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Structure (v.14, #11)

Single-Ring GroEL: An Expanded View by Sharon Grayer Wolf (pp. 1599-1600).
A remarkable structure of an 86 kDa substrate encapsulated in a single-ring GroEL/GroES chaperonin complex is revealed by cryo-electron microscopy in this issue of Structure (). Surprisingly, the protein-folding chamber is 80% larger than that of the double-ring GroEL/ES structure.

RecA Assembly, One Molecule at a Time by Edward H. Egelman (pp. 1600-1602).
Several recent papers have applied optical methods to directly visualize the assembly of individual RecA and Rad51 filaments on DNA. The hope is that application of such methods will shed light on the many mysteries that still surround how these remarkable filaments function in genetic recombination.

The Unmasking of Telomerase by Jason D. Legassie; Michael B. Jarstfer (pp. 1603-1609).
Telomerase is a ribonucleoprotein complex that reverse transcribes a portion of its RNA subunit during the synthesis of G-rich DNA at the 3′ end of each chromosome in most eukaryotes. This activity compensates for the inability of the normal DNA replication machinery to fully replicate chromosome termini. The roles of telomerase in cellular immortality and tumor biology have catalyzed a significant interest in this unusual polymerase. Recently the first structures of two domains, the CR4/CR5 and pseudoknot, of human telomerase RNA (hTR) were reported, offering a structural basis for interpreting biochemical studies and possible roles of hTR mutations in human diseases. Structures of the stem II and stem IV domains of Tetrahymena thermophila TR as well as the N-terminal domain of the T. thermophila telomerase reverse transcriptase have also been determined. These studies complement previous biochemical studies, providing rich insight into the structural basis for telomerase activity.

Complement and the Multifaceted Functions of VWA and Integrin I Domains by Timothy A. Springer (pp. 1611-1616).
The recent crystal structure of complement protein component C2a reveals an interface between its VWA and serine protease domains that could not exist in the zymogen C2. The implied change in VWA domain conformation between C2 and C2a differs from that described for other VWA domains, including the I domains in integrins. Here, the remarkable diversity in both conformational regulation and ligand binding among VWA domains that function in complement, hemostasis, cell adhesion, anthrax toxin binding, vesicle transport, DNA break repair, and RNA quality control is reviewed. Finally, implications for metastability of complement convertases are discussed.

Lysine Methylation as a Routine Rescue Strategy for Protein Crystallization by Thomas S. Walter; Christoph Meier; Rene Assenberg; Kin-Fai Au; Jingshan Ren; Anil Verma; Joanne E. Nettleship; Raymond J. Owens; David I. Stuart; Jonathan M. Grimes (pp. 1617-1622).
Crystallization remains a critical step in X-ray structure determination. Because it is not generally possible to rationally predict crystallization conditions, commercial screens have been developed which sample a wide range of crystallization space. While this approach has proved successful in many cases, a significant number of proteins fail to crystallize despite being soluble and monodispersed. It is established that chemical modification can facilitate the crystallization of otherwise intractable proteins. Here we describe a method for the reductive methylation of lysine residues which is simple, inexpensive, and efficient, and report on its application to ten proteins. We describe the effect of methylation on the physico-chemical properties of these proteins, and show that it led to diffraction-quality crystals from four proteins and structures for three that had hitherto proved refractory to crystallization. The method is suited to both low- and high-throughput laboratories.

Purification and 3D Structural Analysis of Oligomeric Human Multidrug Transporter ABCG2 by Christopher A. McDevitt; Richard F. Collins; Michael Conway; Szabolcs Modok; Janet Storm; Ian D. Kerr; Robert C. Ford; Richard Callaghan (pp. 1623-1632).
ABCG2 is a multidrug efflux pump associated with resistance of cancer cells to a plethora of unrelated drugs. ABCG2 is a “half-transporter,? and previous studies have indicated that it forms homodimers and higher oligomeric species. In this manuscript, electron microscopic structural analysis directly addressed this issue. An N-terminal hexahistidine-tagged ABCG2R482G isoform was expressed to high levels in insect cells. An extensive detergent screen was employed to effect extraction of ABCG2R482G from membranes and identified only the fos-choline detergents as efficient. Soluble protein was purified to >95% homogeneity by a three-step procedure while retaining the ability to bind substrates. Cryonegative stain electron microscopy of purified ABCG2R482G provided 3D structural data at a resolution of ∼18 Å. Single-particle analysis revealed that the complex forms a tetrameric complex (∼180 Å in diameter × ∼140 Å high) with an aqueous central region. We interpret the tetrameric structure as comprising four homodimeric ABCG2R482G complexes.


Pathways and Kinetic Barriers in Mechanical Unfolding and Refolding of RNA and Proteins by Changbong Hyeon; Ruxandra I. Dima; D. Thirumalai (pp. 1633-1645).
Using self-organized polymer models, we predict mechanical unfolding and refolding pathways of ribozymes, and the green fluorescent protein. In agreement with experiments, there are between six and eight unfolding transitions in the Tetrahymena ribozyme. Depending on the loading rate, the number of rips in the force-ramp unfolding of the Azoarcus ribozymes is between two and four. Force-quench refolding of the P4-P6 subdomain of the Tetrahymena ribozyme occurs through a compact intermediate. Subsequent formation of tertiary contacts between helices P5b-P6a and P5a/P5c-P4 leads to the native state. The force-quench refolding pathways agree with ensemble experiments. In the dominant unfolding route, the N-terminal α helix of GFP unravels first, followed by disruption of the N terminus β strand. There is a third intermediate that involves disruption of three other strands. In accord with experiments, the force-quench refolding pathway of GFP is hierarchic, with the rate-limiting step being the closure of the barrel.


Interpreting Correlated Motions Using Normal Mode Analysis by Adam W. Van Wynsberghe; Qiang Cui (pp. 1647-1653).
With the increased popularity of normal mode analyses in structural biology, it is important to carefully consider how to best utilize the results for gaining biological insights without over interpretation. The discussion in this article argues that for the purpose of identifying correlated motions in biomolecules, a case separate from concomitant conformational changes of structural motifs, it is generally important to use a large number of normal modes. This is illustrated through three increasingly complex examples. The simplest case includes two bilinearly coupled harmonic oscillators and serves as a straightforward problem where the important considerations are explicit and transparent. The argument is then generalized to include a system of N-coupled harmonic oscillators and finally to a realistic biomolecule. Although a small number of normal modes are useful for probing structural flexibility, it is clear that a much larger number of modes are required for properly investigating correlated motions in biomolecules.

Capsid Conformational Sampling in HK97 Maturation Visualized by X-Ray Crystallography and Cryo-EM by Lu Gan; Jeffrey A. Speir; James F. Conway; Gabriel Lander; Naiqian Cheng; Brian A. Firek; Roger W. Hendrix; Robert L. Duda; Lars Liljas; John E. Johnson (pp. 1655-1665).
Maturation of the bacteriophage HK97 capsid from a precursor (Prohead II) to the mature state (Head II) involves a 60 Å radial expansion. The mature particle is formed by 420 copies of the major capsid protein organized on a T = 7 laevo lattice with each subunit covalently crosslinked to two neighbors. Well-characterized pH 4 expansion intermediates make HK97 valuable for investigating quaternary structural dynamics. Here, we use X-ray crystallography and cryo-EM to demonstrate that in the final transition in maturation (requiring neutral pH), pentons in Expansion Intermediate IV (EI-IV) reversibly sample 14 Å translations and 6° rotations relative to a fixed hexon lattice. The limit of this trajectory corresponds to the Head II conformation that is secured at this extent only by the formation of the final class of covalent crosslinks. Mutants that cannot crosslink or EI-IV particles that have been rendered incapable of forming the final crosslink remain in the EI-IV state.

Proline and Glycine Control Protein Self-Organization into Elastomeric or Amyloid Fibrils by Sarah Rauscher; Stéphanie Baud; Ming Miao; Fred W. Keeley; Régis Pomès (pp. 1667-1676).
Elastin provides extensible tissues, including arteries and skin, with the propensity for elastic recoil, whereas amyloid fibrils are associated with tissue-degenerative diseases, such as Alzheimer's. Although both elastin-like and amyloid-like materials result from the self-organization of proteins into fibrils, the molecular basis of their differing physical properties is poorly understood. Using molecular simulations of monomeric and aggregated states, we demonstrate that elastin-like and amyloid-like peptides are separable on the basis of backbone hydration and peptide-peptide hydrogen bonding. The analysis of diverse sequences, including those of elastin, amyloids, spider silks, wheat gluten, and insect resilin, reveals a threshold in proline and glycine composition above which amyloid formation is impeded and elastomeric properties become apparent. The predictive capacity of this threshold is confirmed by the self-assembly of recombinant peptides into either amyloid or elastin-like fibrils. Our findings support a unified model of protein aggregation in which hydration and conformational disorder are fundamental requirements for elastomeric function.

Keywords: PROTEINS

Solution Structures of the SURP Domains and the Subunit-Assembly Mechanism within the Splicing Factor SF3a Complex in 17S U2 snRNP by Kanako Kuwasako; Fahu He; Makoto Inoue; Akiko Tanaka; Sumio Sugano; Peter Güntert; Yutaka Muto; Shigeyuki Yokoyama (pp. 1677-1689).
The SF3a complex, consisting of SF3a60, SF3a66, and SF3a120, in 17S U2 snRNP is crucial to spliceosomal assembly. SF3a120 contains two tandem SURP domains (SURP1 and SURP2), and SURP2 is responsible for binding to SF3a60. We found that the SURP2 fragment forms a stable complex with an SF3a60 fragment (residues 71–107) and solved its structure by NMR spectroscopy. SURP2 exhibits a fold of the α1-α2-310-α3 topology, and the SF3a60 fragment forms an amphipathic α helix intimately contacting α1 of SURP2. We also solved the SURP1 structure, which has the same fold as SURP2. The protein-binding interface of SURP2 is quite similar to the corresponding surface of SURP1, except for two amino acid residues. One of them, Leu169, is characteristic of SF3a120 SURP2 among SURP domains. Mutagenesis showed that this single Leu residue is the critical determinant for complex formation, which reveals the protein recognition mechanism in the subunit assembly.

Molecular Architecture and Conformational Flexibility of Human RNA Polymerase II by Seth A. Kostek; Patricia Grob; Sacha De Carlo; J. Slaton Lipscomb; Florian Garczarek; Eva Nogales (pp. 1691-1700).
Transcription by RNA polymerase II (RNAPII) is a central process in eukaryotic gene regulation. While atomic details exist for the yeast RNAPII, characterization of the human complex lags behind, mostly due to the inability to obtain large quantities of purified material. Although the complexes have the same protein composition and high sequence similarity, understanding of transcription and of transcription-coupled DNA repair (TCR) in humans will require the use of human proteins in structural studies. We have used cryo-electron microscopy, image reconstruction, and variance analysis to characterize the structure and dynamics of human RNAPII (hRNAPII). Our studies show that hRNAPII in solution parallels the conformational flexibility of the yeast structures crystallized in different states but also illustrate a more extensive conformational range with potential biological significance. This hRNAPII study will serve as a structural platform to build up higher-order transcription and TCR complexes and to gain information that may be unique to the human RNAPII system.

Keywords: DNA

Structural Basis for Target Protein Recognition by the Protein Disulfide Reductase Thioredoxin by Kenji Maeda; Per Hägglund; Christine Finnie; Birte Svensson; Anette Henriksen (pp. 1701-1710).
Thioredoxin is ubiquitous and regulates various target proteins through disulfide bond reduction. We report the structure of thioredoxin (HvTrxh2 from barley) in a reaction intermediate complex with a protein substrate, barley α-amylase/subtilisin inhibitor (BASI). The crystal structure of this mixed disulfide shows a conserved hydrophobic motif in thioredoxin interacting with a sequence of residues from BASI through van der Waals contacts and backbone-backbone hydrogen bonds. The observed structural complementarity suggests that the recognition of features around protein disulfides plays a major role in the specificity and protein disulfide reductase activity of thioredoxin. This novel insight into the function of thioredoxin constitutes a basis for comprehensive understanding of its biological role. Moreover, comparison with structurally related proteins shows that thioredoxin shares a mechanism with glutaredoxin and glutathione transferase for correctly positioning substrate cysteine residues at the catalytic groups but possesses a unique structural element that allows recognition of protein disulfides.

Keywords: PROTEINS

An Expanded Conformation of Single-Ring GroEL-GroES Complex Encapsulates an 86 kDa Substrate by Dong-Hua Chen; Jiu-Li Song; David T. Chuang; Wah Chiu; Steven J. Ludtke (pp. 1711-1722).
Electron cryomicroscopy reveals an unprecedented conformation of the single-ring mutant of GroEL (SR398) bound to GroES in the presence of Mg-ATP. This conformation exhibits a considerable expansion of the folding cavity, with ∼80% more volume than the X-ray structure of the equivalent cis cavity in the GroEL-GroES-(ADP)7 complex. This expanded conformation can encapsulate an 86 kDa heterodimeric (αβ) assembly intermediate of mitochondrial branched-chain α-ketoacid dehydrogenase, the largest substrate ever observed to be cis encapsulated. The SR398-GroES-Mg-ATP complex is found to exist as a mixture of standard and expanded conformations, regardless of the absence or presence of the substrate. However, the presence of even a small substrate causes a pronounced bias toward the expanded conformation. Encapsulation of the large assembly intermediate is supported by a series of electron cryomicroscopy studies as well as the protection of both α and β subunits of the substrate from tryptic digestion.

Determinants of Bacteriophage ϕ29 Head Morphology by Kyung H. Choi; Marc C. Morais; Dwight L. Anderson; Michael G. Rossmann (pp. 1723-1727).
Bacteriophage ϕ29 requires scaffolding protein to assemble the 450 × 540 Å prolate prohead with T = 3 symmetry end caps. In infections with a temperature-sensitive mutant scaffolding protein, capsids assemble predominantly into 370 Å diameter isometric particles with T = 3 symmetry that lack a head-tail connector. However, a few larger, 430 Å diameter, particles are also assembled. Cryo-electron microscopy shows that these larger particles are icosahedral with T = 4 symmetry. The prolate prohead, as well as the two isometric capsids with T = 3 and T = 4 symmetry, are composed of similar pentamers and differently skewed hexamers. The skewing of the hexamers in the equatorial region of proheads and in the T = 4 isometric particles reflects their different environments. One of the functions of the scaffolding protein, present in the prohead, may be to stabilize skewed hexamers during assembly.
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