Structure (v.18, #7)

In This Issue (v-vi).

The Apo Riboswitch as a Molecular Hydra by Joseph E. Wedekind (757-758).
Riboswitches “sense” metabolites but knowledge is sparse for structures without bound ligand. determined an apo riboswitch structure “closed” to metabolite binding. Further SAXS, biochemical, and computational analyses support ensemble behavior with interconverting open and closed conformations.

Mg2+ Channel Selectivity Probed by EPR by Yeon-Kyun Shin (759-760).
The functionally important extracellular loop is not resolved in the crystal structures of a putative bacterial Mg2+ channel CorA. In this issue, Dalmas et al. use EPR to determine a structural model for this conserved loop, providing new insight into the ion selectivity.

Multiple Conformations of F-actin by Toshiro Oda; Yuichiro Maéda (761-767).
Actin works within eukaryotic cells to facilitate a variety of cellular processes, which are driven by the assembly of G-actin (monomeric form) into F-actin (fibrous form), and the disassembly of F-actin into G-actin. F-actin adopts multiple conformations, which are specified by interactions with various actin-binding proteins. Knowledge of the multiple conformations of actin is the key for understanding its cellular functions. Recently, we published a refined model for F-actin. In this review, based on this model, we discuss the origin, mechanism, and possible physiological significance of the multiple conformations of F-actin.
Keywords: CELLBIO;

In this paper, we review current practices for establishing the resolution in single-particle reconstructions. The classical Raleigh criterion for the resolution is not applicable in this case, and the resolution is commonly defined by a consistency test, whereby the data set is randomly split in half and the two resulting reconstructions are then compared. Such a procedure, however, may introduce statistical dependence between the two half-sets, which leads to a too optimistic resolution estimate. On the other hand, this overestimation is counteracted by the diminished statistical properties of a mere half of the data set. The “true” resolution of the whole data set can be estimated when the functional relationship between the data size and the resolution is known. We are able to estimate this functional by taking into account the B-factor and the geometry of data collection. Finally, the drawbacks of resolution estimation are entirely avoided by computing the correlation of neighboring voxels in the Fourier domain.► Review of current practices for establishing the resolution in single-particle reconstructions ► Drawbacks of the methods of comparison between two half-sets ► Functional relationship between the data size and the resolution revisited ► Fourier neighbor correlation as a new way of establishing the resolution
Keywords: PROTEINS;

We have developed methods for ab initio three-dimensional (3D) structure determination from projection images of randomly oriented single molecules coexisting in multiple functional states, to aid the study of complex samples of macromolecules and nanoparticles by electron microscopy (EM). New algorithms for the determination of relative 3D orientations and conformational state assignment of single-molecule projection images are combined with well-established techniques for alignment and statistical image analysis. We describe how the methodology arrives at homogeneous groups of images aligned in 3D and discuss application to experimental EM data sets of the Escherichia coli ribosome and yeast RNA polymerase II.► New technique for structure determination of large macromolecules ► No a priori information needed to reconstruct coexisting conformational states
Keywords: PROTEINS;

Free State Conformational Sampling of the SAM-I Riboswitch Aptamer Domain by Colby D. Stoddard; Rebecca K. Montange; Scott P. Hennelly; Robert P. Rambo; Karissa Y. Sanbonmatsu; Robert T. Batey (787-797).
Riboswitches are highly structured elements residing in the 5′ untranslated region of messenger RNAs that specifically bind cellular metabolites to alter gene expression. While there are many structures of ligand-bound riboswitches that reveal details of bimolecular recognition, their unliganded structures remain poorly characterized. Characterizing the molecular details of the unliganded state is crucial for understanding the riboswitch's mechanism of action because it is this state that actively interrogates the cellular environment and helps direct the regulatory outcome. To develop a detailed description of the ligand-free form of an S-adenosylmethionine binding riboswitch at the local and global levels, we have employed a series of biochemical, biophysical, and computational methods. Our data reveal that the ligand binding domain adopts an ensemble of states that minimizes the energy barrier between the free and bound states to establish an efficient decision making branchpoint in the regulatory process.Display Omitted► SAXS reveals a dynamic RNA that involves global conformational changes ► Crystal structure of an active unliganded SAM-I riboswitch was determined ► REMD simulations uncover motions in the SAM binding pocket ► The free RNA adopts an ensemble of conformers that includes bound-like states
Keywords: RNA;

Structural and Theoretical Studies Indicate that the Cylindrical Protease ClpP Samples Extended and Compact Conformations by Matthew S. Kimber; Angela Yeou Hsiung Yu; Mikael Borg; Elisa Leung; Hue Sun Chan; Walid A. Houry (798-808).
The highly conserved ClpP protease consists of two heptameric rings that interact by the interdigitation of an α-helix β strand handle domain motif to form a tetradecameric cylinder. We previously proposed that protease dynamics results in the temporary unstructuring of interacting pairs of handle domains, opening transient equatorial side pores that allow for peptide egress. Here, we report the structure of an Escherichia coli ClpP mutant in which each opposing pair of protomers is linked by a disulfide bond. This structure resembles the compact structures of Streptococcus pneumoniae, Mycobacterium tuberculosis, and Plasmodium falciparum ClpPs, rather than the active, extended structures that have previously been determined for E. coli ClpPs. The structural data, along with normal mode analysis, support a model whereby the ClpP cylinder switches dynamically between an active extended state required for substrate degradation and an inactive compact state allowing peptide product release.Display Omitted► The ClpP cylinder is a dynamic structure ► ClpP samples an extended and a compact state ► The extended state is proposed to be required for substrate degradation ► The compact state is proposed to allow for peptide-product exit
Keywords: PROTEINS;

An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis by Elif Eren; Megan Murphy; Jon Goguen; Bert van den Berg (809-818).
The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 Å. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.► We have determined the first high-resolution structure of an active omptin ► The Pla structure shows the active site nucleophilic water for the first time ► Catalytically important residues are linked by an active site water network ► The structure provides an explanation for the activation of omptins by LPS

Thermophilic Adaptation of Protein Complexes Inferred from Proteomic Homology Modeling by Bin-Guang Ma; Alexander Goncearenco; Igor N. Berezovsky (819-828).
As protein complexes must remain in their native conformations at habitat temperatures, thermal adaptation requires adjustment of their parts and interactions between them. Based on independent sets of structural templates and sequences of 127 complete prokaryotic proteomes with optimal growth temperatures from 8°C to 100°C, we performed proteomic homology modeling of complexes and analyzed peculiarities in their traits related to thermal adaptation. We explore compositional determinants of thermostability of protein complexes based on the model of stability including negative and positive components of design. We show that positively charged amino acids play an important role in protein complexes, working in negative design against misfolded conformations and aberrant assemblies and contributing to positive design by stabilizing both the native interface and the overall structure of the complex. Aggregation propensity of interfaces is higher than that of surfaces and the difference between them increases with optimal growth temperature securing native complexes in hot environments.► Proteomic homology modeling ► Role of negative design in thermostability of protein complexes ► Differential aggregation propensity is a new characteristic of protein-protein interfaces ► Negative design is provided by positive charges in protein complexes
Keywords: PROTEINS;

Topoisomerase V is an archaeal type I topoisomerase that is unique among topoisomerases due to presence of both topoisomerase and DNA repair activities in the same protein. It is organized as an N-terminal topoisomerase domain followed by 24 tandem helix-hairpin-helix (HhH) motifs. Structural studies have shown that the active site is buried by the (HhH) motifs. Here we show that the N-terminal domain can relax DNA in the absence of any HhH motifs and that the HhH motifs are required for stable protein-DNA complex formation. Crystal structures of various topoisomerase V fragments show changes in the relative orientation of the domains mediated by a long bent linker helix, and these movements are essential for the DNA to enter the active site. Phosphate ions bound to the protein near the active site helped model DNA in the topoisomerase domain and show how topoisomerase V may interact with DNA.► The N-terminal domain of topoisomerase V has topoisomerase activity ► Movement of the (HhH)2 domains is mediated by a linker helix ► The (HhH)2 domains could help enclose DNA during the swiveling of the DNA ► Phosphate ions bound in the DNA binding groove provide a possible path for the DNA
Keywords: PROTEINS; DNA;

Conformational Changes During the Gating of a Potassium Channel Revealed by Structural Mass Spectrometry by Sayan Gupta; Vassiliy N. Bavro; Rhijuta D'Mello; Stephen J. Tucker; Catherine Vénien-Bryan; Mark R. Chance (839-846).
Potassium channels are dynamic proteins that undergo large conformational changes to regulate the flow of K+ ions across the cell membrane. Understanding the gating mechanism of these channels therefore requires methods for probing channel structure in both their open and closed conformations. Radiolytic footprinting is used to study the gating mechanism of the inwardly-rectifying potassium channel KirBac3.1. The purified protein stabilized in either open or closed conformations was exposed to focused synchrotron X-ray beams on millisecond timescales to modify solvent accessible amino acid side chains. These modifications were identified and quantified using high-resolution mass spectrometry. The differences observed between the closed and open states were then used to reveal local conformational changes that occur during channel gating. The results provide support for a proposed gating mechanism of the Kir channel and demonstrate a method of probing the dynamic gating mechanism of other integral membrane proteins and ion channels.Display Omitted► Radiolytic labeling and mass spectroscopy is used to analyze KirBac3.1 gating ► Results identify conformationally sensitive residues near selectivity filter ► Largest structural changes is observed within the cavity of the membrane pore ► Results indicate direct evidence for dynamic role of G-loop and CD-loop for gating

Dynamics of SecY Translocons with Translocation-Defective Mutations by Ana-Nicoleta Bondar; Coral del Val; J. Alfredo Freites; Douglas J. Tobias; Stephen H. White (847-857).
The SecY/Sec61 translocon complex, located in the endoplasmic reticulum membrane of eukaryotes (Sec61) or the plasma membrane of prokaryotes (SecY), mediates the transmembrane secretion or insertion of nascent proteins. Mutations that permit the secretion of nascent proteins with defective signal sequences (Prl-phenotype), or interfere with the transmembrane orientation of newly synthesized protein segments, can affect protein topogenesis. The crystallographic structure of SecYEβ from Methanococcus jannaschii revealed widespread distribution of mutations causing topogenesis defects, but not their molecular mechanisms. Based upon prolonged molecular dynamics simulations of wild-type M. jannaschii SecYEβ and an extensive sequence-conservation analysis, we show that the closed state of the translocon is stabilized by hydrogen-bonding interactions of numerous highly conserved amino acids. Perturbations induced by mutation at various locations are rapidly relayed to the plug segment that seals the wild-type closed-state translocon, leading to displacement and increased hydration of the plug.Display Omitted► H-bonding amino acids form a network that stabilizes the SecY closed state ► Mutation-induced perturbations are relayed throughout SecY via the network ► Perturbations in plug location and water distribution explain translocation defects ► Conservation pattern of critical H-bonding amino acids depends on the organism

Protein template identification is essential to protein structure and function predictions. However, conventional whole-chain threading approaches often fail to recognize conserved substructure motifs when the target and templates do not share the same fold. We developed a new approach, SEGMER, for identifying protein substructure similarities by segmental threading. The target sequence is split into segments of two to four consecutive or nonconsecutive secondary structural elements, which are then threaded through PDB to identify appropriate substructure motifs. SEGMER is tested on 144 nonredundant hard proteins. When combined with whole-chain threading, the TM-score of alignments and accuracy of spatial restraints of SEGMER increase by 16% and 25%, respectively, compared with that by the whole-chain threading methods only. When tested on 12 free modeling targets from CASP8, SEGMER increases the TM-score and contact accuracy by 28% and 48%, respectively. This significant improvement should have important impact on protein structure modeling and functional inference.Display Omitted► New method for detecting substructure motifs between distant-homologous proteins ► Significant improvement in the ability of substructure identification ► Alignment accuracy increases by 16% as measured by TM-score ► Accuracy of combined residue-residue contact restraints increases by 25%
Keywords: PROTEINS;

Structural Dynamics of the Magnesium-Bound Conformation of CorA in a Lipid Bilayer by Olivier Dalmas; Luis G. Cuello; Vishwanath Jogini; D. Marien Cortes; Benoit Roux; Eduardo Perozo (868-878).
The transmembrane conformation of Thermotoga maritima CorA, a magnesium transport system, has been studied in its Mg2+-bound form by site-directed spin labeling and electron paramagnetic resonance spectroscopy. Probe mobility together with accessibility data were used to evaluate the overall dynamics and relative arrangement of individual transmembrane segments TM1 and TM2. TM1 extends toward the cytoplasmic side creating a water-filled cavity, while TM2 is located in the periphery of the oligomer, contacting the lipid bilayer. A structural model for the conserved extracellular loop was generated based on EPR data and MD simulations, in which residue E316 is located toward the five-fold symmetry axis in position to electrostatically influence divalent ion translocation. Electrostatic analysis of our model suggest that, in agreement with the crystal structure, Mg2+ -bound CorA is in a closed conformation. The present results suggest that long-range structural rearrangements are necessary to allow Mg2+ translocation.Display Omitted► The conformation of Mg2+-bound CorA was evaluated using EPR spectroscopy ► We find overall structural correspondence with the recent crystal structures ► A model for the highly conserved extracellular loop was generated based on EPR data ► The loop might serve as an electrostatic sink and may participate in Mg2+ binding
Keywords: PROTEINS;

Why an A-Loop Phospho-Mimetic Fails to Activate PAK1: Understanding an Inaccessible Kinase State by Molecular Dynamics Simulations by Yuen-Wai Ng; Devanathan Raghunathan; Perry M. Chan; Yohendran Baskaran; Derek J. Smith; Chung-Hung Lee; Chandra Verma; Ed Manser (879-890).
Crystal structures of inactive PAK1(K299R) and the activation (A)-loop phospho-mimetic PAK1(T423E) have suggested that the kinase domain is in an active state regardless of activation loop status. Contrary to a large body of literature, we find that neither is PAK1(T423E) active in cells, nor does it exhibit significant activity in vitro. To explain these discrepancies all-atom molecular dynamics (MD) simulations of PAK1(phospho-T423) in complex with ATP and substrate were performed. These simulations point to a key interaction between PAK1 Lys308, at the end of the αC helix, and the pThr423 phosphate group, not seen in X-ray structures. The orthologous PAK4 Arg359 fulfills the same role in immobilizing the αC helix. These in silico predictions were validated by experimental mutagenesis of PAK1 and PAK4. The simulations explain why the PAK1 A-loop phospho-mimetic is inactive, but also point to a key functional interaction likely found in other protein kinases.► The widely used PAK1(T423E) mutant is revealed to be inactive ► MD simulations of active pThr432-PAK1 uncover new interactions ► The importance of α-C helix Lys308 is validated experimentally ► PAK4 simulation reveal analogous coupling of pS474 and α-C helix
Keywords: SIGNALING;