Structure (v.13, #11)
A New Niche for Notch on Deltex? by Stephen C. Blacklow (1579-1580).
The structure of the Deltex tandem WWE repeats reported by Barrick and colleagues in this issue of Structure reveals an intimate interface between the repeats, which together combine to create a binding site for the ankyrin repeat domain of Notch.
Syn-Full Behavior by T7 DNA Polymerase by William A. Beard; Samuel H. Wilson (1580-1582).
In this issue of Structure, report the structure of a mutant T7 DNA polymerase with a template lesion (8-oxo-7,8-dihydro-2′-deoxyguanosine) in a mutagenic syn conformation. This result provides a structural basis for understanding the role of template positioning during mutagenic DNA synthesis.
SARS Proteomics Reveals Viral Secrets by Elspeth Garman (1582-1583).
Worldwide cooperative efforts to understand the biology of the SARS coronavirus have already born significant fruit. In a further advance, the X-ray structure of a domain of nonstructural protein 3 is reported by in this issue of Structure.
The Ins and Outs of Protein Synthesis by Jamie H. Doudna Cate (1584-1585).
Translation initiation and protein trafficking begin and end the process of protein synthesis. In this issue of Structure, provide the first global model of HCV IRES-80S ribosome interactions. In a second paper, describe the structure of the bacterial 50S subunit with the ribosome binding domain of trigger factor (TF) with surprising conclusions about TF function.
Folding of Small Helical Proteins Assisted by Small-Angle X-Ray Scattering Profiles by Yinghao Wu; Xia Tian; Mingyang Lu; Mingzhi Chen; Qinghua Wang; Jianpeng Ma (1587-1597).
This paper reports a computational method for folding small helical proteins. The goal was to determine the overall topology of proteins given secondary structure assignment on sequence. In doing so, a Monte Carlo protocol, which combines coarse-grained normal modes and a Hamiltonian at a different scale, was developed to enhance sampling. In addition to the knowledge-based potential functions, a small-angle X-ray scattering (SAXS) profile was also used as a weak constraint for guiding the folding. The algorithm can deliver structural models with overall correct topology, which makes them similar to those of 5∼6 Å cryo-EM density maps. The success could contribute to make the SAXS technique a fast and inexpensive solution-phase experimental method for determining the overall topology of small, soluble, but noncrystallizable, helical proteins.
Structure and Notch Receptor Binding of the Tandem WWE Domain of Deltex by Mark E. Zweifel; Daniel J. Leahy; Doug Barrick (1599-1611).
Deltex is a cytosolic effector of Notch signaling thought to bind through its N-terminal domain to the Notch receptor. Here we report the structure of the Drosophila Deltex N-terminal domain, which contains two tandem WWE sequence repeats. The WWE repeats, which adopt a novel fold, are related by an approximate two-fold axis of rotation. Although the WWE repeats are structurally distinct, they interact extensively and form a deep cleft at their junction that appears well suited for ligand binding. The two repeats are thermodynamically coupled; this coupling is mediated in part by a conserved segment that is immediately C-terminal to the second WWE domain. We demonstrate that although the Deltex WWE tandem is monomeric in solution, it forms a heterodimer with the ankyrin domain of the Notch receptor. These results provide structural and functional insight into how Deltex modulates Notch signaling, and how WWE modules recognize targets for ubiquitination.
The Molecular Mechanism for Receptor-Stimulated Iron Release from the Plasma Iron Transport Protein Transferrin by Anthony M. Giannetti; Peter J. Halbrooks; Anne B. Mason; Todd M. Vogt; Caroline A. Enns; Pamela J. Björkman (1613-1623).
Human transferrin receptor 1 (TfR) binds iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes where iron is released in a TfR-facilitated process. Consistent with our hypothesis that TfR binding stimulates iron release from Fe-Tf at acidic pH by stabilizing the apo-Tf conformation, a TfR mutant (W641A/F760A-TfR) that binds Fe-Tf, but not apo-Tf, cannot stimulate iron release from Fe-Tf, and less iron is released from Fe-Tf inside cells expressing W641A/F760A-TfR than cells expressing wild-type TfR (wtTfR). Electron paramagnetic resonance spectroscopy shows that binding at acidic pH to wtTfR, but not W641A/F760A-TfR, changes the Tf iron binding site ≥30 Å from the TfR W641/F760 patch. Mutation of Tf histidine residues predicted to interact with the W641/F760 patch eliminates TfR-dependent acceleration of iron release. Identification of TfR and Tf residues critical for TfR-facilitated iron release, yet distant from a Tf iron binding site, demonstrates that TfR transmits long-range conformational changes and stabilizes the conformation of apo-Tf to accelerate iron release from Fe-Tf.
Mycobacterium tuberculosis Protein Tyrosine Phosphatase PtpB Structure Reveals a Diverged Fold and a Buried Active Site by Christoph Grundner; Ho-Leung Ng; Tom Alber (1625-1634).
Intracellular pathogenic bacteria manipulate host signal transduction pathways to facilitate infection. Mycobacterium tuberculosis protein tyrosine phosphatases (PTPs) PtpA and PtpB are thought to be secreted into host cells and interfere with unidentified signals. To illuminate the mechanisms of regulation and substrate recognition, we determined the 1.7 Å resolution crystal structure of PtpB in complex with the product phosphate. The protein adopts a simplified PTP fold, which combines features of the conventional PTPs and dual-specificity phosphatases. PtpB shows two unusual elaborations—a disordered, acidic loop and a flexible, two-helix lid that covers the active site—that are specific to mycobacterial orthologs. Biochemical studies suggest that substrate mimicry in the lid may protect the phosphatase from oxidative inactivation. The insertion and deletion of large structural elements in PtpB suggest that, outside the active site module, the PTP family is under unusual selective pressure that promotes changes in overall structure.
Activation Process of [NiFe] Hydrogenase Elucidated by High-Resolution X-Ray Analyses: Conversion of the Ready to the Unready State by Hideaki Ogata; Shun Hirota; Asuka Nakahara; Hirofumi Komori; Naoki Shibata; Tatsuhisa Kato; Kenji Kano; Yoshiki Higuchi (1635-1642).
Hydrogenases catalyze oxidoreduction of molecular hydrogen and have potential applications for utilizing dihydrogen as an energy source. [NiFe] hydrogenase has two different oxidized states, Ni-A (unready, exhibits a lag phase in reductive activation) and Ni-B (ready). We have succeeded in converting Ni-B to Ni-A with the use of Na2S and O2 and determining the high-resolution crystal structures of both states. Ni-B possesses a monatomic nonprotein bridging ligand at the Ni-Fe active site, whereas Ni-A has a diatomic species. The terminal atom of the bridging species of Ni-A occupies a similar position as C of the exogenous CO in the CO complex (inhibited state). The common features of the enzyme structures at the unready (Ni-A) and inhibited (CO complex) states are proposed. These findings provide useful information on the design of new systems of biomimetic dihydrogen production and fuel cell devices.
Crystal Structure of a Complex between Protein Tyrosine Phosphatase 1B and the Insulin Receptor Tyrosine Kinase by Shiqing Li; Rafael S. Depetris; David Barford; Jonathan Chernoff; Stevan R. Hubbard (1643-1651).
Protein tyrosine phosphatase 1B (PTP1B) is a highly specific negative regulator of insulin receptor signaling in vivo. The determinants of PTP1B specificity for the insulin receptor versus other receptor tyrosine kinases are largely unknown. Here, we report a crystal structure at 2.3 Å resolution of the catalytic domain of PTP1B (trapping mutant) in complex with the phosphorylated tyrosine kinase domain of the insulin receptor (IRK). The crystallographic asymmetric unit contains two PTP1B-IRK complexes that interact through an IRK dimer interface. Rather than binding to a phosphotyrosine in the IRK activation loop, PTP1B binds instead to the opposite side of the kinase domain, with the phosphorylated activation loops sequestered within the IRK dimer. The crystal structure provides evidence for a noncatalytic mode of interaction between PTP1B and IRK, which could be important for the selective recruitment of PTP1B to the insulin receptor.
A Lysine Residue in the Fingers Subdomain of T7 DNA Polymerase Modulates the Miscoding Potential of 8-Oxo-7,8-Dihydroguanosine by Luis G. Brieba; Robert J. Kokoska; Katarzyna Bebenek; Thomas A. Kunkel; Tom Ellenberger (1653-1659).
8-Oxo-7,8-dihydroguanosine (8oG) is a highly mutagenic DNA lesion that stably pairs with adenosine, forming 8oG(syn)·dA(anti) Hoogsteen base pairs. DNA polymerases show different propensities to insert dCMP or dAMP opposite 8oG, but the molecular mechanisms that determine faithful or mutagenic bypass are poorly understood. Here, we report kinetic and structural data providing evidence that, in T7 DNA polymerase, residue Lys536 is responsible for attenuating the miscoding potential of 8oG. The Lys536Ala polymerase shows a significant increase in mutagenic 8oG bypass versus wild-type polymerase, and a crystal structure of the Lys536Ala mutant reveals a closed complex with an 8oG(syn)·dATP mismatch in the polymerase active site, in contrast to the unproductive, open complex previously obtained by using wild-type polymerase. We propose that Lys536 acts as a steric and/or electrostatic filter that attenuates the miscoding potential of 8oG by normally interfering with the binding of 8oG in a syn conformation that pairs with dATP.
Bovine Mitochondrial Peroxiredoxin III Forms a Two-Ring Catenane by Zhenbo Cao; Aleksander W. Roszak; Louise J. Gourlay; J. Gordon Lindsay; Neil W. Isaacs (1661-1664).
A crystal structure is reported for the C168S mutant of a typical 2-Cys peroxiredoxin III (Prx III) from bovine mitochondria at a resolution of 3.3 Å. Prx III is present as a two-ring catenane comprising two interlocking dodecameric toroids that are assembled from basic dimeric units. Each ring has an external diameter of 150 Å and encompasses a central cavity that is 70 Å in width. The concatenated dodecamers are inclined at an angle of 55°, which provides a large contact surface between the rings. Dimer-dimer contacts involved in toroid formation are hydrophobic in nature, whereas the 12 areas of contact between interlocked rings arise from polar interactions. These two major modes of subunit interaction provide important insights into possible mechanisms of catenane formation.
Structural Basis of Severe Acute Respiratory Syndrome Coronavirus ADP-Ribose-1″-Phosphate Dephosphorylation by a Conserved Domain of nsP3 by Kumar Singh Saikatendu; Jeremiah S. Joseph; Vanitha Subramanian; Tom Clayton; Mark Griffith; Kin Moy; Jeffrey Velasquez; Benjamin W. Neuman; Michael J. Buchmeier; Raymond C. Stevens; Peter Kuhn (1665-1675).
The crystal structure of a conserved domain of nonstructural protein 3 (nsP3) from severe acute respiratory syndrome coronavirus (SARS-CoV) has been solved by single-wavelength anomalous dispersion to 1.4 Å resolution. The structure of this “X” domain, seen in many single-stranded RNA viruses, reveals a three-layered α/β/α core with a macro-H2A-like fold. The putative active site is a solvent-exposed cleft that is conserved in its three structural homologs, yeast Ymx7, Archeoglobus fulgidus AF1521, and Er58 from E. coli. Its sequence is similar to yeast YBR022W (also known as Poa1P), a known phosphatase that acts on ADP-ribose-1″-phosphate (Appr-1″-p). The SARS nsP3 domain readily removes the 1″ phosphate group from Appr-1″-p in in vitro assays, confirming its phosphatase activity. Sequence and structure comparison of all known macro-H2A domains combined with available functional data suggests that proteins of this superfamily form an emerging group of nucleotide phosphatases that dephosphorylate Appr-1″-p.
An Experimental Investigation of Conformational Fluctuations in Proteins G and L by Richard B. Tunnicliffe; Joe L. Waby; Ryan J. Williams; Mike P. Williamson (1677-1684).
The B1 domains of streptococcal proteins G and L are structurally similar, but they have different sequences and they fold differently. We have measured their NMR spectra at variable temperature using a range of concentrations of denaturant. Many residues have curved amide proton temperature dependence, indicating that they significantly populate alternative, locally unfolded conformations. The results, therefore, provide a view of the locations of low-lying, locally unfolded conformations. They indicate approximately 4–6 local minima for each protein, all within ca. 2.5 kcal/mol of the native state, implying a locally rough energy landscape. Comparison with folding data for these proteins shows that folding involves most molecules traversing a similar path, once a transition state containing a β hairpin has been formed, thereby defining a well-populated pathway down the folding funnel. The hairpin that directs the folding pathway differs for the two proteins and remains the most stable part of the folded protein.
The Binding Mode of the Trigger Factor on the Ribosome: Implications for Protein Folding and SRP Interaction by Frank Schlünzen; Daniel N. Wilson; Pingsheng Tian; Jörg M. Harms; Stuart J. McInnes; Harly A.S. Hansen; Renate Albrecht; Jörg Buerger; Sigurd M. Wilbanks; Paola Fucini (1685-1694).
This study presents the X-ray structure of the N-terminal binding domain of the D. radiodurans trigger factor (TF) in complex with the D. radiodurans large ribosomal subunit. At 3.35 Å, a complete description of the interactions with ribosomal proteins L23, L29, and 23S rRNA are disclosed, many of which differ from those found previously for a heterologous bacterial-archaeal TF-ribosome complex. The β hairpin loop of eubacterial L24, which is shorter in archaeal ribosomes, contacts the TF and severely diminishes the molecular cradle proposed to exist between the TF and ribosome. Bound to the ribosome, TF exposes a hydrophobic crevice large enough to accommodate the nascent polypeptide chain. Superimposition of the full-length TF and the signal-recognition particle (SRP) onto the complex shows that simultaneous cohabitation is possible, in agreement with biochemical data, and suggests a model for the interplay of TF, SRP, and the nascent chain during translation.
Structure of the Hepatitis C Virus IRES Bound to the Human 80S Ribosome: Remodeling of the HCV IRES by Daniel Boehringer; Rolf Thermann; Antje Ostareck-Lederer; Joe D. Lewis; Holger Stark (1695-1706).
Initiation of translation of the hepatitis C virus (HCV) polyprotein is driven by an internal ribosome entry site (IRES) RNA that bypasses much of the eukaryotic translation initiation machinery. Here, single-particle electron cryomicroscopy has been used to study the mechanism of HCV IRES-mediated initiation. A HeLa in vitro translation system was used to assemble human IRES-80S ribosome complexes under near physiological conditions; these were stalled before elongation. Domain 2 of the HCV IRES is bound to the tRNA exit site, touching the L1 stalk of the 60S subunit, suggesting a mechanism for the removal of the HCV IRES in the progression to elongation. Domain 3 of the HCV IRES positions the initiation codon in the ribosomal mRNA binding cleft by binding helix 28 at the head of the 40S subunit. The comparison with the previously published binary 40S-HCV IRES complex reveals structural rearrangements in the two pseudoknot structures of the HCV IRES in translation initiation.
Substrate-Induced Conformational Changes in Bacillus subtilis Glutamate Racemase and Their Implications for Drug Discovery by Sergey N. Ruzheinikov; Makie A. Taal; Svetlana E. Sedelnikova; Patrick J. Baker; David W. Rice (1707-1713).
D-glutamate is an essential building block of the peptidoglycan layer in bacterial cell walls and can be synthesized from L-glutamate by glutamate racemase (RacE). The structure of a complex of B. subtilis RacE with D-glutamate reveals that the glutamate is buried in a deep pocket, whose formation at the interface of the enzyme's two domains involves a large-scale conformational rearrangement. These domains are related by pseudo-2-fold symmetry, which superimposes the two catalytic cysteine residues, which are located at equivalent positions on either side of the α carbon of the substrate. The structural similarity of these two domains suggests that the racemase activity of RacE arose as a result of gene duplication. The structure of the complex is dramatically different from that proposed previously and provides new insights into the RacE mechanism and an explanation for the potency of a family of RacE inhibitors, which have been developed as novel antibiotics.
The N-Terminal End of the Catalytic Domain of Src Kinase Hck Is a Conformational Switch Implicated in Long-Range Allosteric Regulation by Nilesh K. Banavali; Benoît Roux (1715-1723).
Signal transduction in cell growth and proliferation involves regulation of kinases through long-range allostery between remote protein regions. Molecular dynamics free energy calculations are used to clarify the coupling between the catalytic domain of Src kinase Hck and its N-terminal end connecting to the regulatory SH2 and SH3 modules. The N-terminal end is stable in the orientation required for the regulatory modules to remain properly bound only in the inactive catalytic domain. In the active catalytic domain, the N-terminal end prefers a different conformation consistent with dissociation of the regulatory modules. The free energy surface shows that the N-terminal end acts as a reversible two-state conformational switch coupling the catalytic domain to the regulatory modules. Structural analogy with insulin receptor kinase and c-Src suggests that such reversible conformational switching in a critical hinge region could be a common mechanism in long-range allosteric regulation of protein kinase activity.
A Structure of the Human Apoptosome at 12.8 Å Resolution Provides Insights into This Cell Death Platform by Xinchao Yu; Devrim Acehan; Jean-François Ménétret; Christopher R. Booth; Steven J. Ludtke; Stefan J. Riedl; Yigong Shi; Xiaodong Wang; Christopher W. Akey (1725-1735).
Apaf-1 and cytochrome c coassemble in the presence of dATP to form the apoptosome. We have determined a structure of the apoptosome at 12.8 Å resolution by using electron cryomicroscopy and single-particle methods. We then docked appropriate crystal structures into the map to create an accurate domain model. Thus, we found that seven caspase recruitment domains (CARDs) form a central ring within the apoptosome. At a larger radius, seven copies of the nucleotide binding and oligomerization domain (NOD) associate laterally to form the hub, which encircles the CARD ring. Finally, an arm-like helical domain (HD2) links each NOD to a pair of β propellers, which bind a single cytochrome c. This model provides insights into the roles of dATP and cytochrome c in assembly. Our structure also reveals how a CARD ring and the central hub combine to create a platform for procaspase-9 activation.