Structure (v.19, #3)

In This Issue (v-vi).

In this issue, reveal the structural basis for degenerate RNA recognition by human Pumilio proteins. The structures suggest an appealing model where nucleotides that do not contribute to binding affinity define Pumilio regulatory activity by adopting distinctive conformations with differential ability to recruit regulatory cofactors.

In this issue, show that the 3′ end processing factor CFIm interacts with RNA in manner that facilitates RNA looping, suggesting mechanistic roles for this factor in the regulation of poly(A) site selection.

Mechanism-Based Strategies for Trapping and Crystallizing Complexes of RNA-Modifying Enzymes by Amandine Guelorget; Béatrice Golinelli-Pimpaneau (282-291).
Posttranscriptional chemical modifications of RNA are maturation steps necessary for their correct functioning in translation during protein synthesis. Various structures of RNA-modifying enzymes complexed with RNA fragments or full-length tRNA have been obtained, mimicking several stages along the catalytic cycle such as initial RNA binding, covalent intermediate formation, or RNA-product binding. We summarize here the strategies that have been used to trap and crystallize these stable complexes. Absence of the cosubstrate transferring the chemical group leads to the Michaelis complex, whereas use of a cosubstrate analog to a ternary complex. 5-fluoro-pyrimidine-containing mini RNAs have been used as a general means to trap RNA m5U methyltransferase covalent complexes and RNA product/pseudouridine synthase complexes. Altogether, these structures have brought key information about enzyme/RNA recognition and highlighted the details of several catalytic steps of the reactions.► Posttranscriptional chemical modification ensures correct functioning of RNAs ► Several RNA-modifying enzyme structures in complex with RNA are known ► The enzymatic mechanism has been exploited to crystallize reaction snapshots ► The complex structures explain the RNA-recognition mode and catalytic mechanism

ePMV Embeds Molecular Modeling into Professional Animation Software Environments by Graham T. Johnson; Ludovic Autin; David S. Goodsell; Michel F. Sanner; Arthur J. Olson (293-303).
Increasingly complex research has made it more difficult to prepare data for publication, education, and outreach. Many scientists must also wade through black-box code to interface computational algorithms from diverse sources to supplement their bench work. To reduce these barriers we have developed an open-source plug-in, embedded Python Molecular Viewer (ePMV), that runs molecular modeling software directly inside of professional 3D animation applications (hosts) to provide simultaneous access to the capabilities of these newly connected systems. Uniting host and scientific algorithms into a single interface allows users from varied backgrounds to assemble professional quality visuals and to perform computational experiments with relative ease. By enabling easy exchange of algorithms, ePMV can facilitate interdisciplinary research, smooth communication between broadly diverse specialties, and provide a common platform to frame and visualize the increasingly detailed intersection(s) of cellular and molecular biology.Display Omitted► ePMV provides molecular viewer functionality inside of professional animation software ► ePMV interoperates molecular modeling tools on the same data and from a single interface ► ePMV enables production of high-quality molecular visualizations, both static and dynamic ► ePMV facilitates communication between researchers and scientific visualization specialists

Our ability to infer the protein quaternary structure automatically from atom and lattice information is inadequate, especially for weak complexes, and heteromeric quaternary structures. Several approaches exist, but they have limited performance. Here, we present a new scheme to infer protein quaternary structure from lattice and protein information, with all-around coverage for strong, weak and very weak affinity homomeric and heteromeric complexes. The scheme combines naive Bayes classifier and point group symmetry under Boolean framework to detect quaternary structures in crystal lattice. It consistently produces ≥90% coverage across diverse benchmarking data sets, including a notably superior 95% coverage for recognition heteromeric complexes, compared with 53% on the same data set by current state-of-the-art method. The detailed study of a limited number of prediction-failed cases offers interesting insights into the intriguing nature of protein contacts in lattice. The findings have implications for accurate inference of quaternary states of proteins, especially weak affinity complexes.Display Omitted► Boolean framework for quaternary structure detection ► Naive Bayes classifier for discriminating biological and nonbiological interfaces ► Scheme to detect weak affinity quaternary structures in crystalline state ► Using point-group symmetry to aid quaternary structure detection

Two Structural and Functional Domains of MESD Required for Proper Folding and Trafficking of LRP5/6 by Jianglei Chen; Chia-Chen Liu; Qianqian Li; Christian Nowak; Guojun Bu; Jianjun Wang (313-323).
How the endoplasmic reticulum (ER) folding machinery coordinates general and specialized chaperones during protein translation and folding remains an important unanswered question. Here, we show two structural domains in MESD, a specialized chaperone for LRP5/6, carry out dual functions. The chaperone domain forms a complex with the immature receptor, maintaining the β-propeller (BP) domain in an interaction competent state for epidermal growth factor-repeat binding. This promotes proper folding of the BP domain, causing a binding switch from the chaperone domain to the escort domain. The escort complex ensures LRP5/6 safe-trafficking from the ER to the Golgi by preventing premature ligand-binding. Inside the Golgi, the BP domain may contain a histidine switch, regulating MESD dissociation and retrieval. Together, we generate a plausible cell biology picture of the MESD/LRP5/6 pathway, suggesting that it is the specialized chaperones, MESD, that serves as the folding template to drive proper folding and safe trafficking of large multidomain proteins LRP5/6.Display Omitted► MESD contains two structural/functional domains: a chaperone and an escort domain ► The two domains of MESD regulate proper folding and safe-trafficking of LRP5/6 ► The BP domain of LRP5/6 mediates interaction switches between MESD and LRP5/6 ► The ER folding machinery uniquely coordinates specialized and general chaperones

Folding and trafficking of low-density lipoprotein receptor (LDLR) family members, which play essential roles in development and homeostasis, are mediated by specific chaperones. The Boca/Mesd chaperone family specifically promotes folding and trafficking of the YWTD β propeller-EGF domain pair found in the ectodomain of all LDLR members. Limited proteolysis, NMR spectroscopy, analytical ultracentrifugation, and X-ray crystallography were used to define a conserved core composed of a structured domain that is preceded by a disordered N-terminal region. High-resolution structures of the ordered domain were determined for homologous proteins from three metazoans. Seven independent protomers reveal a novel ferrodoxin-like superfamily fold with two distinct β sheet topologies. A conserved hydrophobic surface forms a dimer interface in each crystal, but these differ substantially at the atomic level, indicative of nonspecific hydrophobic interactions that may play a role in the chaperone activity of the Boca/Mesd family.Display Omitted► Boca/Mesd chaperone family has a conserved but unstructured N-terminal region ► Four crystals of conserved structured domain (SD) from three species, distinct from NMR SD ► Slippery hydrophobic dimer interface implies putative SD-substrate interaction site ► Conformational change within SD β sheet may act in the chaperone mechanism

The Structure of MESD45–184 Brings Light into the Mechanism of LDLR Family Folding by Christian Köhler; Janet K. Lighthouse; Tobias Werther; Olav M. Andersen; Annette Diehl; Peter Schmieder; Jianguang Du; Bernadette C. Holdener; Hartmut Oschkinat (337-348).
Mesoderm development (MESD) is a 224 amino acid mouse protein that acts as a molecular chaperone for the low-density lipoprotein receptor (LDLR) family. Here, we provide evidence that the region 45–184 of MESD is essential and sufficient for this function and suggest a model for its mode of action. NMR studies reveal a β−α−β−β−α−β core domain with an α-helical N-terminal extension that interacts with the β sheet in a dynamic manner. As a result, the structural ensemble contains open (active) and closed (inactive) forms, allowing for regulation of chaperone activity through substrate binding. The mutant W61R, which is lethal in Drosophila, adopts only the open state. The receptor motif recognized by MESD was identified by in vitro-binding studies. Furthermore, in vivo functional evidence for the relevance of the identified contact sites in MESD is provided.► Solution NMR structure of the conserved part of MESD ► Measurements of internal dynamics reveal a two-state conformer ► Identification of MESD's contact site to LRP's by in vitro and in vivo data ► Suggestion of a mechanism for the MESD-assisted folding of LDLR family members

Cooperativity is a defining feature of protein folding, but its thermodynamic and structural origins are not completely understood. By constructing consensus ankyrin repeat protein arrays that have nearly identical sequences, we quantify cooperativity by resolving stability into intrinsic and interfacial components. Heteronuclear NMR and CD spectroscopy show that these constructs adopt ankyrin repeat structures. Applying a one-dimensional Ising model to a series of constructs chosen to maximize information content in unfolding transitions, we quantify stabilities of the terminal capping repeats, and resolve the effects of denaturant into intrinsic and interfacial components. Reversible thermal denaturation resolves interfacial and intrinsic free energies into enthalpic, entropic, and heat capacity terms. Intrinsic folding is entropically disfavored, whereas interfacial interaction is entropically favored and attends a decrease in heat capacity. These results suggest that helix formation and backbone ordering occurs upon intrinsic folding, whereas hydrophobic desolvation occurs upon interfacial interaction, contributing to cooperativity.► Ankyrin repeats are entropically destabilized but enthalpically stabilized ► Ankyrin interfaces are entropically stabilized but enthalpically destabilized ► Ankyrin repeats show high guanidine dependence, low heat capacity change ► Consensus ankyrin repeats adopt ankyrin tertiary structure in solution

Alternate Modes of Cognate RNA Recognition by Human PUMILIO Proteins by Gang Lu; Traci M. Tanaka Hall (361-367).
Human PUMILIO1 (PUM1) and PUMILIO2 (PUM2) are members of the PUMILIO/FBF (PUF) family that regulate specific target mRNAs posttranscriptionally. Recent studies have identified mRNA targets associated with human PUM1 and PUM2. Here, we explore the structural basis of natural target RNA recognition by human PUF proteins through crystal structures of the RNA-binding domains of PUM1 and PUM2 in complex with four cognate RNA sequences, including sequences from p38α and erk2 MAP kinase mRNAs. We observe three distinct modes of RNA binding around the fifth RNA base, two of which are different from the prototypical 1 repeat:1 RNA base binding mode previously identified with model RNA sequences. RNA-binding affinities of PUM1 and PUM2 are not affected dramatically by the different binding modes in vitro. However, these modes of binding create structurally variable recognition surfaces that suggest a mechanism in vivo for recruitment of downstream effector proteins defined by the PUF:RNA complex.► PUM1 and PUM2 bind to cognate RNAs with three different recognition modes ► Binding affinities are equivalent despite the different binding modes ► Different modes present alternative recognition surfaces for downstream effector proteins

Cleavage factor Im (CFIm) is a highly conserved component of the eukaryotic mRNA 3′ processing machinery that functions in sequence-specific poly(A) site recognition through the collaboration of a 25 kDa subunit containing a Nudix domain and a larger subunit of 59, 68, or 72 kDa containing an RNA recognition motif (RRM). Our previous work demonstrated that CFIm25 is both necessary and sufficient for sequence-specific binding of the poly(A) site upstream element UGUA. Here, we report the crystal structure of CFIm25 complexed with the RRM domain of CFIm68 and RNA. The CFIm25 dimer is clasped on opposite sides by two CFIm68 RRM domains. Each CFIm25 subunit binds one UGUA element specifically. Biochemical analysis indicates that the CFIm68 RRMs serve to enhance RNA binding and facilitate RNA looping. The intrinsic ability of CFIm to direct RNA looping may provide a mechanism for its function in the regulation of alternative poly(A) site selection.Display Omitted► The two subunits of cleavage factor Im (CFIm) form an unexpected heterotetramer ► The CFIm25 homodimer specifically recognizes two UGUA cis-elements simultaneously ► CFIm68 facilitates looping of the intervening RNA between two UGUA elements ► CFIm-mediated RNA looping may regulate alternative poly(A) site selection

The Mechanisms of HAMP-Mediated Signaling in Transmembrane Receptors by Hedda U. Ferris; Stanislaw Dunin-Horkawicz; Laura García Mondéjar; Michael Hulko; Klaus Hantke; Jörg Martin; Joachim E. Schultz; Kornelius Zeth; Andrei N. Lupas; Murray Coles (378-385).
HAMP domains mediate signal transduction in over 7500 enzyme-coupled receptors represented in all kingdoms of life. The HAMP domain of the putative archaeal receptor Af1503 has a parallel, dimeric, four-helical coiled coil structure, but with unusual core packing, related to canonical packing by concerted axial rotation of the helices. This has led to the gearbox model for signal transduction, whereby the alternate packing modes correspond to signaling states. Here we present structures of a series of Af1503 HAMP variants. We show that substitution of a conserved small side chain within the domain core (A291) for larger residues induces a gradual transition in packing mode, involving both changes in helix rotation and bundle shape, which are most prominent at the C-terminal, output end of the domain. These are correlated with activity and ligand response in vitro and in vivo by incorporating Af1503 HAMP into mycobacterial adenylyl cyclase assay systems.► Residue substitutions in the HAMP domain core induce axial helix rotation ► Helix rotation can be correlated with activity in vitro and in vivo ► HAMP domain output includes both changes in rotation state and bundle radius ► Supports the gearbox model of signal transduction via axial helix rotation

Heat Maps for Intramolecular Communication in an RNP Enzyme Encoding Glutamine by Annia Rodríguez-Hernández; John J. Perona (386-396).
Allosteric signaling within large ribonucleoproteins modulates both catalytic function and biological specificity, but the spatial extent and quantitative magnitudes of long-distance free-energy couplings have yet to be well characterized. Here, we employ pre-steady-state kinetics to generate a comprehensive mapping of intramolecular communication in the glutaminyl-tRNA synthetase:tRNAGln complex. Alanine substitution at 29 positions across the protein-RNA interface reveals distinct coupling amplitudes for glutamine binding and aminoacyl-tRNA formation on the enzyme, respectively, implying the existence of multiple signaling pathways. Structural models suggest that long-range signal propagation from the tRNA anticodon is dynamically driven, whereas shorter pathways are mediated by induced-fit rearrangements. Seven protein contacts with the distal tRNA vertical arm each weaken glutamine binding affinity across distances up to 40 Å, demonstrating that negative allosteric coupling plays a key role in enforcing the selective RNA-amino acid pairing at the heart of the genetic code.► Indirect readout of RNA sequence by a tRNA synthetase dominates direct readout ► Negative allostery between RNA and amino acid determines genetic code fidelity ► Signaling in the GlnRS RNP likely occurs by both induced-fit and dynamic mechanisms

X-ray Crystal Structure of the UCS Domain-Containing UNC-45 Myosin Chaperone from Drosophila melanogaster by Chi F. Lee; Arthur V. Hauenstein; Jonathan K. Fleming; William C. Gasper; Valerie Engelke; Banumathi Sankaran; Sanford I. Bernstein; Tom Huxford (397-408).
UCS proteins, such as UNC-45, influence muscle contraction and other myosin-dependent motile processes. We report the first X-ray crystal structure of a UCS domain-containing protein, the UNC-45 myosin chaperone from Drosophila melanogaster (DmUNC-45). The structure reveals that the central and UCS domains form a contiguous arrangement of 17 consecutive helical layers that arrange themselves into five discrete armadillo repeat subdomains. Small-angle X-ray scattering data suggest that free DmUNC-45 adopts an elongated conformation and exhibits flexibility in solution. Protease sensitivity maps to a conserved loop that contacts the most carboxy-terminal UNC-45 armadillo repeat subdomain. Amino acid conservation across diverse UCS proteins maps to one face of this carboxy-terminal subdomain, and the majority of mutations that affect myosin-dependent cellular activities lie within or around this region. Our crystallographic, biophysical, and biochemical analyses suggest that DmUNC-45 function is afforded by its flexibility and by structural integrity of its UCS domain.Display Omitted► The UCS and central domains of UNC-45 consist of five armadillo repeat subdomains ► The N-terminal TPR domain exhibits flexibility relative to central and UCS domains ► UNC-45 is more elongated and exhibits conformational flexibility in solution ► Sequence identity and deleterious mutations identify a myosin-interaction surface

Intrinsic Bending of Microtubule Protofilaments by Andrea Grafmüller; Gregory A. Voth (409-417).
The complex polymerization dynamics of the microtubule (MT) plus end are closely linked to the hydrolysis of the GTP nucleotide bound to the β-tubulin. The destabilization is thought to be associated with the conformational change of the tubulin dimers from the straight conformation in the MT lattice to a curved conformation. It remains under debate whether this transformation is directly related to the nucleotide state, or a consequence of the longitudinal or lateral contacts in the MT lattice. Here, we present large-scale atomistic simulations of short tubulin protofilaments with both nucleotide states, starting from both extreme conformations. Our simulations indicate that both interdimer and intradimer contacts in both GDP and GTP-bound tubulin dimers and protofilaments in solution bend. There are no observable differences between the mesoscopic properties of the contacts in GTP and GDP-bound tubulin or the intradime and interdimer interfaces.Display Omitted► GDP and GTP-bound tubulin protofilaments converge to a curved conformation ► No difference in mesoscopic properties of GDP and GTP filaments ► Rotation of the intermediate domain is not prevented by GTP ► Conformations resemble the 1SA2 structure closely with some additional contacts

Interferon-Inducible Protein 16: Insight into the Interaction with Tumor Suppressor p53 by Jack C.C. Liao; Robert Lam; Vaclav Brazda; Shili Duan; Mani Ravichandran; Justin Ma; Ting Xiao; Wolfram Tempel; Xiaobing Zuo; Yun-Xing Wang; Nickolay Y. Chirgadze; Cheryl H. Arrowsmith (418-429).
IFI16 is a member of the interferon-inducible HIN-200 family of nuclear proteins. It has been implicated in transcriptional regulation by modulating protein-protein interactions with p53 tumor suppressor protein and other transcription factors. However, the mechanisms of interaction remain unknown. Here, we report the crystal structures of both HIN-A and HIN-B domains of IFI16 determined at 2.0 and 2.35 Å resolution, respectively. Each HIN domain comprises a pair of tightly packed OB-fold subdomains that appear to act as a single unit. We show that both HIN domains of IFI16 are capable of enhancing p53-DNA complex formation and transcriptional activation via distinctive means. HIN-A domain binds to the basic C terminus of p53, whereas the HIN-B domain binds to the core DNA-binding region of p53. Both interactions are compatible with the DNA-bound state of p53 and together contribute to the effect of full-length IFI16 on p53-DNA complex formation and transcriptional activation.► Structures of a HIN domain revealing a pair of tandem OB folds in a rigid orientation relative to one another ► Structures reveal functional differences between the two homologous HIN domains of IFI16 ► Enhancement of p53 DNA binding and transactivation by both HIN domains of IFI16 ► SAXS reveals a shape of full-length IFI16 consistent with potential function as a scaffold protein

Quaternary Structure of SecA in Solution and Bound to SecYEG Probed at the Single Molecule Level by Ilja Kusters; Geert van den Bogaart; Alexej Kedrov; Victor Krasnikov; Faizah Fulyani; Bert Poolman; Arnold J.M. Driessen (430-439).
Dual-color fluorescence-burst analysis (DCFBA) was applied to measure the quaternary structure and high-affinity binding of the bacterial motor protein SecA to the protein-conducting channel SecYEG reconstituted into lipid vesicles. DCFBA is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. SecA binds to SecYEG as a dimer with a nucleotide- and preprotein-dependent dissociation constant. One of the SecA protomers binds SecYEG in a salt-resistant manner, whereas binding of the second protomer is salt sensitive. Because protein translocation is salt sensitive, we conclude that the dimeric state of SecA is required for protein translocation. A structural model for the dimeric assembly of SecA while bound to SecYEG is proposed based on the crystal structures of the Thermotoga maritima SecA-SecYEG and the Escherichia coli SecA dimer.Display Omitted► DCFBA allows assessment of the stoichiometry of ligands bound to membrane receptors ► Dimeric SecA binds asymmetrically to the protein-conducting membrane channel SecYEG ► Monomeric SecA binds SecYEG, but dimeric SecA is required for protein translocation ► Protein translocation depends on receptor cycling of the dimeric SecA