Although the HPr:EIsugar interaction has been well characterized, involving multiple complexes and transient interactions, the exact nature of the NPr:EINtr complex was unknown. We set out to identify the key features of the interaction by performing binding assays and elucidating the structure of NPr in complex with the phosphorylation domain of EINtr (EINNtr), using a hybrid approach involving X-ray, homology, and sparse nuclear magnetic resonance. We found that the overall fold and active-site structure of the two complexes are conserved in order to maintain productive phosphorylation, however, the interface surface potential differs between the two complexes, which prevents cross-reaction.

The TonB-ExbB-ExbD molecular motor harnesses the proton motive force across the bacterial inner membrane to couple energy to transporters at the outer membrane, facilitating uptake of essential nutrients such as iron and cobalamine. TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Until recently, no high-resolution structural information was available for this unique molecular motor. We published the first crystal structure of ExbB-ExbD in 2016 and showed that five copies of ExbB are arranged as a pentamer around a single copy of ExbD. However, our spectroscopic experiments clearly indicated that two copies of ExbD are present in the complex. To resolve this ambiguity, we used single-particle cryo-electron microscopy to show that the ExbB pentamer encloses a dimer of ExbD in its transmembrane pore, and not a monomer as previously reported. The revised stoichiometry has implications for motor function.

Diagram of PEZ dispenser and analogy to Lpt system.

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.

In Gram-negative bacteria, outer membrane transporters import nutrients by coupling to an inner membrane protein complex called the Ton complex. The Ton complex consists of TonB, ExbB, and ExbD, and uses the proton motive force at the inner membrane to transduce energy to the outer membrane via TonB. Here, we structurally characterize the Ton complex from Escherichia coli using X-ray crystallography, electron microscopy, double electron-electron resonance (DEER) spectroscopy, and crosslinking. Our results reveal a stoichiometry consisting of a pentamer of ExbB, a dimer of ExbD, and at least one TonB. Electrophysiology studies show that the Ton subcomplex forms pH-sensitive cation-selective channels and provide insight into the mechanism by which it may harness the proton motive force to produce energy.

Toll-like receptors (TLRs) recognize conserved molecular patterns in invading pathogens and trigger innate immune responses. TLR3 recognizes dsRNA, a molecular signature of most viruses via its ectodomain (ECD). The TLR3-ECD structure consists of a 23 turn coil bent into the shape of a horseshoe with specialized domains capping the N and C terminal ends of the coil. TLR3-ECDs bind as dimeric units to dsRNA oligonucleotides of at least 45 bp in length, the minimal length required for signal transduction. X-ray analysis has shown that each TLR3-ECD of a dimer binds dsRNA at two sites located at opposite ends of the TLR3 “horseshoe” on the one lateral face that lacks N-linked glycans. Intermolecular contacts between the C-terminal domains of two TLR3-ECDs stabilizes the dimer and positions the C-terminal residues within 20–25Å of each other, which is thought to be essential for transducing a signal across the plasma membrane in intact TLR3 molecules. Interestingly, in TLRs 1, 2 and 4, which bind lipid ligands using very different interactions from TLR3, the ligands nevertheless promote the formation of a dimer in which the same two lateral surfaces as in the TLR3-ECD:dsRNA complex face each other, bringing their C-termini in close proximity. Thus, a pattern is emerging in which pathogen-derived substances bind to TLR-ECDs, thereby promoting the formation of a dimer in which the glycan-free ligand-binding surfaces face each other and the two C-termini are brought in close proximity for signal transduction.

We carried out chymotryptic digestion of multimeric ATP-dependent Lon protease from Escherichia coli. Four regions sensitive to proteolytic digestion were located in the enzyme and several fragments corresponding to the individual structural domains of the enzyme or their combinations were isolated. It was shown that (i) unlike the known AAA(+) proteins, the ATPase fragment (A) of Lon has no ATPase activity in spite of its ability to bind nucleotides, and it is monomeric in solution regardless of the presence of any effectors; (ii) the monomeric proteolytic domain (P) does not display proteolytic activity; (iii) in contrast to the inactive counterparts, the AP fragment is an oligomer and exhibits both the ATPase and proteolytic activities. However, unlike the full-length Lon, its AP fragment oligomerizes into a dimer or a tetramer only, exhibits the properties of a non-processive protease, and undergoes self-degradation upon ATP hydrolysis. These results reveal the crucial role played by the non-catalytic N fragment of Lon (including its coiled-coil region), as well as the contribution of individual domains to creation of the quaternary structure of the full-length enzyme, empowering its function as a processive protease.

Serine hydrolases use a hydroxyl of a serine, assisted by one or more other residues, to cleave peptide bonds. They belong to several different families whose general mechanism is well known. However, the subtle structural differences that have recently been observed across a variety of families shed light on their functional diversity, including variations in mechanism of action, differences in the modes of substrate binding, and substrate-assisted orientation of catalytic residues. Of particular interest are the Rhomboid family serine proteinases that are active within the plasma membrane, for which several new structures have been reported. Because these enzymes are involved in biological and pathological processes, many are becoming important targets of drug design.

Toll-like receptors (TLRs), type I integral membrane receptors, recognize pathogen associated molecular patterns (PAMPs). PAMP recognition occurs via the N-terminal ectodomain (ECD) which initiates an inflammatory response that is mediated by the C-terminal cytosolic signaling domain. To understand the molecular basis of PAMP recognition, we have begun to define TLR—ECD structurally. We have solved the structure of TLR3-ECD, which recognizes dsRNA, a PAMP associated with viral pathogens. TLR3-ECD is a horseshoe-shaped solenoid composed of 23 leucine-rich repeats (LRRs). The regular LRR surface is disrupted by two insertions at LRR12 and LRR20 and 11 N-linked carbohydrates. Of note, one side of the ECD is carbohydrate-free and could form an interaction interface. We have shown that TLR3-ECD binds directly to pI:pC, a synthetic dsRNA ligand, but not to p(dI):p(dC). Without a TLR3—dsRNA complex structure, we can only speculate how ligand binds. Analysis of the unliganded structure reveals two patches of basic residues and two binding sites for phosphate backbone mimics, sulfate ions, that may be capable of recognizing ligand. Mutational and co-crystallization studies are currently underway to determine how TLR3 binds its ligand at the molecular level.

Scytovirin (SVN) is a novel anti-human immunodeWciency virus (HIV) protein isolated from aqueous extracts of the cultured cyanobacterium Scytonema varium. The protein consists of a single 95-amino acid chain with signiWcant internal sequence duplication and 10 cysteines forming Wve intrachain disulWde bonds. A synthetic gene that encodes scytovirin was constructed, and expressed in Escherichia coli, with thioredoxin (TRX) fused to its N-terminius (TRX-SVN). Most of the expressed protein was in soluble form, which was puriWed by a polyhistidine tag aYnity puriWcation step. SVN was then cleaved from TRX with enterokinase and separated from the TRX partner by C18 reversed-phase HPLC. This production method has proven superior to earlier synthetic attempts and recombinant procedures using a standard expression system. The current system resulted in yields of 5–10 mg/L of puriWed SVN for structural studies and for preclinical development of SVN as a topical microbicide for HIV prophylaxis. Published by Elsevier Inc.

A broad range of proteins bind high-mannose carbohydrates found on the surface of the envelope protein gp120 of the human immunodeficiency virus and thus interfere with the viral life cycle, providing a potential new way of controlling HIV infection. These proteins interact with the carbohydrate moieties in different ways. A group of them interacts as typical C-type lectins via a Ca2+ ion. Another group interacts with specific single, terminal sugars, without the help of a metal cation. A third group is involved in more intimate interactions, with multiple carbohydrate rings and no metal ion. Finally, there is a group of lectins for which the interaction mode has not yet been elucidated. This review summarizes, principally from a structural point of view, the current state of knowledge about these high-mannose binding proteins and their mode of sugar binding.

ATP-dependent Lon proteases belong to the superfamily of AAA+ proteins. Until recently, the identity of the residues involved in their proteolytic active sites was not elucidated. However, the putative catalytic Ser-Lys dyad was recently suggested through sequence comparison of more than 100 Lon proteases from various sources. The presence of the catalytic dyad was experimentally confirmed by site-directed mutagenesis of the Escherichia coli Lon protease and by determination of the crystal structure of its proteolytic domain. Furthermore, this extensive sequence analysis allowed the definition of two subfamilies of Lon proteases, LonA and LonB, based on the consensus sequences in the active sites of their proteolytic domains. These differences strictly associate with the specific characteristics of their AAA+ modules, as well as with the presence or absence of an N-terminal domain.

ATP-dependent Lon protease degrades specific short-lived regulatory proteins as well as defective and abnormal proteins in the cell. The crystal structure of the proteolytic domain (P domain) of the Escherichia coli Lon has been solved by single-wavelength anomalous dispersion and refined at 1.75-A resolution. The P domain was obtained by chymotrypsin digestion of the full-length, proteolytically inactive Lon mutant (S679A) or by expression of a recombinant construct encoding only this domain. The P domain has a unique fold and assembles into hexameric rings that likely mimic the oligomerization state of the holoenzyme. The hexamer is dome-shaped, with the six N termini oriented toward the narrower ring surface, which is thus identified as the interface with the ATPase domain in full-length Lon. The catalytic sites lie in a shallow concavity on the wider distal surface of the hexameric ring and are connected to the proximal surface by a narrow axial channel with a diameter of approximately 18 A. Within the active site, the proximity of Lys(722) to the side chain of the mutated Ala(679) and the absence of other potential catalytic side chains establish that Lon employs a Ser(679)-Lys(722) dyad for catalysis. Alignment of the P domain catalytic pocket with those of several Ser-Lys dyad peptide hydrolases provides a model of substrate binding, suggesting that polypeptides are oriented in the Lon active site to allow nucleophilic attack by the serine hydroxyl on the si-face of the peptide bond.

Cyanovirin-N (CV-N) is a potent 11 kDa HIV-inactivating protein that binds with high affinity to the HIV surface envelope protein gp120. A double mutant P51S/S52P of CV-N was engineered by swapping two critical hinge-region residues Pro51 and Ser52. This mutant has biochemical and biophysical characteristics equivalent to the wild-type CV-N and its structure resembles that of wild-type CV-N. However, the mutant shows a different orientation in the hinge region that connects two domains of the protein. The observation that this double mutant crystallizes under a wide variety of conditions challenges some of the current hypotheses on domain swapping and on the role of hinge-region proline residues in domain orientation. The current structure contributes to the understanding of domain swapping in cyanovirins, permitting rational design of domain-swapped CV-N mutants.

The crystal structure of the semireduced form of cyclic nucleotide phosphodiesterase (CPDase) from Arabidopsis thaliana has been solved by molecular replacement and refined at the resolution of 1.8 A. We have previously reported the crystal structure of the native form of this enzyme, whose main target is ADP-ribose 1",2"-cyclic phosphate, a product of the tRNA splicing reaction. CPDase possesses six cysteine residues, four of which are involved in forming two intra-molecular disulfide bridges. One of these bridges, between Cys-104 and Cys-110, is opened in the semireduced CPDase, whereas the other remains intact. This change of the redox state leads to a conformational rearrangement in the loop covering the active site of the protein. While the native structure shows this partially disordered loop in a coil conformation, in the semireduced enzyme the N-terminal lobe of this loop winds up and elongates the preceding alpha-helix. The semireduced state of CPDase also enabled co-crystallization with a putative inhibitor of its enzymatic activity, 2',3'-cyclic uridine vanadate. The ligand is bound within the active site, and the mode of binding is in agreement with the previously proposed enzymatic mechanism. Selected biophysical properties of the oxidized and the semireduced CPDase are also discussed.

The bacterial outer membrane contains phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. Both proteins and LPS must be frequently inserted into the outer membrane to preserve its integrity. The protein complex that inserts LPS into the outer membrane is called LptDE, and consists of an integral membrane protein, LptD, with a separate globular lipoprotein, LptE, inserted in the barrel lumen. The protein complex that inserts newly synthesized outer-membrane proteins (OMPs) into the outer membrane is called the BAM complex, and consists of an integral membrane protein, BamA, plus four lipoproteins, BamB, C, D and E. Recent structural and functional analyses illustrate how these two complexes insert their substrates into the outer membrane by distorting the membrane component (BamA or LptD) to directly access the lipid bilayer. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

The South American imported fire ant (Solenopsis invicta), without natural enemies in the United States, widely infests the southern United States, causing more than a half billion dollars in health and agriculture-related damage annually in Texas alone. Fire ants are resistant to most insecticides, so control will require a more fundamental understanding of their biochemistry and metabolism leading to the design of selective, ecologically safe insecticides. The 4th instar larvae play a crucial role in the nutrition of the colony by secreting proteinases (especially chymotrypsin) which digest food products for the entire colony. The first structure of an ant proteolytic enzyme, fire ant chymotrypsin, was determined to atomic resolution (1.7 A). A structural comparison of the ant and mammalian structures confirms the "universality" of the serine proteinase motif and reveals a difference at residues 147-148, which are proteolytically removed in the bovine enzyme but are firmly intact in the ant chymotrypsin, suggesting a different activation mechanism for the latter. Likewise, the absence of the covalently attached propeptide domain (1-15) further suggests an uncharacteristic activation mechanism. The presence of Gly189 in the S1 site is an atypical feature of this chymotrypsin and is comparable only to human leukocyte elastase, hornet chymotrypsin and fiddler crab collagenase. Binding studies confirm the chymotrypsin nature of this novel enzyme.

Matrix metalloproteinase enzymes have been implicated in degenerative processes like tumor cell invasion, metastasis, and arthritis. Specific metalloproteinase inhibitors have been used to block tumor cell proliferation. We have examined the interaction of batimastat (BB-94) with a metalloproteinase [atrolysin C (Ht-d), EC 3.4.24.42] active site at 2.0-angstroms resolution (R = 16.8%). The title structure exhibits an unexpected binding geometry, with the thiophene ring deeply inserted into the primary specificity site. This unprecedented binding geometry dramatizes the significance of the cavernous primary specificity site, pointing the way for the design of a new generation of potential antitumor drugs.

From simple to highly complex molecules, examples are cited of ‘backward’ or retro-binding. In some cases, symmetry dictates the directions observed and in cases not treated here, statistical disorder reveals averaged forward/backward binding. In most of the cases found, both the receptor and the ligand(s) are asymmetric, so local interactions dictate the preferred binding mode. A general rationale is presented to explain some of these observations and a hypothesis derived from this review can be tested experimentally.

A theoretical study was performed on the structure of both the native and inhibited metalloproteinase Ht-d (E.C. 3.4.24.42) solved at 2.0/~ resolution. The energy maps calculated by program GRID clearly showed the extended binding site of Ht-d and allowed localization and characterization of the pockets S1-$3 and S1'-$3'. The GRID energy contour maps point out the particular shape of the S I' pocket in agreement with experimental density maps and inhibited Ht-d structures. Based on the high degree of sequence homology of the Ht-d active site to that of mammalian metalloproteinases, the characterization of active site pockets was extended to neutrophil collagenase, fibroblast collagenase, stromelysin 1 and 2. Thirty residues of the Ht-d propeptide were modeled and optimized with reference to the Ht-d structure, giving insight to the mechanism of natural inhibition in metalloproteinase proenzymes. Kinetic measurements of Ht-d inhibition by a series of synthetic peptides show, in agreement with our Ht-d propeptide model, the crucial role of cysteine and adjacent residues in the specificity of Ht-d propeptide. This study suggests the structural link between Ht-d and mammalian metalloproteinases, contributing to the understanding of the mechanism of natural and synthetic inhibitor binding to metalloproteinases. Therefore, Ht-d is a good model system for the design of novel inhibitors against these enzymes with enhanced potency and specificity.

The structure of the metalloproteinase and hemorrhagic toxin atrolysin C form d (EC 3.4.24.42), from the venom of the western diamondback rattlesnake Crotalus atrox, has been determined to atomic resolution by x-ray crystallographic methods. This study illuminates the nature of inhibitor binding with natural (< Glu-Asn-Trp, where < Glu is pyroglutamic acid) and synthetic (SCH 47890) ligands. The primary specificity pocket is exceptionally deep; the nature of inhibitor and productive substrate binding is discussed. Insights gained from the study of these complexes facilitate the design of potential drugs to treat diseases where matrix metalloproteinases have been implicated, e.g., arthritis and tumor metastasis.

Publisher Summary The innate immune system provides a rapid first line of defense against pathogen attack. One of its principal components is the array of pathogen-associated molecular pattern detectors known as the Toll-Like Receptors (TLRs)—germline-encoded type one transmembrane receptors of ancient lineage that are expressed on numerous cell types, and are the focus of this chapter. This chapter begins by describing the structure and function of TLRs. TLR ectodomains contain about 18–25 leucine-rich repeats (LRRs) and recognize molecules that are as diverse as double-stranded RNA, lipopolysaccharide, peptidoglycan, and unmethylated CpG DNA. TLRs rapidly trigger the production of a variety of cytokines, producing inflammation, and also activate the adaptive immune response by up-regulating co-stimulatory molecules and antigen-presenting cells. Under the structure, this chapter discusses mutational analysis to identify the dsRNA, and TLR4. It also deals with the structure of human MD-2 (hMD-2) and its complex with antiendotoxic lipid IVa. Furthermore, it considers the phenomenon of TLR1–TLR2 dimerization by atri-acylated lipopeptide. Finally, it explains the concept of signaling, stating that ample evidence in the literature points to an accepted universal mechanism in which, similar to most other type 1 transmembrane receptors, ligand-induced dimerization or crosslinking initiates signal transduction through the membrane. Dimerization of the TLR cytoplasmic TIR domains is a prerequisite for the binding of other TIR-domain containing adaptor molecules.

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA), a molecular signature of most viruses, and triggers inflammatory responses that prevent viral spread. TLR3 ectodomains (ECDs) dimerize on oligonucleotides of at least 40 to 50 base pairs in length, the minimal length required for signal transduction. To establish the molecular basis for ligand binding and signaling, we determined the crystal structure of a complex between two mouse TLR3-ECDs and dsRNA at 3.4 angstrom resolution. Each TLR3-ECD binds dsRNA at two sites located at opposite ends of the TLR3 horseshoe, and an intermolecular contact between the two TLR3-ECD C-terminal domains coordinates and stabilizes the dimer. This juxtaposition could mediate downstream signaling by dimerizing the cytoplasmic Toll interleukin-1 receptor (TIR) domains. The overall shape of the TLR3-ECD does not change upon binding to dsRNA.

We carried out chymotryptic digestion of multimeric ATP-dependent Lon protease from Escherichia coli. Four regions sensitive to proteolytic digestion were located in the enzyme and several fragments corresponding to the individual structural domains of the enzyme or their combinations were isolated. It was shown that (i) unlike the known AAA(+) proteins, the ATPase fragment (A) of Lon has no ATPase activity in spite of its ability to bind nucleotides, and it is monomeric in solution regardless of the presence of any effectors; (ii) the monomeric proteolytic domain (P) does not display proteolytic activity; (iii) in contrast to the inactive counterparts, the AP fragment is an oligomer and exhibits both the ATPase and proteolytic activities. However, unlike the full-length Lon, its AP fragment oligomerizes into a dimer or a tetramer only, exhibits the properties of a non-processive protease, and undergoes self-degradation upon ATP hydrolysis. These results reveal the crucial role played by the non-catalytic N fragment of Lon (including its coiled-coil region), as well as the contribution of individual domains to creation of the quaternary structure of the full-length enzyme, empowering its function as a processive protease.

The crystal structures of the natural and recombinant antiviral lectin scytovirin (SVN) were solved by single-wavelength anomalous scattering and refined with data extending to 1.3 A and 1.0 A resolution, respectively. A molecule of SVN consists of a single chain 95 amino acids long, with an almost perfect sequence repeat that creates two very similar domains (RMS deviation 0.25 A for 40 pairs of Calpha atoms). The crystal structure differs significantly from a previously published NMR structure of the same protein, with the RMS deviations calculated separately for the N- and C-terminal domains of 5.3 A and 3.7 A, respectively, and a very different relationship between the two domains. In addition, the disulfide bonding pattern of the crystal structures differs from that described in the previously published mass spectrometry and NMR studies.

Innate immunity is the first line of defense against invading pathogens. Toll-like receptors (TLRs) act as sentinels of the innate immune system, sensing a variety of ligands from lipopolysaccharide to flagellin to dsRNA through their ligand-binding domain that is composed of leucine-rich repeats (LRRs). Ligand binding initiates a signaling cascade that leads to the up-regulation of inflammation mediators. In this study, we have expressed and crystallized the ectodomain (ECD) of human TLR3, which recognizes dsRNA, a molecular signature of viruses, and have determined the molecular structure to 2.4-Å resolution. The overall horseshoe-shaped structure of the TLR3-ECD is formed by 23 repeating LRRs that are capped at each end by specialized non-LRR domains. The extensive β-sheet on the molecule's concave surface forms a platform for several modifications, including insertions in the LRRs and 11 N -linked glycans. The TLR3-ECD structure indicates how LRR loops can establish distinct pathogen recognition receptors.

The atomic-resolution crystal structure of the proteolytic domain (P-domain, residues 415–621) of Archaeoglobus fulgidus B-type Lon protease (wtAfLonB) and the structures of several mutants have revealed significant differences in the conformation of the active-site residues when compared to other known Lon P-domains, despite the conservation of the overall fold. The catalytic Ser509 is facing the solvent and is distant from Lys552, the other member of the catalytic dyad. Instead, the adjacent Asp508 forms an ion pair with the catalytic lysine residue. Glu506, an analog of the putative third catalytic residue from a related Methanococcus jannaschii LonB, also faces the solvent and does not interact with the catalytic dyad. We have established that full-length wtAfLonB is proteolytically active in an ATP-dependent manner. The loss of enzymatic activity of the S509A mutant confirms the functional significance of this residue, while retention of considerable level of activity by the D508A and E506A mutants rules out their critical involvement in catalysis. In contrast to the full-length enzymes, all individually purified P-domains (wild-type and mutants) were inactive, and the mutations had no influence on the active-site structure. These findings raise the possibility that, although isolated proteolytic domains of both AfLonB and E. coli LonA are able to assemble into expected functional hexamers, the presence of the other domains, as well as substrate binding, may be needed to stabilize the productive conformation of their active sites. Thus, the observed conformational variability may reflect the differences in the stability of active-site structures for the proteolytic counterparts of single-chain Lon versus independently folded proteolytic subunits of two-chain AAA+ proteases.

Although macromolecular crystallography is rapidly becoming largely routine owing to advances in methods of data collection, structure solution and refinement, difficult cases are still common. To remind structural biologists about the kinds of crystallographic difficulties that might be encountered, case studies of several successfully completed structure determinations that utilized less than perfect crystals are discussed here. The structure of the proteolytic domain of Archaeoglobus fulgidus Lon was solved with crystals that contained superimposed orthorhombic and monoclinic lattices, a case not previously described for proteins. Another hexagonal crystal form of this protein exhibited an unusually high degree of non-isomorphism. Crystals of A. fulgidus Rio1 kinase exhibited both pseudosymmetry and twinning. Ways of identifying the observed phenomena and approaches to solving and refining macromolecular structures when only less than perfect crystals are available are discussed here.

The crystal structure of the small, mostly helical alpha domain of the AAA+ module of the Escherichia coli ATP-dependent protease Lon has been solved by single isomorphous replacement combined with anomalous scattering and refined at 1.9A resolution to a crystallographic R factor of 17.9%. This domain, comprising residues 491-584, was obtained by chymotrypsin digestion of the recombinant full-length protease. The alpha domain of Lon contains four alpha helices and two parallel strands and resembles similar domains found in a variety of ATPases and helicases, including the oligomeric proteases HslVU and ClpAP. The highly conserved "sensor-2" Arg residue is located at the beginning of the third helix. Detailed comparison with the structures of 11 similar domains established the putative location of the nucleotide-binding site in this first fragment of Lon for which a crystal structure has become available.

The development of anti-human immunodeficiency virus (HIV) microbicides for either topical or ex vivo use is of considerable interest, mainly due to the difficulties in creating a vaccine that would be active against multiple clades of HIV. Cyanovirin-N (CV-N), an 11-kDa protein from the cyanobacterium (blue-green algae) Nostoc ellipsosporum with potent virucidal activity, was identified in the search for such antiviral agents. The binding of CV-N to the heavily glycosylated HIV envelope protein gp120 is carbohydrate-dependent. Since previous CV-N-dimannose structures could not fully explain CV-N-oligomannose binding, we determined the crystal structures of recombinant CV-N complexed to Man-9 and a synthetic hexamannoside, at 2.5- and 2.4-A resolution, respectively. CV-N is a three-dimensional domain-swapped dimer in the crystal structures with two primary sites near the hinge region and two secondary sites on the opposite ends of the dimer. The binding interface is constituted of three stacked alpha1-->2-linked mannose rings for Man-9 and two stacked mannose rings for hexamannoside with the rest of the saccharide molecules pointing to the solution. These structures show unequivocally the binding geometry of high mannose sugars to CV-N, permitting a better understanding of carbohydrate binding to this potential new lead for the design of drugs against AIDS.

The structure of the potent HIV-inactivating protein cyanovirin-N was previously found by NMR to be a monomer in solution and a domain-swapped dimer by X-ray crystallography. Here we demonstrate that, in solution, CV-N can exist both in monomeric and in domain-swapped dimeric form. The dimer is a metastable, kinetically trapped structure at neutral pH and room temperature. Based on orientational NMR constraints, we show that the domain-swapped solution dimer is similar to structures in two different crystal forms, exhibiting solely a small reorientation around the hinge region. Mutation of the single proline residue in the hinge to glycine significantly stabilizes the protein in both its monomeric and dimeric forms. By contrast, mutation of the neighboring serine to proline results in an exclusively dimeric protein, caused by a drastic destabilization of the monomer.

The crystal structure of a cyclic form of a mutant of bovine pancreatic trypsin inhibitor has been solved at 1.0 Å resolution. The protein was synthesized by native chemical ligation and its structure is almost indistinguishable from the previously described recombinant form of the same mutant; however, the new loop containing the former termini became much better ordered.

The matrix metalloproteinases are crucial in the physiological and pathological degradation of the mammalian extracellular matrix, including breast tumours, and osteoarthritic cartilage. These enzymes are classified according to their matrix substrate specificity. Collagenase-3 (MMP-13) is a member of this family and preferentially cleaves type II collagen, cartilage, fibronectin and aggrecan. Collagenase-3 is normally expressed in hypertrophic chondrocytes, periosteal cells, and osteoblasts during bone development. The structure of the catalytic domain of recombinant mouse collagenase-3, complexed to the hydroxamate inhibitor (RS-113456), is reported at 2.0 A resolution. Molecular replacement and weak phasing information from a single derivative determined the structure. Neither molecular replacement nor derivative methods had a sufficient radius of convergence to yield a refinable structure. The structure illuminates the atomic zinc ion interactions with functional groups in the active site, emphasizing zinc ligation and the very voluminous hydrophobic P1′ group for the inhibitor potency. The structure provides insight into the specificity of this enzyme, facilitating design of specific inhibitors to target various diseases.

The membrane-bound Toll-like receptors (TLRs) trigger innate immune responses following recognition of a wide variety of pathogen-derived compounds. Despite the wide range of ligands recognized by TLRs, the receptors share a common structural framework in their extracellular, ligand-binding domains. These domains all adopt horseshoe-shaped structures built from leucine-rich repeat motifs. Typically, upon ligand binding, two extracellular domains form an “m”-shaped dimer sandwiching the ligand molecule bringing the transmembrane and cytoplasmic domains in close proximity and triggering a downstream signalling cascade. Although the ligand-induced dimerization of these receptors has many common features, the nature of the interactions of the TLR extracellular domains with their ligands varies markedly between TLR paralogs.

Deletion of the transmembrane domain (TM-domain) of Archaeoglobus fulgidus LonB protease (Archaeoglobus fulgidus (AfLon)) was shown to result in uncontrollable activation of the enzyme proteolytic site and in vivo autolysis yielding a stable and functionally inactive fragment consisting of both α-helical and proteolytic domains (αP). The ΔTM-AfLon-S509A enzyme form, obtained by site-directed mutagenesis of the catalytic Ser residue, is capable of recombination with the αP fragment. The mixed oligomers were shown to be proteolytically active, which indicates a crucial role of subunit interactions in the activation of the AfLon proteolytic site. The thermophilic nature of AfLon protease was found to be due to the special features of the enzyme activity regulation, the structure of ATPase domain, and the quaternary structure.