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Strains of the Burkholderia cepacia complex (Bcc) are Gram-negative opportunisitic bacteria that are capable of causing serious diseases, mainly in immunocompromised individuals. Bcc pathogens are intrinsically resistant to multiple antibiotics, including β-lactams, aminoglycosides, fluoroquinolones, and polymyxins. They are major pathogens in patients with cystic fibrosis (CF) and can cause severe necrotizing pneumonia, which is often fatal. Hopanoid biosynthesis is one of the major mechanisms involved in multiple antimicrobial resistance of Bcc pathogens. The hpnN gene of B. multivorans encodes an integral membrane protein of the HpnN family of transporters, which is responsible for shuttling hopanoids to the outer membrane. Here, we report crystal structures of B. multivorans HpnN, revealing a dimeric molecule with an overall butterfly shape. Each subunit of the transporter contains 12 transmembrane helices and two periplasmic loops that suggest a plausible pathway for substrate transport. Further analyses indicate that HpnN is capable of shuttling hopanoid virulence factors from the outer leaflet of the inner membrane to the periplasm. Taken together, our data suggest that the HpnN transporter is critical for multidrug resistance and cell wall remodeling in Burkholderia.

A bacterial defense mechanism Polymyxins are antibiotics that disrupt the bacterial cell membrane and are used to treat multidrug-resistant infections. A bacterial enzyme called ArnT can mediate resistance to polymyxins by transferring a sugar group from a lipid carrier to lipid A, a component of the bacterial outer membrane. Petrou et al. described structures of ArnT alone and in complex with a lipid carrier and identified a cavity where lipid A probably binds. Insights into the enzyme mechanism could be exploited to design drugs that combat polymyxin resistance. Science , this issue p. 608

Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5'-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process.

The group II intron and the spliceosome share a common active site architecture and are thought to be evolutionarily related. Here we report the 3.7 Å crystal structure of a eukaryotic group II intron in the lariat-3′ exon form, immediately preceding the second step of splicing, analogous to the spliceosomal P complex. This structure reveals the location of the intact 3′ splice site within the catalytic core of the group II intron. The 3′-OH of the 5′ exon is positioned in close proximity to the 3′ splice site for nucleophilic attack and exon ligation. The active site undergoes conformational rearrangements with the catalytic triplex having different configurations before and after the second step of splicing. We describe a complete model for the second step of group II intron splicing that incorporates a dynamic catalytic triplex being responsible for creating the binding pocket for 3′ splice site capture., Group II introns are large, self-splicing RNAs that catalyze their own excision from pre-mRNA molecules. Here the authors determine the 3.7 Å crystal structure of the group II intron in the stage immediately before the second step of splicing and present a complete model for the second step of group II intron splicing.

The high-resolution structure of glucan binding protein C (GbpC) at 1.14 Å, a sucrose-dependent virulence factor of the dental caries pathogen Streptococcus mutans , has been determined. GbpC shares not only structural similarities with the V regions of AgI/II and SspB but also functional adherence to salivary agglutinin (SAG) and its scavenger receptor cysteine-rich domains (SRCRs). This is not only a newly identified function for GbpC but also an additional fail-safe binding mechanism for S. mutans . Despite the structural similarities with S. mutans antigen I/II (AgI/II) and SspB of Streptococcus gordonii , GbpC remains unique among these surface proteins in its propensity to adhere to dextran/glucans. The complex crystal structure of GbpC with dextrose (β- d -glucose; Protein Data Bank ligand BGC) highlights exclusive structural features that facilitate this interaction with dextran. Targeted deletion mutant studies on GbpC's divergent loop region in the vicinity of a highly conserved calcium binding site confirm its role in biofilm formation. Finally, we present a model for adherence to dextran. The structure of GbpC highlights how artfully microbes have engineered the lectin-like folds to broaden their functional adherence repertoire.

The mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the MmpL (mycobacterial membrane protein large) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complex regulatory network, including the TetR family transcriptional regulators Rv3249c and Rv1816. Here we report the crystal structures of these two regulators, revealing dimeric, two-domain molecules with architecture consistent with the TetR family of regulators. Buried extensively within the C-terminal regulatory domains of Rv3249c and Rv1816, we found fortuitous bound ligands, which were identified as palmitic acid (a fatty acid) and isopropyl laurate (a fatty acid ester), respectively. Our results suggest that fatty acids may be the natural ligands of these regulatory proteins. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by these proteins. Binding of palmitic acid renders these regulators incapable of interacting with their respective operator DNAs, which will result in derepression of the corresponding mmpL genes. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.

Mycobacterium tuberculosis is a pathogenic bacterial species, which is neither Gram positive nor Gram negative. It has a unique cell wall, making it difficult to kill and conferring resistance to antibiotics that disrupt cell wall biosynthesis. Thus, the mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the mycobacterial membrane protein large (MmpL) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complicated regulatory network system. Here we report crystallographic structures of two forms of the TetR‐family transcriptional regulator Rv0302, which participates in regulating the expression of MmpL proteins. The structures reveal a dimeric, two‐domain molecule with architecture consistent with the TetR family of regulators. Comparison of the two Rv0302 crystal structures suggests that the conformational changes leading to derepression may be due to a rigid body rotational motion within the dimer interface of the regulator. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by this protein. In addition, our isothermal titration calorimetry and electrophoretic mobility shift experiments indicate that fatty acids may be the natural ligand of this regulator. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.

Summary ZTP, the pyrophosphorylated analog of ZMP (5-amino-4-imidazole carboxamide ribose-5′-monophosphate), was identified as an alarmone that senses 10-formyl-tetrahydroflate deficiency in bacteria. Recently, a pfl riboswitch was identified that selectively binds ZMP and regulates genes associated with purine biosynthesis and one-carbon metabolism. We report on the structure of the ZMP-bound Thermosinus carboxydivorans pfl riboswitch sensing domain, thereby defining the pseudoknot-based tertiary RNA fold, the binding-pocket architecture, and principles underlying ligand recognition specificity. Molecular recognition involves shape complementarity, with the ZMP 5-amino and carboxamide groups paired with the Watson-Crick edge of an invariant uracil, and the imidazole ring sandwiched between guanines, while the sugar hydroxyls form intermolecular hydrogen bond contacts. The burial of the ZMP base and ribose moieties, together with unanticipated coordination of the carboxamide by Mg 2+ , contrasts with exposure of the 5′-phosphate to solvent. Our studies highlight the principles underlying RNA-based recognition of ZMP, a master regulator of one-carbon metabolism.

Cells use specific mechanisms such as histone chaperones to abrogate the inherent barrier that the nucleosome poses to transcribing polymerases. The current model postulates that nucleosomes can be transiently disrupted to accommodate passage of RNA polymerases and that histones H3 and H4 possess their own chaperones dedicated to the recovery of nucleosomes. Here, we determined the crystal structure of the conserved C terminus of human Suppressors of Ty insertions 2 (hSpt2C) chaperone bound to an H3/H4 tetramer. The structural studies demonstrate that hSpt2C is bound to the periphery of the H3/H4 tetramer, mimicking the trajectory of nucleosomal-bound DNA. These structural studies have been complemented with in vitro binding and in vivo functional studies on mutants that disrupt key intermolecular contacts involving two acidic patches and hydrophobic residues on Spt2C. We show that contacts between both human and yeast Spt2C with the H3/H4 tetramer are required for the suppression of H3/H4 exchange as measured by H3K56ac and new H3 deposition. These interactions are also crucial for the inhibition of spurious transcription from within coding regions. Together, our data indicate that Spt2 interacts with the periphery of the H3/H4 tetramer and promotes its recycling in the wake of RNA polymerase.

Stearoyl-CoA desaturase (SCD) is conserved in all eukaryotes and introduces the first double bond into saturated fatty acyl-CoAs1–4. Since the monounsaturated products of SCD are key precursors of membrane phospholipids, cholesterol esters, and triglycerides, SCD is pivotal in fatty acid metabolism. Humans have two SCD homologs (SCD1 and SCD5), while mice have four (SCD1–SCD4). SCD1-deficient mice do not become obese or diabetic when fed a high-fat diet because of improved lipid metabolic profiles and insulin sensitivity5,6. Thus, SCD1 is a pharmacological target in the treatment of obesity, diabetes, and other metabolic diseases7. SCD1 is an integral membrane protein located in the endoplasmic reticulum, and catalyzes the formation of a cis-double bond between the 9th and 10th carbons of stearoyl- or palmitoyl-CoA8,9. The reaction requires molecular oxygen, which is activated by a diiron center, and cytochrome b5, which regenerates the diiron center10. To better understand the structural basis of these characteristics of SCD function, we crystallized and solved the structure of mouse SCD1 bound to stearoyl-CoA at 2.6 Å resolution. The structure shows a novel fold comprising four transmembrane helices capped by a cytosolic domain, and a plausible pathway for lateral substrate access and product egress. The acyl chain of the bound stearoyl-CoA is enclosed in a tunnel buried in the cytosolic domain, and the geometry of the tunnel and configuration of the bound acyl chain provide a structural basis for the regioselectivity and stereospecificity of the desaturation reaction. The dimetal center is coordinated by a unique configuration of nine conserved histidine residues that implies a potentially novel metal center and mechanism for oxygen activation. The structure also illustrates a possible route for electron transfer from cytochrome b5 to the diiron center.

Fattening up proteins Many eukaryotic proteins are modified by the attachment of lipids, and these modifications can alter how proteins interact with cellular membranes. Rana et al. present x-ray crystal structures of an integral membrane enzyme that appends a fatty acyl chain onto a cysteine residue of target proteins. The enzyme active site is situated at the membrane surface, thus explaining the enzyme's preference for substrates that are already membrane-associated. The structure of a fatty acid-like inhibitor bound within a hydrophobic cavity elucidates the mechanism for the enzyme's acyl chain specificity. Science , this issue p. eaao6326

DHHC (Asp-His-His-Cys) palmitoyltransferases are eukaryotic integral membrane enzymes that catalyze protein palmitoylation, which is important in a range of physiological processes, including small guanosine triphosphatase (GTPase) signaling, cell adhesion, and neuronal receptor scaffolding. We present crystal structures of two DHHC palmitoyltransferases and a covalent intermediate mimic. The active site resides at the membrane-cytosol interface, which allows the enzyme to catalyze thioester-exchange chemistry by using fatty acyl-coenzyme A and explains why membrane-proximal cysteines are candidates for palmitoylation. The acyl chain binds in a cavity formed by the transmembrane domain. We propose a mechanism for acyl chain-length selectivity in DHHC enzymes on the basis of cavity mutants with preferences for shorter and longer acyl chains.

Resistance-nodulation-cell division efflux pumps are integral membrane proteins that catalyze the export of substrates across cell membranes. Within the hydrophobe-amphiphile efflux subfamily, these resistance-nodulation-cell division proteins largely form trimeric efflux pumps. The drug efflux process has been proposed to entail a synchronized motion between subunits of the trimer to advance the transport cycle, leading to the extrusion of drug molecules. Here we use X-ray crystallography and single-molecule fluorescence resonance energy transfer imaging to elucidate the structures and functional dynamics of the Campylobacter jejuni CmeB multidrug efflux pump. We find that the CmeB trimer displays a very unique conformation. A direct observation of transport dynamics in individual CmeB trimers embedded in membrane vesicles indicates that each CmeB subunit undergoes conformational transitions uncoordinated and independent of each other. On the basis of our findings and analyses, we propose a model for transport mechanism where CmeB protomers function independently within the trimer., Multidrug efflux pumps significantly contribute for bacteria resistance to antibiotics. Here the authors present the structure of Campylobacter jejuni CmeB pump combined with functional FRET assays to propose a transport mechanism where each CmeB protomers is functionally independent from the trimer.

Sulfur, most abundantly found in the environment as sulfate (SO42-), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. However, current understanding of the cellular delivery of SO42- at the molecular level is limited. CysZ has been described as a SO42- permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO42- binding and flux experiments, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO42- across membranes. CysZ structures from three different bacterial species display a hitherto unknown fold and have subunits organized with inverted transmembrane topology. CysZ from Pseudomonas denitrificans assembles as a trimer of antiparallel dimers and the CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues.

CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids, using an RNA-mediated interference mechanism. Interference in type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine, Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Thermobifida fusca Cas3 bound to single-stranded (ss) DNA substrate and show that it is an obligate 3'-to-5' ssDNase that preferentially accepts substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ssDNA cleavage. We demonstrate ATP coordination and conformational flexibility of the SF2-type helicase domain. Cas3 is specifically guided toward Cascade-bound target DNA by a PAM sequence, through physical interactions with both the nontarget substrate strand and the CasA protein. The sequence of recognition events ensures well-controlled DNA targeting and degradation of foreign DNA by Cascade and Cas3.

Dissecting how signaling directs axon growth During development of the nervous system, nerve cells send out projections called axons that must be guided to their proper targets. Netrins are secreted proteins that bind to receptors to either attract or repel the growing axons. Xu et al. present x-ray structures that show that complexes of netrin with two different receptors, neogenin and DCC, have different architectures. How netrin signals remains to be understood in detail, but netrin's ability to create different assemblies probably plays a role in the diverse signaling outcomes it mediates. Science , this issue p. 1275

As one of the world's most prevalent enteric pathogens, Campylobacter jejuni is a major causative agent of human enterocolitis and is responsible for more than 400 million cases of diarrhea each year. The impact of this pathogen on children is of particular significance. Campylobacter has developed resistance to many antimicrobial agents via multidrug efflux machinery. The CmeABC tripartite multidrug efflux pump, belonging to the resistance-nodulation-cell division (RND) superfamily, plays a major role in drug resistant phenotypes of C. jejuni. This efflux complex spans the entire cell envelop of C. jejuni and mediates resistance to various antibiotics and toxic compounds. We here report the crystal structure of C. jejuni CmeC, the outer membrane component of the CmeABC tripartite multidrug efflux system. The structure reveals a possible mechanism for substrate export.

The Rv1217c-Rv1218c multidrug efflux system, which belongs to the ATP-binding cassette superfamily, recognizes and actively extrudes a variety of structurally unrelated toxic chemicals and mediates the intrinsic resistance to these antimicrobials in Mycobacterium tuberculosis. The expression of Rv1217c-Rv1218c is controlled by the TetR-like transcriptional regulator Rv1219c, which is encoded by a gene immediately upstream of rv1218c. To elucidate the structural basis of Rv1219c regulation, we have determined the crystal structure of Rv1219c, which reveals a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. The N-terminal domains of the Rv1219c dimer are separated by a large center-to-center distance of 64 A. The C-terminal domain of each protomer possesses a large cavity. Docking of small compounds to Rv1219c suggests that this large cavity forms a multidrug binding pocket, which can accommodate a variety of structurally unrelated antimicrobial agents. The internal wall of the multidrug binding site is surrounded by seven aromatic residues, indicating that drug binding may be governed by aromatic stacking interactions. In addition, fluorescence polarization reveals that Rv1219c binds drugs in the micromolar range.

Strategies for phasing nucleic acid structures by molecular replacement, using both experimental and de novo designed models, are discussed., Structured RNA molecules are key players in ensuring cellular viability. It is now emerging that, like proteins, the functions of many nucleic acids are dictated by their tertiary folds. At the same time, the number of known crystal structures of nucleic acids is also increasing rapidly. In this context, molecular replacement will become an increasingly useful technique for phasing nucleic acid crystallographic data in the near future. Here, strategies to select, create and refine molecular-replacement search models for nucleic acids are discussed. Using examples taken primarily from research on group II introns, it is shown that nucleic acids are amenable to different and potentially more flexible and sophisticated molecular-replacement searches than proteins. These observations specifically aim to encourage future crystallographic studies on the newly discovered repertoire of noncoding transcripts.

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 77 people, with a fatality rate of more than 50%. Alarmingly, the virus demonstrates the capability of human-to-human transmission, raising the possibility of global spread and endangering world health and economy. Here we have identified the receptor-binding domain (RBD) from the MERS-CoV spike protein and determined its crystal structure. This study also presents a structural comparison of MERS-CoV RBD with other coronavirus RBDs, successfully positioning MERS-CoV on the landscape of coronavirus evolution and providing insights into receptor binding by MERS-CoV. Furthermore, we found that MERS-CoV RBD functions as an effective entry inhibitor of MERS-CoV. The identified MERS-CoV RBD may also serve as a potential candidate for MERS-CoV subunit vaccines. Overall, this study enhances our understanding of the evolution of coronavirus RBDs, provides insights into receptor recognition by MERS-CoV, and may help control the transmission of MERS-CoV in humans.

Angiogenesis is a complex cellular process involving multiple regulatory growth factors and growth factor receptors. Among them, the ligands for the endothelial-specific tunica intima endothelial receptor tyrosine kinase 2 (Tie2) receptor kinase, angiopoietin-1 (Ang1) and Ang2, play essential roles in balancing vessel stability and regression during both developmental and tumor-induced angiogenesis. Despite possessing a high degree of sequence identity, Ang1 and Ang2 have distinct functional roles and cell-signaling characteristics. Here, we present the crystal structures of Ang1 both unbound and in complex with the Tie2 ectodomain. Comparison of the Ang1-containing structures with their Ang2-containing counterparts provide insight into the mechanism of receptor activation and reveal molecular surfaces important for interactions with Tie2 coreceptors and associated signaling proteins. Using structure-based mutagenesis, we identify a loop within the angiopoietin P domain, adjacent to the receptor-binding interface, which confers the specific agonist/antagonist properties of the molecule. We demonstrate using cell-based assays that an Ang2 chimera containing the Ang1 loop sequence behaves functionally similarly to Ang1 as a constitutive Tie2 agonist, able to efficiently dissociate the inhibitory Tie1/Tie2 complex and elicit Tie2 clustering and downstream signaling.

Analysis of crystal structure elucidates the mechanism by which a human enzyme acts as both a primase and a DNA polymerase., PrimPol is a novel human enzyme that contains both DNA primase and DNA polymerase activities. We present the first structure of human PrimPol in ternary complex with a DNA template-primer and an incoming deoxynucleoside triphosphate (dNTP). The ability of PrimPol to function as a DNA primase stems from a simple but remarkable feature—almost complete lack of contacts to the DNA primer strand. This, in turn, allows two dNTPs to bind initiation and elongation sites on the enzyme for the formation of the first dinucleotide. PrimPol shows the ability to synthesize DNA opposite ultraviolet (UV) lesions; however, unexpectedly, the active-site cleft of the enzyme is constrained, which precludes the bypass of UV-induced DNA lesions by conventional translesion synthesis. Together, the structure addresses long-standing questions about how DNA primases actually initiate synthesis and how primase and polymerase activities combine in a single enzyme to carry out DNA synthesis.

Entry to sporulation in bacilli is governed by a histidine kinase phosphorelay, a variation of the predominant signal transduction mechanism in prokaryotes. Sda directly inhibits sporulation histidine kinases in response to DNA damage and replication defects. We determined a 2.0-A-resolution X-ray crystal structure of the intact cytoplasmic catalytic core [comprising the dimerization and histidine phosphotransfer domain (DHp domain), connected to the ATP binding catalytic domain] of the Geobacillus stearothermophilus sporulation kinase KinB complexed with Sda. Structural and biochemical analyses reveal that Sda binds to the base of the DHp domain and prevents molecular transactions with the DHp domain to which it is bound by acting as a simple molecular barricade. Sda acts to sterically block communication between the catalytic domain and the DHp domain, which is required for autophosphorylation, as well as to sterically block communication between the response regulator Spo0F and the DHp domain, which is required for phosphotransfer and phosphatase activities.

CRISPR-Cas9 and CRISPR-Cpf1 systems have been successfully harnessed for genome editing. In the CRISPR-Cas9 system, the preordered A-form RNA seed sequence and preformed protein PAM-interacting cleft are essential for Cas9 to form a DNA recognition-competent structure. Whether the CRISPR-Cpf1 system employs a similar mechanism for target DNA recognition remains unclear. Here, we have determined the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with crRNA and target DNA. Structural comparison between the AsCpf1-crRNA-DNA ternary complex and the recently reported Lachnospiraceae bacterium Cpf1 (LbCpf1)-crRNA binary complex identifies a unique mechanism employed by Cpf1 for target recognition. The seed sequence required for initial DNA interrogation is disordered in the Cpf1-cRNA binary complex, but becomes ordered upon ternary complex formation. Further, the PAM interacting cleft of Cpf1 undergoes an "open-to-closed" conformational change upon target DNA binding, which in turn induces structural changes within Cpf1 to accommodate the ordered A-form seed RNA segment. This unique mechanism of target recognition by Cpf1 is distinct from that reported previously for Cas9.

The Ltn1 E3 ligase (listerin in mammals) has emerged as a paradigm for understanding ribosome-associated ubiquitylation. Ltn1 binds to 60S ribosomal subunits to ubiquitylate nascent polypeptides that become stalled during synthesis; among Ltn1's substrates are aberrant products of mRNA lacking stop codons [nonstop translation products (NSPs)]. Here, we report the reconstitution of NSP ubiquitylation in Neurospora crassa cell extracts. Upon translation in vitro, ribosome-stalled NSPs were ubiquitylated in an Ltn1-dependent manner, while still ribosome-associated. Furthermore, we provide biochemical evidence that the conserved N-terminal domain (NTD) plays a significant role in the binding of Ltn1 to 60S ribosomal subunits and that NTD mutations causing defective 60S binding also lead to defective NSP ubiquitylation, without affecting Ltn1's intrinsic E3 ligase activity. Finally, we report the crystal structure of the Ltn1 NTD at 2.4-Å resolution. The structure, combined with additional mutational studies, provides insight to NTD's role in binding stalled 60S subunits. Our findings show that Neurospora extracts can be used as a tool to dissect mechanisms underlying ribosome-associated protein quality control and are consistent with a model in which Ltn1 uses 60S subunits as adapters, at least in part via its NTD, to target stalled NSPs for ubiquitylation.

Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis., The validation and analysis of X-ray crystallographic data is essential for reproducibility and the development of crystallographic methods. Here, the authors describe a repository for crystallographic datasets and demonstrate some of the ways it could serve the crystallographic community.

Interleukin 20 (IL-20) is a pleotropic IL-10 family cytokine that protects epithelial surfaces from pathogens. However, dysregulated IL-20 signaling is implicated in several human pathologies including psoriasis, rheumatoid arthritis, atherosclerosis, and osteoporosis. IL-20, and related cytokines IL-19 and IL-24, designated IL-20 subfamily cytokines (IL-20SFCs), induce cellular responses through an IL-20R1/IL-20R2 (type I) receptor heterodimer, whereas IL-20 and IL-24 also signal through the IL-22R1/IL-20R2 (type II) receptor complex. The crystal structure of the IL-20/IL-20R1/IL-20R2 complex reveals how type I and II complexes discriminate cognate from noncognate ligands. The structure also defines how the receptor–cytokine interfaces are affinity tuned to allow distinct signaling through a receptor complex shared by three different ligands. Our results provide unique insights into the complexity of IL-20SFC signaling that may be critical in the design of mechanistic-based inhibitors of IL-20SFC–mediated inflammatory disease.

Significant advances in our understanding of RNA architecture, folding and recognition have emerged from structure–function studies on riboswitches, non-coding RNAs whose sensing domains bind small ligands and whose adjacent expression platforms contain RNA elements involved in the control of gene regulation. We now report on the ligand-bound structure of the Thermotoga petrophila fluoride riboswitch, which adopts a higher-order RNA architecture stabilized by pseudoknot and long-range reversed Watson–Crick and Hoogsteen A•U pair formation. The bound fluoride ion is encapsulated within the junctional architecture, anchored in place through direct coordination to three Mg2+ ions, which in turn are octahedrally coordinated to water molecules and five inwardly pointing backbone phosphates. Our structure of the fluoride riboswitch in the bound state shows how RNA can form a binding pocket selective for fluoride, while discriminating against larger halide ions. The T. petrophila fluoride riboswitch probably functions in gene regulation through a transcription termination mechanism. A riboswitch that binds fluoride was identified recently, which is surprising because both RNA and fluoride are negatively charged; here it is shown that the fluoride ion is coordinated to three positively charged magnesium ions, which are further encased in a negatively charged shell of RNA backbone phosphates and water molecules. Riboswitches are untranslated, structured regions of messenger RNA molecules that adopt and bind a ligand to affect its expression. The recent identification of a riboswitch that binds fluoride was a surprise, because both fluoride and RNA are negatively charged. Dinshaw Patel and colleagues have now determined the structure of the fluoride riboswitch from Thermotoga petrophila, and show how this unlikely contribution can occur. The fluoride ligand is coordinated to three positively charged magnesium ions, which are further encased in a negatively charged shell of backbone phosphates and water molecules. The size of the binding pocket excludes the binding of larger halide ions.

Interleukin-20 (IL-20) is an IL-10-family cytokine that regulates innate and adaptive immunity in skin and other tissues. In addition to protecting the host from various external pathogens, dysregulated IL-20 signaling has been shown to contribute to the pathogenesis of human psoriasis. IL-20 signals through two cell-surface receptor heterodimers, IL-20R1-IL-20R2 and IL-22R1-IL-20R2. In this report, crystals of the IL-20-IL-20R1-IL-20R2 ternary complex have been grown from polyethylene glycol solutions. The crystals belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 111, c = 135 Å, and diffracted X-rays to 3 Å resolution. The crystallographic asymmetric unit contains one IL-20-IL-20R1-IL-20R2 complex, corresponding to a solvent content of approximately 54%.

Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same β-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein–protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the β-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

The Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein that adheres to salivary components and extracellular matrix molecules. Here we report the 2.5 Å resolution crystal structure of the complete C-terminal region of AgI/II. The C-terminal region is comprised of three major domains: C(1), C(2), and C(3). Each domain adopts a DE-variant IgG fold, with two β-sheets whose A and F strands are linked through an intramolecular isopeptide bond. The adherence of the C-terminal AgI/II fragments to the putative tooth surface receptor salivary agglutinin (SAG), as monitored by surface plasmon resonance, indicated that the minimal region of binding was contained within the first and second DE-variant-IgG domains (C(1) and C(2)) of the C terminus. The minimal C-terminal region that could inhibit S. mutans adherence to SAG was also confirmed to be within the C(1) and C(2) domains. Competition experiments demonstrated that the C- and N-terminal regions of AgI/II adhere to distinct sites on SAG. A cleft formed at the intersection between these C(1) and C(2) domains bound glucose molecules from the cryo-protectant solution, revealing a putative binding site for its highly glycosylated receptor SAG. Finally, electron microscopy images confirmed the elongated structure of AgI/II and enabled building a composite tertiary model that encompasses its two distinct binding regions.

Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.

The TrkH/TrkG/KtrB proteins mediate K+ uptake in bacteria and probably evolved from simple K+ channels by multiple gene duplications or fusions. Here we present the crystal structure of a TrkH from Vibrio parahaemolyticus. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to those of K+ channels but significantly shorter, is lined by backbone and side-chain oxygen atoms. Functional studies showed that TrkH is selective for permeation of K+ and Rb+ over smaller ions such as Na+ or Li+. Immediately intracellular to the selectivity filter are an intramembrane loop and an arginine residue, both highly conserved, which constrict the permeation pathway. Substituting the arginine with an alanine significantly increases the rate of K+ flux. These results reveal the molecular basis of K+ selectivity and suggest a novel gating mechanism for this large and important family of membrane transport proteins. All living cells accumulate potassium ions, and a high intracellular potassium concentration is essential for many physiological processes. In bacteria, yeast and plants, potassium ion uptake is achieved by a superfamily of transporters, the SKT proteins. These are thought to have evolved from simple potassium channels, but the mechanism of potassium ion selectivity and transport is unknown. The crystal structure of a bacterial SKT protein, the TrkH potassium transporter from Vibrio parahaemolyticus, has now been determined. Its selectivity filter is similar to that of potassium ion channels, but it is shorter. Biochemical studies suggest that K+ selectivity depends on a novel gating mechanism that excludes smaller ions such as Na+ and Li+ in favour of the larger K+ and Rb+. Here, the X-ray crystal structure of TrkH is solved, a protein that mediates potassium uptake in Vibrio parahaemolyticus, a bacterium. The selectivity filter of the ion transporter is very similar to what is seen in (much simpler) potassium ion channels, but it is significantly shorter. Biochemical studies were performed to explore the molecular basis of K+ selectivity, and it is believed that a novel gating mechanism is involved.

Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 Å) crystal structure of the A 3 VP 1 fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 Å) through the interaction of two noncontiguous regions in the primary sequence. The A 3 repeat of the alanine-rich domain adopts an extended α-helix that intertwines with the P 1 repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A 3 and P 1 helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

Gram-negative bacteria, such as Escherichia coli, use tripartite efflux complexes in the resistance-nodulation-division family to expel diverse toxic compounds from the cell. The CusCBA system is responsible for extruding biocidal Cu(I) and Ag(I) ions from the cell. The X-ray crystal structure of CusA, the inner membrane transporter, has now been determined in the absence and presence of bound Cu(I) or Ag(I). The structures suggest that the metal binding site, formed by a three-methionine cluster, is located within the cleft region of the periplasmic domain. The authors propose a potential pathway for ion export in which CusA is capable of picking up the metal ion from the cytosol, with this transporter utilizing the methionine pairs and clusters to bind and export metal ions. Gram-negative bacteria, such as Escherichia coli, use tripartite efflux complexes in the resistance-nodulation-cell division family to expel toxic compounds from the cell. The CusCBA system is responsible for removing biocidal Cu(I) and Ag(I) ions. Here, the X-ray crystal structure is reported of CusA in the absence and presence of bound Cu(I) or Ag(I). The structures reveal that the metal-binding sites are located within the cleft region of the periplasmic domain. A potential pathway for ion export is proposed. Gram-negative bacteria, such as Escherichia coli, frequently use tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel various toxic compounds from the cell1,2. The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions3,4. No previous structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner-membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide new structural information about the HME subfamily of RND efflux pumps. The structures suggest that the metal-binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. This cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal-binding site, four methionine pairs—three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, using these methionine pairs or clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites.

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance-nodulation-division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein that is critical for substrate transport. We here present the x-ray structures of the CusB membrane fusion protein from the copper/silver efflux system of E. coli. This is the first structure of any membrane fusion proteins associated with heavy-metal efflux transporters. CusB bridges the inner membrane efflux pump CusA and outer membrane channel CusC to mediate resistance to Cu+ and Ag+ ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly β-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of membrane fusion proteins, the α-helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N and C-termini of CusB form the first β-strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic details of how this efflux protein binds Cu+ and Ag+ were revealed by the crystals of the CusB-Cu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of a membrane fusion protein in the resistance-nodulation-division efflux system, and provide evidence that this protein specifically interacts with transported substrates.

In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of beta-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct beta-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

Peripheral membrane proteins of the Bin/amphiphysin/Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) family participate in cellular membrane trafficking and have been shown to generate membrane tubules. The degree of membrane bending appears to be encoded in the structure and immanent curvature of the particular protein domains, with BAR and F-BAR domains inducing high- and low-curvature tubules, respectively. In addition, oligomerization and the formation of ordered arrays influences tubule stabilization. Here, the F-BAR domain-containing protein Pacsin was found to possess a unique activity, creating small tubules and tubule constrictions, in addition to the wide tubules characteristic for this subfamily. Based on crystal structures of the F-BAR domain of Pacsin and mutagenesis studies, vesiculation could be linked to the presence of unique structural features distinguishing it from other F-BAR proteins. Tubulation was suppressed in the context of the full-length protein, suggesting that Pacsin is autoinhibited in solution. The regulated deformation of membranes and promotion of tubule constrictions by Pacsin suggests a more versatile function of these proteins in vesiculation and endocytosis beyond their role as scaffold proteins.

Ephrin (Eph) receptor tyrosine kinases fall into two subclasses (A and B) according to preferences for their ephrin ligands. All published structural studies of Eph receptor/ephrin complexes involve B-class receptors. Here, we present the crystal structures of an A-class complex between EphA2 and ephrin-A1 and of unbound EphA2. Although these structures are similar overall to their B-class counterparts, they reveal important differences that define subclass specificity. The structures suggest that the A-class Eph receptor/ephrin interactions involve smaller rearrangements in the interacting partners, better described by a 'lock-and-key'-type binding mechanism, in contrast to the 'induced fit' mechanism defining the B-class molecules. This model is supported by structure-based mutagenesis and by differential requirements for ligand oligomerization by the two subclasses in cell-based Eph receptor activation assays. Finally, the structure of the unligated receptor reveals a homodimer assembly that might represent EphA2-specific homotypic cell adhesion interactions.

Nuclear pore complexes (NPCs) facilitate nucleocytoplasmic transport. These massive assemblies comprise an eightfold symmetric scaffold of architectural proteins and central-channel phenylalanine-glycine-repeat proteins forming the transport barrier. We determined the nucleoporin 85 (Nup85)⚫Seh1 structure, a module in the heptameric Nup84 complex, at 3.5 angstroms resolution. Structural, biochemical, and genetic analyses position the Nup84 complex in two peripheral NPC rings. We establish a conserved tripartite element, the ancestral coatomer element ACE1, that reoccurs in several nucleoporins and vesicle coat proteins, providing structural evidence of coevolution from a common ancestor. We identified interactions that define the organization of the Nup84 complex on the basis of comparison with vesicle coats and confirmed the sites by mutagenesis. We propose that the NPC scaffold, like vesicle coats, is composed of polygons with vertices and edges forming a membrane-proximal lattice that provides docking sites for additional nucleoporins.

DNA sliding clamps form an oligomeric ring encircling DNA and serve as a moving platform for DNA-processing proteins. The opening and closing of a sliding-clamp ring is essential to load the clamp onto DNA in order to perform its functions. The molecular details of how clamp rings open and enclose DNA are still not clear. Three PCNA homologues have been found in Sulfolobus solfataricus which form a heterotrimer. Taking advantage of their hetero-oligomeric nature, the structures of the PCNAs in monomeric PCNA3, dimeric PCNA1-PCNA2 and trimeric PCNA1-PCNA2-PCNA3 forms were determined at resolutions of 2.6-1.9 A. The distinct oligomeric structures represent different stages in ring formation, which were verified in solution by ultracentrifugation analysis. The heterodimer opens in a V-shape of 130 degrees , while the heterotrimers form a ring with a 120 degrees rotation between monomers. The association of a rigid PCNA3 monomer with an opened PCNA1-PCNA2 heterodimer closes the ring and introduces a spring tension in the PCNA1-PCNA2 interface, thus bending the nine-stranded intermolecular beta-sheet to fit the 120 degrees rotation. The release of the spring tension as PCNA3 dissociates from the ring may facilitate ring opening. The structural features in different assemblies present a molecular model for clamp ring assembly and opening.